KR20160147055A - Sustained release angiogenesis modulating compositions and methods for induction and modulation of angiogenesis - Google Patents

Abstract

The present disclosure relates to compositions comprising human placenta extract and biodegradable microparticles, compositions for the controlled release angiogenesis, compositions and methods for the targeted release of placental extract over a period of time, and methods for inducing and / or regulating angiogenesis , And a method for identifying a regulator of angiogenesis. The present disclosure also provides a method of making a composition comprising a placenta extract capable of inducing and / or modulating angiogenesis.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for sustained angiogenesis regulation and a method for inducing and regulating angiogenesis,

Cross-reference to related application

This application claims the benefit of U.S. Provisional Application Serial No. 61 / 990,256 filed May 8, 2014 entitled "Compositions and Methods for Identification of Compositions and Methods for the Induction and Regulation of Angiogenesis and Angiogenesis Modulators " " for < / RTI > Induction and Modulation of Angiogenesis and Methods and Assays for Identifying Angiogenesis Modulators "), which application is incorporated herein by reference.

Reference to federal funding research or development

The present invention was made with government support under subsidy number HL088207 granted by the National Institutes of Health. The government has certain rights in the invention.

Angiogenesis can form blood vessels from physiological and pathological conditions, from wound healing to cancer. Angiogenesis regulation depends on both location and stimulation, and may in each case involve a unique combination of regulatory molecules.

The lack of ability to vascularize in engineered organs and to re-vascularize at infarct sites has been a major obstacle to successful regenerative medicine therapy in clinical applications. The ability to regulate angiogenesis in a limited manner has a significant impact on a wide range of clinical applications, from defining normal and pathological vascular physiology, tissue / organ regeneration, wound healing, infarct tissue repair, and inhibition of cancer. A variety of approaches have been considered to initiate angiogenesis and to induce greater angiogenesis, including direct cell seeding (single culture or co-culture), stem cell use, and a combination of human derived regulators / growth factors. To date, such in vitro approaches, which typically utilize non-human animal compounds, have been shown to be clinically transformed due to their individual protein organization, non-human origin, tumor origin, or lack of genetic control, Have not been successful.

Current methods for inducing angiogenesis in vitro consist of a simple combination of human-derived regulators, which are wholly dependent on using animal-derived stimulants or using live animals for evaluation. Using such current in vitro models consisting of a simple combination of human and / or animal-derived regulators to test for potent angiostatic inhibitors does not represent a broad set of human in vivo molecular interactions, Limit the process. Since the regulation of angiogenesis requires the induction of many metabolic pathways, the regulation of the only selected molecular pathway also restricts attempts to prevascularize the engineered organ. Also, at present, the most common and successful approach is to use Matrigel (TM), a material derived from Engelbreth-Holm-Swarm mouse sarcoma cells, which is considered inappropriate for human therapy have. Thus, as a method of inducing and modulating angiogenesis, an improved human-based method can be a driving force for both pharmaceutical development and regenerative medicine.

summary

Briefly described, embodiments of the present disclosure provide a human placenta extract composition comprising human placental extract, a composition for the controlled release of angiogenesis, an angiogenesis inducing method, and an anti-inflammatory composition and method.

The present disclosure provides a composition comprising a human placenta extract coupled to a biodegradable microparticle such that the human placental extract can be released from the microparticles after exposure to the cells in vivo or in vitro. The human placenta extract is obtained from a human placenta sample in which blood and solids are substantially removed from the extract, and the extract includes placental proteins, including cytokines and growth factors, present in placental samples.

An embodiment of the method of inducing angiogenesis of the present disclosure comprises contacting a cell with a composition for the controlled release angiogenesis wherein the composition for the controlled release angiogenesis is administered to a cell of a microparticle in vivo or in vitro Biodegradable microparticles coupled to the human placenta extract of the present disclosure so that the human placenta extract can be released from the microparticles over a period of time after exposure of the human placenta extract. In an embodiment, the method further comprises coupling the sustained angiogenesis-regulating composition to the biomaterial to induce vascularization of the biomaterial.

The present disclosure also provides a method of reducing inflammation in a subject by administering a human placenta extract of the present disclosure and / or a sustained / human placenta extract composition of the disclosure to a subject in need of treatment of inflammation.

Other methods, compositions, features, and advantages of the disclosure will be apparent to those skilled in the art upon examination of the following figures and detailed description. All of the above additional compositions, methods, features and advantages are included within the scope of this disclosure and are intended to be included within the scope of this disclosure.

Brief Description of Drawings
Additional aspects of the present disclosure will become more readily appreciated as the same becomes better understood by reference to the following detailed description of various embodiments thereof, when taken in conjunction with the accompanying drawings.
Figures la-lk show the formation of human placental extract (hPE), also referred to herein as human placental matrix or h PM (human placenta matrix), characterization of hPE membranes, and formation of blood vessels And characterization of the emerging networks. Figure 1A shows an embodiment of the step of homogenizing the placental ECM followed by urea solubilization and dialysis to obtain hPM. FIGS. 1B-1C are SEM images showing the surface morphology of hPE, and FIGS. 1D-1E show the formation of angiogenic networks when HUVECS was seeded at ⁴ × 10 ⁴ cells / cm 2 on h Pm thin film and cultured for 3 days . Figure 1f shows Rhodamine Phalloidin (denoted by a "red" -grey basal pathway) and DAPI ("blue" -brush), which show a basal cell hypoplasia during angiogenic germination one day after the placenta extract Indicated by a lighter gray dot in the path). Fig. 1g shows the results of rhodamine paloidine (indicated by the "red" -grey basal pathway) and DAPI (represented by the "blue" -branch path), which shows a mature angiogenic network with extensive cellular cording after 3 days Indicated by gray dots). Figure 1h shows that Calcein (labeled "green" -grey branch) and DAPI ("blue" -lighter gray point within the branch during the early stage cell coding and angiogenesis network formation on day 1 after placenta extract) Lt; RTI ID = 0.0 > HUVEC). ≪ / RTI > Figure 1I shows DAPI (indicated by the "blue" -gray dashed path) staining showing cell coding of HUVEC after 3 days on placenta extract. Figure 1J shows HUVECs seeded on tissue culture plates at ⁴ x 10 ⁴ cells / cm 2 and incubated in endothelial cell culture for 3 days. Figure 1k shows the angiogenic network formed by HUVEC cultured for 3 days in endothelial cell medium after seeding at ⁴ x 10 cells / cm2 on placental extract attached to the surface of tissue culture plate with 100 占 PE PE / cm2 It is.
Figures 2a-2c show biochemical analysis of hPE and gene analysis of HUVEC seeded on hPE. 2A is a bar graph depicting a cytokine assay performed using a sandwich-based human angiogenesis antibody array; Data were normalized from a negative control value (0%) to a scale ranging from a positive control value (100%) (data represent three biopsy experiments). Figures 2b and 2c are bar graphs showing the normalized spectral exoscience factor (%) of immuno-related (2b) and angiogenesis-associated (2c) BM related proteins measured using LC-MS / MS. The fibrinogen normalized spectral exponent factor value is presented as the sum of the FGA and FGG values, and the laminin is presented as the sum of LAMA2, LAMA4, LAMA5, LAMB1, LAMB2, LAMB3, and LAMC1 values. Figure 2d shows gene analysis performed for 3 days in HUVEC seeded at a density of 80,000 cells / cm < 2 &gt; on 100 [mu] l PE / cm2. Some angiogenesis-related proteins, including VEGFAs, that are not present in the lysate were up-regulated by HUVEC when seeded onto hPE. The data represent 4 biological repeat experiments. P values were calculated using the Student t test of 2 ^ (- delta Ct) values repeatedly tested for each gene in the control and treatment groups.
Figures 3a-3d illustrate an in vitro neovascularization network formed on hPE and Matrigel coated tissue culture flasks. Figure 3a shows the chalcene stained HUVECS on hPE and matrigel at various cell seeding densities after 1d, 3d, and 5d. The rate of maturation of the angiogenic network, defined as the osmotic time to the maximum number of tubules per mm2, was controlled in the hPE sample by changing the cell seeding density. Quantitative analysis showed that at 40,000 cells / cm 2, the angiogenic network took up to 3 days to reach its maximum tubular density (tubule / mm 2), but at 80,000 cells / cm 2, It has been shown that maximum tubing density has been reached (data not shown). In the Matrigel sample, the network of angiogenesis was not evident after day 1. Scale bar, 200 탆. Figure 3b shows that WPMY-1 myofibroblasts did not show angiogenesis when seeded onto the placenta extract (Figure 3b), but angiogenesis when seeded on matrigel (Figure 3b. It is. Figure 3c shows quantitative image analysis (masking) measuring angiogenic network parameters, including mean tubulin length (mm), tubulin density, number of bifurcations, number of meshes, and tubing width. 3c is a series of bar graphs showing a comparison of hPE and Matrigel inducible angiogenic network parameters, showing that for samples seeded at 80,000 cells / cm2, the Martrigel sample had a maximum mean tubulin length, Bifurcation, and mesh counts were reached, whereas apoptotic ball formation was achieved by day 3, while hPM network parameters were more stable over 5 days of culture.
4A-4C show screening of anti-antiangiogenic tumor suppressor protein thrombospondin 1 using hPE-based angiogenesis screening assay. Figure 4a shows the HUVECs seeded for one day on stained hPE, Matrigel, and control culture flasks (uncoated) with Calcine AM after the addition of TSP-1 to the culture medium was done . Scale bar, 200 탆. The graph of Figure 4b shows that in hPE coated flasks, both the mean total tubule length [mm] and the average number of bifurcations, both decreased linearly with increasing TSP-1 concentration. The graph of Figure 4c shows a comparison of normalized angiogenic network reach area reduction rates (%); hPE coated culture plates were significantly more susceptible to TSP-1 concentration than matrigel coated plates, where the R ² values were 0.97 and 0.36, respectively.
Figures 5a-5c illustrate in vitro angiogenesis on 3D tissue structures. FIG. 5A is a schematic drawing showing placenta-derived cells, scaffolds, and cytokines that induce in vitro angiogenesis in hPP-immersed (human, intravenous) biocapsules after seeding and incubation for 3 days. Figure 5b shows that HUVEC seeded tissue scaffolds that were not immersed in hPE did not form an angiogenic network. Figure 5c shows an image of a representative series of images of hPE-immersed bioscapes showing both germination and intestinal hyperplasia of angiogenesis 3 days after incubation. At a HUVEC cell seeding density of 20,000 cells / cm 2, germination was most prevalent (Figs. 5c.i-5c.ii). At seeding density of 40,000 cells / cm 2, germination and intestinal angiogenesis were both seen (Fig. 5c.iii.-5c.iv.), while at a density of 60,000 cells / cm 2 .v.-5c.vi.) Angiogenic tubules were formed through intestinal superposition.
Figures 6a-6b illustrate examples of in vivo angiogenesis in hPE incubated bioscalf. 6A is a schematic diagram depicting a cell-free HUV scaffold incubated in PE, martrigel, or phosphate buffered saline (control) for 2 hr prior to transplantation into the rat model between fascia and muscle layer (control). FIG. 6B shows a series of images depicting the scaffolds removed for analysis on day 5 (d) after implantation. Significantly more fibrocyte encapsulation was achieved in the control and Matrigel incubated bioscalves as compared to the hPE incubated scaffold (Fig. 6b.i.-6b.iii.). Bright field images captured through the front two sides of the translucent bioscapable sheet were significantly improved in Martrigel and hPE incubated scaffolds (Fig. 6b.iv.-6b.vi.) compared to the control group Vein network formation, and the most mature capillary blood vessel image in the hPE scaffold, which shows the formation of the vascular structures in the capillaries and then into the venous blood vessels in the arterioles (Fig. 6b.vi Indicated by a circle)). Hematoxylin and eosin staining showed that scaffold remodeling was the most abundant in the hPE incubated scaffold (Figure 6b.vii.-6b.vi.) when compared to the control and Matrigel scaffolds. In the control scaffold (Fig. 6b.vi), remodeling of the original fiber orientation was scarcely occurred, and the cell migration from the HUV's outer surface of the biofold lumen to the scaffold was minimal (in italicized ' l '), . In the Matrigel incubated scaffold, there was slightly less cell migration from the intrinsic surface of the HUV as compared to the hPE scaffold (Fig. 6b.vii.-6b.ix); Compared with the control group, the Matrigel incubated scaffold also had less uniform cell distribution and fewer scaffold remodeling than the hPE incubated scaffold showing new collagen fiber orientation and more uniform cell distribution.
Figures 7a-7e illustrate embodiments of angiogenesis network formation in a human umbilical vein (HUV) cultured using dynamic cell culture conditions. As shown in the schematic diagram of Figures 7a and 7b, tubular HUV scaffolds were incubated in placental extracts for 2 hours prior to cell seeding and the constructs were cultured for 5 days in a dual perfusion bioreactor under standard cell culture conditions. The cells remained intact in the scaffold lumen and no migration (Fig. 7c). Cell coding, an early stage of tubule formation, was sporadic (Figures 7d and 7e).
Fig. 8 shows a circle-size image (left side) and a zoom image (right side) of the tube and cell network showing growth on the PE. The zoom enlarged image shows how each tube (dotted line) is identified and measured. In the right image, the circle mark represents a branch point, which is identified by the numbered point on the image indicated by the arrow. Mesh (dotted circle) is confirmed by each hexagonal array of the custom tube.
Figures 9a-b are an image series (Figure 9a) and a bar graph (Figure 9b) illustrating the angiogenic potential of PE. The panel of Figure 9a shows the morphology of microvessel tubules formed by HUVECs seeded on PE and cultured for 1, 3, and 5 days, as shown by comparison with HUVEC seeded on tissue culture plates. The capillary network began to form on day 1 and reached a more mature form on day 3 (see magnified image at lower right). On day 5, the network was regressed: the number of meshes decreased and some isolated cells (white dotted) were present. The graph of Figure 9b shows the morphological and local anatomical characteristics of the tubular-like network formed on PE after days 1, 3, and 5 of culture. The number of tubules, number of BPs, and number of meshes were compared between days 1, 3 and 5. There was a statistical difference (marked with an asterisk) in all three parameters between cells on day 1 and cells on day 5. Statistical analysis was performed using dual bilateral t-test with this variance at p <0.05.
Figures 10a-9b show the cell morphology of the microvascular network formed by HUVECs seeded on PE and cultured for 5 days, as shown by comparison with HUVEC (control) seeded on tissue culture plates. The control group is presented as an illustration image in the upper right corner of the image of FIG. 10A; On the 1st day from the upper left corner, PE single dose, 2 doses on day 1 and 3, and 3 doses on days 1, 3, and 4. As the number of inoculations increases (from one to three times), it can be observed that the capillary network evolved into a more mature, long-lasting form. The comparison of the tubule length, BP number and mesh number between the 1st and 5th days is made in the histogram of FIG. 10b. There was a statistical difference (marked with an asterisk) in all three parameters between the cells inoculated (inoculated) and cells inoculated three times (inoculated three times). Statistical analysis was performed using dual bilateral t-test with this variance at p <0.05.
Figure 11 shows a series of images showing when HUVECs incubated with PE for 3 days were stored in a 37 ° C humidified CO ₂ incubator from day 1 up to 20 days. The number in each image represents the incubation period (days). PE retained its ability to induce angiogenesis even after 15 days of incubation.
Figures 12A-12B illustrate the characteristics of gelatin microparticles. In Fig. 12A, the upper line shows a SEM image of blank particles of approximately 40 mu m in size. It has a regular shape and a smooth surface. The lower line in FIG. 12A shows a SEM image of particles loaded with PE. Surface morphology change, and size increase may be due to PE sorption. The graph of FIG. 12B shows the analysis of the size distribution of gelatin microparticles by optical microscopy.
Fig. 13 shows the decomposition profile of the non-crosslinked fine particles. The cumulative degradation rate (%) of microparticles in vitro crosslinked is presented as a function of the dry weight of the microparticles. The total cumulative degradation (%) after 20 days was 18.65% ± 0.09. The plot shows the degradation after a 6 hour incubation (6.45% ± 0.12). The error bars represent the mean ± standard deviation (n = 3).
Figures 14a-14b show the protein release kinetic behavior of blank microparticles and loaded microparticles. Figure 14a shows the cumulative release rate (%) from in-vitro blank microparticles (without PE) and loaded (PE) microparticles, respectively, as a function of the dry weight and the loaded weight of the particles. The cumulative release rates (%) after 22 days were 4.65% ± 0.11 and 4.65% ± 0.07, respectively. The error bars represent the mean ± standard deviation (n = 3). FIG. 14B shows the in vitro release difference (%) between the loaded microparticles and the blank microparticles. The first peak may represent PE release from the particle surface, while the second peak may be due to bulk corrosion.
Figure 15 shows PE delivery using gelatin microparticles. The images show the response to PE-loaded microparticles of HUVEC seeded at a density of 20,000 cells / cm 2 (D5) after 5 days of culture, as compared to HUVEC (control) where blank microparticles were seeded . The arrows represent cells in different states: this shows the shape difference. After 5 days of incubation with particles loaded with PE, some germination was present but no network formation was observed. The larger spheres are fine particles.
Figures 16a-16g illustrate the effect of continuous delivery of hPE on HUVEC. 16A shows a control in which HUVEC was cultured at 20,000 cells / cm &lt; 2 &gt; in angiogenic media; 16B and 16D show HUVEC (Fig. 16B) in which single immunization of hPM was performed on day 1, HUVEC (Fig. 16C) in which inoculation was performed twice on days 1 and 3, and inoculation on three occasions on day 1, Lt; / RTI &gt; (FIG. 16D). Figures 16e-16g are bar graphs showing the angiogenesis quantification performed on the images of Figures 16b-16d after staining. The results are presented as means ± mean standard error between measurements of triplicate samples for each condition.
Figures 17A-17F illustrate fine particle evaluation. Figures 17a-17c illustrate the three protocols evaluated: homogenization (Fig. 17a), double (2 min / 1 min) homogenization (Fig. 17b), and dual (2 min / 20 sec) homogenization FIG. 3 shows an optical microscope image of FIG. The histograms in Figures 17d-17f illustrate the size ([mu] m) distribution for particles formed in each of the three protocols (each batch is made in triplicate).
Figures 18a-18f illustrate the use of three different protocols: single (Figures 18a and 18d), 2/1 duplex (Figures 18b and 18e), and 2/20 duplex (Figures 18c and 18d) It is a scanning electron microscope (SEM) image. Figs. 18A-18C show the shape of the fine particle, and Figs. 18D-18F show the surface porosity.
Figures 19a and 19f provide a graph of loading rates (Figure 19a) for each protocol (P2: single, P3: 2/1 duplex and P4 2/20 duplex) and a graph of the rate of release from particles (Figure 19b) . The curve represents the cumulative release rate for up to 21 days for each protocol.
Figure 20 shows an SDS page analysis of the supernatant of fine particles. The image is a gel image after staining showing the BSA loaded and hPM loaded microparticle supernatant and the relative initial content loaded during microparticle preparation.
Figure 21 shows that when pure hPM was added to the alginate matrix (top row), the sustained release of hPM from the PLGA microparticles embedded in the large matrix resulted in 7,14,21 , And endothelial cells stained with calcein AM after 28 days of culture. The bottom row shows two control groups, one with blank PLGA microparticles embedded in an alginate matrix and the second with no microparticles. HUVEC maintained the generally circular shape at all time points for both control groups.

Explanation

Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to the particular embodiments described, and thus, of course, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Values are provided as ranges, each intermediate value between the upper and lower limits of the range, up to one tenth of the lower limit unless otherwise explicitly specified in the context, and any other stated Or intermediate values are included in the scope of the present disclosure. The upper and lower limits of the smaller ranges may independently be included in a smaller range depending on any limit specifically excluded within the stated range, and this is also included in the scope of this disclosure. Where the stated range includes both limits or both limits, ranges excluding one or both of the included limits are also included in the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

Any of the published documents and patents incorporated herein by reference are incorporated herein by reference to disclose and describe methods and / or materials related to the cited publications. Any citation of an open publication is for its disclosure prior to the filing date and such citations should not be construed as an admission that the present disclosure is not entitled to precede the open publication by prior disclosure. In addition, the disclosure date provided may be different from the actual disclosure date, which may need to be independently verified.

Upon reading the present disclosure, it will be readily apparent to one skilled in the art, that each individual embodiment described and illustrated herein can be readily separated from the features of any of the several embodiments without departing from the scope or spirit of the present disclosure. Or have individual components and characteristics that can be combined therewith. Any of the methods mentioned may be performed in the order of the events mentioned, or in any other order possible logically.

Embodiments of the disclosure will employ, within the skill of the art, medical, biochemical, molecular biology, biology, pharmacology techniques, etc., unless otherwise specified. These techniques are described in detail in the literature.

As used in the specification and the appended claims, the singular forms " a "and" an ", as used herein, Be careful. Thus, for example, reference to "one cell" includes multiple cells. Many terms referred to in the following specification and embodiments should be defined to have the following meanings, unless the context clearly indicates otherwise.

Before describing the various embodiments, the following definitions are provided and should be used unless otherwise specified.

Justice

In describing the disclosed subject matter, the following terms will be used in accordance with the definitions set forth below.

As used herein, the terms "polypeptide" and "protein" refer to an amino acid polymer consisting of three or more amino acids in the form of a continuous array connected via a peptide bond. The term "polypeptide" includes proteins, protein fragments, protein analogs, oligopeptides, and the like. The term "polypeptide" encompasses polypeptides as defined above that are encoded by nucleic acids, produced by recombinant techniques (e. G., Isolated from suitable sources such as algae), or synthesized. The term "polypeptide" further contemplates polypeptides as defined above that include amino acids covalently or noncovalently linked to chemically modified amino acids or a labeled ligand.

The terms "polynucleotide," "oligonucleotide," and "nucleic acid sequence" are used interchangeably herein and refer to a coding sequence And polynucleotide (s) or nucleic acid sequence (s) that are translated into the polypeptide); (E.g., translation start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, etc.); And a regulatory sequence (DNA sequence in which the transcription factor (s) binds thereto and which positively (induces) or negatively (represses) the promoter activity of the gene). No limitations on length or synthetic origin are suggested by the terms described herein.

As used herein, the term "gene" or "genes" refers to a nucleic acid sequence (RNA or DNA, both) encoding the entire RNA, the entire protein, or the genetic information for synthesizing any part of the whole RNA or the entire protein. All included). A "gene" typically refers to a genetic unit corresponding to a DNA sequence that occupies a specific position on a chromosome and contains a genetic indication for the feature (s) or trait (s) in the organism. The term "gene product" refers to an RNA or protein encoded by a gene.

The terms "treat," " treating, "and" treatment "are approaches for obtaining beneficial or desired clinical results. In particular, beneficial or desired clinical results, whether detectable or not, include relief of symptoms, reduction in disease severity, stabilization (e.g., not worsening) of disease, delay or slowing of disease progression, , Improvement or alleviation of the disease state, and remission (partial or complete remission). In addition, "treating," " treating, "and" treatment "may also be therapeutic in that they are partial or complete healing of the side effects that may be caused by the disease and / or disease. As used herein, the term "prophylactically cured," or "prophylactically treated" means to completely, substantially or partially prevent a disease / condition or one or more symptoms thereof in a host. Likewise, "delayed onset of a condition" may also be included in "prophylactically treating " and refers to the act of prolonging the time prior to the actual onset of the condition in a patient suffering from the condition. Lt; RTI ID = 0.0 &gt; before &lt; / RTI &gt;

"Administration" means introducing a compound of the disclosure into a subject; This may also mean providing the composition of the present disclosure (e. G., By prescription) to the subject.

The term "organism," " subject, "or" host, "refers to humans, mammals (e.g., cats, dogs, horses, mice, rats, pigs, , And other living species in need of treatment, all of which require treatment. In particular, the term "host" includes human beings. As used herein, the term "human host" or "human subject" is generally used to refer to a human host. In this disclosure, the term " host "typically refers to a human host, and when used singly in this disclosure, the word " host" means that, unless the intent to specify non- This means human host. A host that is "well on" the condition (s) does not exhibit any obvious symptoms of the one or more conditions, but may be defined as a host that is at risk of developing into one or more of the conditions, genetically, physiologically, or otherwise.

As used herein, the term "expression" describes a process that takes place on a structural gene that produces a polynucleotide. Expression is a combination of transcription and translation. Expression generally refers to "expression" of a nucleic acid that produces a polynucleotide, but it is also generally accepted to mean "expression" of a polynucleotide, which means that the polynucleotide is produced through the expression of the corresponding nucleic acid .

"Angiogenesis" is a physiological process involving the growth of new blood vessels. Angiogenesis is an important part of biological processes such as growth and development, wound healing, embryogenesis, and the like. Excessive angiogenesis may occur when the affected cells produce abnormal amounts of angiogenic growth factors that overwhelm the effects of natural angiogenesis inhibitors. An imbalance between the angiogenic growth factor and angiostatic inhibitor production may inappropriately control the growth or inhibition of blood vessels. Angiogenesis-dependent or related diseases can occur when new blood vessels grow excessively or insufficiently. Angiogenesis-related diseases include, but are not limited to, cancer, precancerous tissue, tumor, myocardial infarction, and stroke. Excessive angiogenesis can include cancer, diabetic blindness, age related macular degeneration, rheumatoid arthritis, psoriasis, and over 70 other conditions. Insufficient angiogenesis can involve delays in coronary artery disease, stroke, and wound healing, which is also a factor in tissue engineering, which is discussed in more detail in this disclosure.

As used herein, the term "modulate" and / or "modulator" generally refers to a cell / organism that directly or indirectly promotes / activates / induces / increases or interferes with a particular function and / / Inhibiting / reducing activity. In some cases, the modulator may increase or decrease a particular activity or function relative to its natural state, or relative to the average level of activity generally expected. The modulation may involve causing overexpression or lack of expression of the peptide (e.g., by the action of upregulating or downregulating the expression of the peptide), or may interact directly with the subject peptide that increases and / or decreases activity . Regulation also includes increasing or decreasing a biological phenomenon associated with a particular biological activity or biological phenomenon, such as angiogenesis or angiogenesis.

As used herein, "up-regulating" refers to the act of increasing the expression and / or activity of a protein or other gene product. "Downregulation" means reducing the expression and / or activity of a protein or other gene product.

As used herein, the term "isolated cell or cell population" refers to an isolated cell or a plurality of cells that have been incised from tissue or grown in vitro by tissue culture techniques. The term "cell or cell population" may mean an isolated cell as described above, or may also refer to an in vivo cell of an animal or human tissue.

The term "tissue" generally refers to a group of cells (e.g., connective tissue, connective tissues, organs) that are orchestrated to perform biological functions in common cooperatively and / or to serve a biological purpose, such as forming all or part of an organ in an organism. Endothelial tissue). "Tissue" generally encompasses a population of pseudopods, or all of the same type of cell population, and the tissue may also include cells of more than one type when the population of cells is generally responsible for a common purpose.

As used herein, the term "biocompatibility" refers to a biological material and / or a biological system (e.g., a cell, a cellular component, a living tissue, an organ And so on).

The term "bioscaffold" refers to any biocompatible substrate (natural or synthetic) with sufficient structural stability to support the growth of living biological material (e.g., living cells). In an embodiment of the disclosure, the biocompatible scaffold material is a naturally occurring substrate (e.g., obtained from a living organism, but may be subjected to further processing and processing, or produced from a material derived from a natural source) It is not, but a human, vein scaffold with cells removed. In an embodiment, the bioscaffold of this disclosure has a three-dimensional structure (rather than a two-dimensional structure of a plane) to support three-dimensional growth of living cells.

As used herein, the term "biodegradable" refers to a substance that dissolves, degrades, or otherwise degrades over time (e.g., in a living organism or living culture) And ceases to exist in its original form of structure. In an embodiment of the present disclosure, the biodegradable material dissolves and decomposes over time in the host organism.

As used herein, the term "engineered" indicates that the object being manipulated is produced and / or altered by a human. The manipulated object may comprise a naturally occurring material, but the object itself is altered in some way by human intervention and design.

As used herein, the term "test compound" may include peptides, peptide mimetics, small molecules, nucleic acid sequences or other compounds capable of affecting living cells or organisms. In some embodiments, a "test compound" may be a compound, such as a chemical substance or a peptide, suspected of having a modulatory effect on biological activity, function, or response to another compound. For example, in the context of this disclosure, a "test compound" is a compound that modulates angiogenesis, such as increasing angiogenic activity and / or reducing angiogenic activity and / or modulating the effects of different angiogenic regulatory elements. It may be a compound suspected of having an effect.

As used herein, the term "removed" or "substantially removed" indicates that a certain amount of a substance or compound is separated from another composition, but all traces of the removed material are also absent Thus, this does not mean that the removed material should not be completely undetectable. For example, if blood is "removed" or "substantially removed " from the composition, this will remove a significant proportion of the blood in the composition, but some blood or blood components may still be detected in trace amounts during thorough screening (E.g., "substantially removed" does not mean that the composition should not contain 100% of the "removed" component; instead, the composition or material will contain no more than about 99% of the " About 95% free, or about 90% free, or may be absent in a range of any ratio or exemplary ratio given above.

Argument

Embodiments of the present disclosure include methods and compositions for inducing angiogenesis, and methods and compositions for modulating angiogenesis, and methods for preparing compositions for modulating angiogenesis. The disclosure also includes a method for identifying an angiogenesis modulator and a method for identifying an angiogenesis modulator. Embodiments of the disclosure further include methods and compositions for delivering a composition for modulating angiogenesis. In an embodiment, the disclosure is directed to a method of treating and / or preventing placental extracts and tissue structures that can be used to induce and / or regulate angiogenesis in vitro and / or in vivo in tissue structures and / Methods and compositions for delivering placental extract to a cell. This disclosure also includes placenta extracts that can be used in assays to identify compounds that modulate angiogenesis. Additionally, the present disclosure includes a delivery vehicle composition loaded with a placenta extract for release of the extract in a controlled release manner in vivo or in vitro cell populations inducing angiogenesis.

Angiogenesis is a complex process that depends on both location and stimulation, and in each case the ability to regulate the process may involve a complex combination of regulatory molecules. Control of angiogenesis is further complicated by different mechanisms of formation, the two most well understood mechanisms being superposition and germination. The present invention is characterized by insertion of an epileptic cell column into the lumen of an existing blood vessel, wherein germination is characterized by germination of endothelial cells against angiogenic stimulation in a tissue previously free of microvessels. Many molecules have been shown to regulate angiogenesis, and these are likely to be studied further. Due to the diversity of angiogenesis inducers, angiogenesis factors for use in the study of angiogenesis, drug screening and regenerative medicine have been continuously studied and developed.

Conventional models for studying angiogenesis depend entirely on the use of animal-derived stimulants or on the use of live animals for evaluation. In vivo animal studies provide a more accurate model for comparing the complexity of biomolecular pathways and mechanisms that occur during the formation of human blood vessels. Standard in vivo angiogenesis models include rabbit corneal neovascularization assays, in vivo / in vitro chick chorioamnion assays, and rat mesenteric window assays. Where possible, in vitro angiogenesis models are selected to better control the complex biological phenomena; However, this usually limits the study on a limited number of molecular species, such as VEGF. If a more complex or multifunctional "mix" may be needed to promote sufficient vascularization, the result of using a single molecule (or several molecules) for the complex cascade may itself be limited.

For the in vivo angiogenesis model, a murine derived basement membrane matrix (BMM) or 'matrigel' assay has been the preferred model because it causes in vivo complexity in vitro models, In vivo results. However, this model is derived from Angel Bres-Holm-Swarm (EHS) mouse sarcoma cells and is not suitable for clinical use because it requires a large number of animal sacrifices ¹¹ . Numerous in vitro human-derived regulators have been used to generate angiogenic models. Historically, they were based on a single regulatory factor (FGF, TGF-beta, VEGF) and do not involve the various cytokines and chemical gradients present in the natural in vivo ¹² . Given the complexity of deriving multiprotein formulations from species differences ^{13 , 14} and human recombinant proteins associated with animal-derived models, a robust, human-derived approach (a more complex mix of multiple proteins at a ratio close to the physiological ratio ) Will have a significant impact on the potential for enhancing the clinical transformation of mechanical studies, screening of angiogenic drugs, and regenerative medicine therapy. In addition, the ability to regulate angiogenic processes indicative of different formation mechanisms and steps will provide an improved platform for characterizing molecular and molecular pathways critical in the vascularization process.

The present disclosure provides methods of inducing and modulating angiogenesis in vitro and in vivo. In addition to inducing in vitro and in vivo angiogenesis, this model not only modulates the rate of maturation of the microvascular network, but also enables selective modeling of germination and intestinal superficial angiogenesis. Human placenta extract (PE) in vivo has been shown to significantly increase capillary blood vessel formation by removing fibrosis using administered collagen-based bioscaffolds.

This disclosure describes a method of inducing and modulating in vitro and in vivo angiogenesis using a complex set of adjustable, fully human biomolecules derived from the human placenta. This approach utilizes the specified fractionation and separation techniques to obtain complexes of active human biomolecules isolated from the human placenta. In addition to inducing and modulating in vitro angiogenesis and in vivo angiogenesis, the methods and compositions of the present disclosure are capable of modulating the rate of microvascular network maturation as well as enabling selective modeling of germination and intestinal angiogenesis . In an embodiment, the methods and compounds of the present disclosure also induce and modulate angiogenesis in both polymeric and in vitro derived tissue scaffolds. These methods can control the rate of microvascular network maturation. In vivo, human placenta extracts of this disclosure have been shown to significantly enhance capillary blood vessel formation while removing fibrosis using administered collagen-based bioscaffolds.

Continuous delivery of growth factors that affect angiogenesis is also a challenge faced in successfully controlling angiogenesis to promote vascularization for tissue manipulation approaches. The present disclosure also provides methods and compositions for modulating the compositions of the present disclosure in a controlled-release mode to regulate angiogenesis both in vitro and in vivo.

Human placenta extract

The present disclosure provides compositions and methods for angiogenesis induction and / or modulation comprising human placenta extract (PE or hPE). In an embodiment of the present disclosure, PE is obtained from a human placenta, the placenta crude extract (crude PE) is removed by removing blood from the placenta sample, and the crude PE is mixed with urea or other protein solubilizing agent Solubilising the existing protein and removing residual solids from the crude extract; Dialysing the urea placenta extract mixture to remove significant amounts of urea from the mixture to produce human PE. Human PE is a matrix-like compound, which is often referred to herein as the human placental matrix (hPM).

In an embodiment, the manufacturing process of human PE is performed at a temperature of about -86 캜 to about 5 캜. In an embodiment, the human placenta extract is prepared at a temperature of about 4 DEG C or less.

In an embodiment, the process of removing blood from a placenta sample to produce a placenta crude extract comprises homogenizing the human placenta sample with a buffer, centrifuging the homogenized sample, and discarding the supernatant containing blood . To prepare crude PE, the process is substantially complete until all of the blood is removed from the sample (e.g., there is no about 99% of the blood in the sample, no about 95% of the blood, about 90% of the blood, etc.) (E.g., two, three or more times). In one embodiment, the buffer is a sodium chloride solution (NaCl).

In an embodiment, the protein in the placenta crude extract is solubilized by mixing the placental crude extract with a protein solubilizing agent. In embodiments, the protein solubilizing agent may be any compound or mixture of compounds capable of solubilizing (e. G., Denaturing) the protein without permanently destroying the protein or otherwise rendering the protein permanently inactive. (For example, solubilization should denature the protein reversibly, whereby the protein may be refolded, for example, when the protein solubilizer is removed). In an embodiment, the protein solubilizing agent may be, but is not limited to, urea, guanidine-HCl, or other similar compounds. In an embodiment, the protein solubilizing agent is urea, and the crude extract is mixed with the urea composition by homogenizing the crude extract with urea. In an embodiment, urea is mixed with the crude extract for a period of about 12 to about 36 hours. In an embodiment, the urea is mixed with the crude extract for about 24 hours. In an embodiment, the urea solution is a urea buffer having a urea concentration of about 0.5 M or greater. In embodiments, the urea is at least about 2 M, the urea is at least about 4 M, and the maximum is about 15 M. In an embodiment, the urea solution may have a concentration of from about 0.5 M to about 15 M. [ In another embodiment, the protein solubilizing agent is guanidine-HCl at a concentration of about 0.5 M to about 15 M. In an embodiment, the guanidine-HCL has a concentration of about 6 M. Although the methods and compositions described below are described as using urea as a solubilizing agent, it should be understood that other suitable solubilizing agents such as, but not limited to, those discussed above may replace urea.

In an embodiment, after mixing with urea, or other protein solubilizing agent, the solids are removed from the solubilized protein-crude extract mixture (e.g., urea-crude extract mixture). In an embodiment, the solids are removed by centrifuging the PE mixture and discarding the pellet (containing solids). This step can be repeated over multiple times. After removing the solids, the PE mixture (e.g., supernatant) is dialyzed to remove urea or other protein solubilizing agent from the placenta extract. In an embodiment, the dialysis fluid is TBS. In an embodiment, the dialysis fluid is exchanged after a period of time (e.g., 1 hour, 2 hours, 3 hours, etc.) has elapsed and dialysis is repeated over multiple (e.g., 2, 3, 4 times) Remove all urea (eg, placenta extract does not contain about 99% of urea, about 95% of urea, etc.). In an embodiment, the PE is centrifuged again to remove residual solids (e.g., polymerized proteins, etc.). In an embodiment, the residual PE is a viscous material that is transparent to pink. A further detailed description of embodiments of the process of this disclosure for preparing placental extracts of the present disclosure can be found in the following examples.

Accordingly, embodiments of the present disclosure also include PEs produced by the methods of this disclosure. In an embodiment, the present disclosure provides a method for preparing crude PE by removing blood from a sample obtained from a human placenta sample; The placenta crude extract is mixed with a protein solubilizing agent (such as, but not limited to, urea, guanidine-HCl, and the like) to solubilize the protein in the crude extract; Separating the solid material from the solubilized protein-PE mixture; PE &lt; / RTI &gt; mixture by dialysis to remove the protein solubilizing agent (e. G., Urea) from the mixture to produce human PE.

Thus, the present disclosure provides a method of treating a human placenta extract, including extracts obtained from a human placenta (e. G., From a human placenta sample) that has substantially removed blood and solids and has placental proteins (some or all) . In an embodiment, the placental protein comprises cytokines and growth factors.

As a result of the PE analysis of this disclosure, it has been found that PE contains many proteins, including cytokines and growth factors. In an embodiment of the placenta extract of the present disclosure, the extract comprises at least 20 different cytokines. In some embodiments, up to 40 different cytokines are included. Other embodiments include more than 50 cytokines. Some cytokines that may be present in the PEs of this disclosure include those enumerated in the following examples. For example, some of the cytokines that may be present in the PE of this disclosure include, but are not limited to, angiogenin, Acrp30Ag, IGFBP-1, NAP-2, and Fas / TNFGSF6, and RANTES, and MIF .

The cytokines and growth factors and other placental compounds present in the placental extracts of this disclosure can induce angiogenesis in endothelial cell cultures, tissues, tissue structures, engineered bioscaffolds, and the like. Placenta extracts of this disclosure can induce angiogenesis in vitro and in vivo. The placenta extract of the present disclosure can stimulate the growth of endothelial cells. In an embodiment, the human PE of the present disclosure is capable of modulating angiogenesis. Compared to other conventional compounds used to induce angiogenesis, such as compounds comprising BMM (a single purified angiogenic regulator, such as purified VEGF-alpha or SDF-1) and purified fibrin, The PEs of the present disclosure stimulate an increase in the angiogenic growth of endothelial cells (e.g., tubule and network formation) and a decrease in angiogenic growth (e.g., tubulogenesis) of myofibroblasts relative to BMM. The PEs of the present disclosure also stimulate different growth and / or differentiation patterns for various cell lines (e.g., stem cells, smooth muscle cells, etc.) as compared to BMM, whereby the growth / Can be distinguished.

The PEs of this disclosure can also upregulate various genes in endothelial cells when compared to endothelial cells grown under the absence of PE. Some of these genes include angiogenesis-related genes, extracellular matrix remodeling genes, and angiogenic genes. Some angiogenesis related genes include, but are not limited to, ANGPTL4, CXCL3, human growth factor (HGF), ANGPT2, PGF, TYMP, VEGFA, HIF1A, and FGF1. Some extracellular matrix remodeling genes that can be induced by the placenta extract of this disclosure include, but are not limited to, MMP2, MMP9, COL4A3, and LAMA5. The angiogenic genes include, but are not limited to, CDH2, HAND2, LECT1, and MDK.

Methods for regulating angiogenesis

The disclosure also includes a method of inducing angiogenesis in a cell culture comprising the step of growing endothelial cells in the presence of the human placenta extract of the present disclosure. In an embodiment, the cell cultures are treated to remove blood and solids, mixed with urea, and grown in the presence of placenta extract of the present disclosure obtained from dialyzed human placenta samples to remove urea, Placental extracts include placental proteins including cytokines and growth factors. In an embodiment, the endothelial cells are human endothelial cells; In another embodiment, the cell is a human umbilical vein endothelial cell (HUVEC). In an embodiment of the method for inducing angiogenesis in a cell culture, the cells are seeded at a density of at least about 40,000 cells / cm2. In an embodiment, the cells are seeded at a density of at least about 80,000 cells / cm2. In an embodiment, the cell culture can be grown on a plate comprising the growth medium and the placental extract of the present disclosure.

The present disclosure also encompasses methods of inducing vascularization of a biomaterial in vivo, including incubating the biomaterial in a composition comprising human placenta extract of the present disclosure, and transplanting the biomaterial into the host do. In an embodiment, the biomaterial comprises a naturally occurring material and / or cell. In an embodiment, the biomaterial comprises a manipulated bioscaffold comprising a human derived matrix material. In an embodiment, the engineered bioscaffold comprises a human vein scaffold. In an embodiment, the human vein scaffold is one in which the cells have been removed. In some embodiments, the biomaterial is seeded with endothelial cells, such as, but not limited to, human endothelial cells (e.g., HUVEC). In an embodiment of the present disclosure, the biomaterial comprises a manipulated scaffolding material comprising a human vein scaffold seeded with HUVEC. In some embodiments, the HUVEC is seeded on the bioscaffold with a cell density above about 40,000 cells / cm2. In an embodiment, the HUVEC is seeded at a density of at least about 80,000 cells / cm2. In an embodiment, the biomaterial is incubated in the placenta extract for at least about 2 hours.

Biomaterials and engineered Bioscapable Vascularization

The present disclosure also encompasses methods of vascularizing biomaterials including, but not limited to, engineered biomaterials, naturally occurring biomaterials, and other biomaterials implanted in the host. In addition to the vascularization of the biomaterial, treatment of the biomaterial with the placenta extract of the present disclosure also provides for bioavailability, reduces biomaterials for in vivo use, such as reducing inflammation, reducing rejection and scar formation, Lt; / RTI &gt; Thus, a composition comprising a placenta extract of the present disclosure and a placenta extract of the present disclosure may be used to "administer" any of a number of biomaterials to improve the outcome of the transplant.

This disclosure also specifically encompasses engineered biomaterials, including, for example, implantable, engineered bioscalves, including incubated human-derived substrate material in a composition comprising the human placenta extract of the present disclosure. The bioscapes of this disclosure can be implanted in mammals, such as humans. The bioscaffolds of this disclosure may comprise any biomaterial suitable for implantation into a host. Examples of bioscapes for use in the present disclosure include, but are not limited to, tissue, matrix material, any number of naturally occurring biomaterials, and the like. In an embodiment, the bioscapes are 2D or 3D bioscapes. In an embodiment, the bioscaffold comprises a human derived substrate material. In an embodiment, the bioscaffold comprises a human vein scaffold from which cells have been removed. In an embodiment, cells in a biocathode include, but are not limited to, human cells, human endothelial cells (e.g., human endothelial cells (HUVEC)), stem cells, other multipotent cells, and the like.

As described in the Examples below, the biocapsule of the present disclosure incubated in the placenta extract of this disclosure contains more vascularization than the incubated bioscaffold in the angiogenesis inducing compound BMM or the control compound (e.g., angiogenesis ) And less fibrosis. Among the placenta extracts of the present disclosure, the incubated bioscaffold can also be used for immunosuppression and angiogenesis-inducing positive macrophages (e.g., CD205 (M2)) versus inflammation-inducible Macrophages such as (CD86 (M1)) at higher rates.

Angiogenesis Screening Assay

Since the placenta extract of this disclosure induces angiogenesis in cell cultures, it provides an excellent assay for identification and screening of angiogenesis factors. Accordingly, the disclosure also includes methods and assays for identifying angiogenesis regulatory factors.

In an embodiment, the method comprises culturing a culture of human endothelial cells in the presence of a compound comprising human placenta extract of the present disclosure, and contacting the human endothelial cell culture with the test compound. Since the placenta extract induces angiogenesis in cell cultures, if the angiogenesis is less or more than expected, the test compound may be identified as an angiogenesis modulator. Thus, the method also comprises the steps of measuring the amount of angiogenesis in the culture and, if the angiogenesis in the cell culture is more or less than the amount of angiogenesis in the culture grown in the absence of the test compound, RTI ID = 0.0 &gt; regulatory &lt; / RTI &gt; In an embodiment, an increase in angiogenesis relative to the culture grown in the absence of the test compound indicates that the test compound induces angiogenesis. The decrease in angiogenesis relative to the culture grown in the absence of the test compound indicates that the test compound inhibits angiogenesis. As described in the following examples, compound thrombospondin 1 (TSP-I) according to the method of the present disclosure was found to be an inhibitor of angiogenesis through the screen. The present disclosure also provides test compound screening assays for identifying angiogenesis regulators, including endothelial cell cultures grown in the presence of human placenta extract of the present disclosure. Assays of this disclosure may be used in conjunction with the methods of this disclosure to identify angiogenesis regulatory factors.

Continuous delivery methods and compositions

Since the angiogenic effect of the placenta extract can not be sustained in vivo or in a biological material (e.g., tissue, cell culture, bioscaffold, etc.) for a prolonged period of time, Are provided in the disclosure.

Embodiments of the present disclosure include a composition comprising human PE (hPE) of the present disclosure coupled to biodegradable microparticles to provide a sustained / human PE composition. PE-loaded biodegradable microparticles provide a composition for controlled release angiogenesis. Thus, in an embodiment, the composition for the controlled release angiogenesis comprises human PE and biodegradable microparticles of the present disclosure in which human PE is coupled to the microparticles such that the human PE can be released from the microparticles. In an embodiment, the human PE is obtained from a human placenta sample, wherein blood and solids are substantially removed from the extract, wherein the extract comprises proteins present in a placenta sample, including cytokines and growth factors. Human PE used in the sustained release angiogenesis modulating composition may be any embodiment of human PE as described above.

In an embodiment, the human PE is released from the microparticles upon exposure to the cell in vivo or in vitro. In embodiments, when the biodegradable microparticle is in a host in vivo, or in contact with a biocidal cell where the cell culture or cell has been seeded in vitro, the biodegradable microparticle will migrate over time to PE Decompose, and release into surrounding cells, tissues, and the like. In an embodiment, the biodegradable microparticles slowly release the PE over an extended period of time (e. G., Days, days, etc.) after releasing the initial burst of PE. The release profile of the fine particles can be controlled by varying the size of the fine particles and the degree of crosslinking. In an embodiment, the microparticles are crosslinked microparticles and the degree of cross-linking can be controlled to modify the release parameters of the particles.

In an embodiment, the biodegradable microparticles are made of gelatin. A further detailed description of embodiments of gelatin biodegradable microparticles is described in Example 2 below. In an embodiment, the biodegradable microparticles comprise a mixture of microparticles of different sizes. It is believed that particles of different sizes emit PE at different rates, so that it is believed that the inclusion of fine particles of varying sizes will release PE continuously.

In an embodiment, the biodegradable microparticles are poly (lactic-co-glycolic acid) (PLGA) microparticles. In an embodiment, the hPE is loaded (e.g., encapsulated therein) in PLGA microparticles. In an embodiment, the average particle size of the hPE loaded PLGA microparticles of the present disclosure is from about 10 to about 1,000 microns. In an embodiment, the PLGA microparticles are prepared by an oil-in-water emulsion process comprising a double homogenization step. In an embodiment, PLGA microparticles are prepared by mixing a PLGA oil solution with a first aqueous solution (W1) comprising hPE of the present disclosure, homogenizing PLGA and W1 at a first microbial disease stage to remove the first emulsion, The first emulsion is added to a second aqueous solution (W2) comprising a solvent (e.g., and alcohol, such as, but not limited to, polyvinyl alcohol) in water to form a second emulsion , The solvent is prepared by evaporation. In an embodiment, a second homogenization step is used to homogenize the second emulsion. In an embodiment, the first homogenization takes about 1 to about 2 minutes. In some embodiments, no other bacterial disease stage is used. In another embodiment, a second homogenization step is added. In an embodiment, the second homogenization takes from about 20 seconds to about 1 minute. A further detailed description of embodiments of the process for preparing hPE loaded PLGA microparticles is provided in Example 3 below. In an embodiment, the size of the PLGA microparticles loaded with hPE, produced in a single homogenization step, is of a size from about 100 to about 1,000 microns in diameter. In embodiments, when a single homogenization step takes about 2 minutes, the size of the resulting microparticles ranges in size from about 50 to about 500 microns in diameter and averages from about 260 to about 290 microns in diameter. In embodiments, the size of the PLGA microparticles loaded with hPE, made with a second homogenization step of about 1 min, ranges from less than about 20 microns in diameter to about 100 microns in diameter and has an average diameter of from about 36 to about 40 microns. In embodiments, the size of the PLGA microparticles loaded with hPE, prepared in a second homogenization step of about 20 sec, is in the range of less than 20 [mu] m to about 200 [mu] m and an average diameter of about 85 to about 95 [mu] m.

The present disclosure also encompasses methods of using the sustained release angiogenesis modulating compositions described above for sustained release of PE. In embodiments, so that the method can be continuously released into the human PE in vivo or in vitro over time, for example, but are not limited to, the biological material of human PE in vivo or in vitro (e. G., Cells Culture composition, culture, tissue, tissue structure, bioscaffold, etc.). In an embodiment, a method of the present disclosure is a method of coupling a cell (e. G., A cell in a culture, an in vivo cell, a cell in a biomaterial, or a cell in contact with a manipulated biomaterial, etc.) Comprising contacting the human placental extract with a sustained release angiogenesis-controlling composition comprising the biodegradable microparticles as described above, so that the human placenta extract is released from the microparticles into the biomaterial over a period of time after exposure of the microparticles to the cells . In an embodiment, the cell is an endothelial cell, such as, but not limited to, human endothelial cell (HUVEC).

In an embodiment, the sustained release angiogenesis control composition is coupled to a biomaterial. In an embodiment, the biomaterial is a cell culture. In an embodiment, the biomaterial is a tissue construct and / or tissue matrix comprising a cell culture. In embodiments, the biomaterial is an alginate matrix comprising cells that are embedded in an alginate matrix (e. G., A human cell, such as HUVEC), and may include a composition for controlled release angiogenesis such as biodegradable microparticles loaded with hPE ) Is also embedded in, or in contact with, the alginate matrix. In an embodiment, the biomaterial coupled to the composition for the controlled release angiogenesis is implanted in the subject and induces vascularization of the biomaterial. In an embodiment, the biomaterial is a manipulated bioscaffold comprising a human-derived substrate material, for example, a human-made vein scaffold with cells removed, including, but not limited to, human endothelial cells seeded. In an embodiment, the subject is a mammal; In an embodiment, the subject is a human. Other modified methods for inducing angiogenesis using the sustained angiostatic composition of this disclosure are also possible, and exemplary embodiments of the method are described in further detail in the following examples.

Anti-inflammatory compositions and methods of use

As described in the following examples, it has also been found that the human PE of this disclosure and the sustained release / human PE composition of this disclosure exerted an anti-inflammatory effect on the surrounding cells and tissues. Thus, the compositions of the present disclosure also include anti-inflammatory compositions comprising human PE or a sustained / human PE composition of the present disclosure. The methods of the present disclosure also include methods of treating inflammation in a subject or tissue of a subject by exposing the subject or tissue to a human PE or a sustained human / PE composition of the present disclosure (e.g., Neutralizing, preventing, etc.).

Further details of the tests and methods of this disclosure are provided in the following examples. It is to be understood that the following specific embodiments are merely illustrative and are not to be construed as limiting the remainder of the disclosure in any case. Without further elaboration, it is believed that one skilled in the art can, based on the teachings herein, utilize the present disclosure to its fullest extent. All publications cited herein are incorporated herein by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, and in particular any "preferred" embodiments, are merely illustrative and are illustrative of the principles of the disclosure. Many variations and modifications may be made to the embodiment (s) of the present disclosure described above without departing substantially from the spirit and principles of the present disclosure. All such variations and modifications are included herein within the scope of this disclosure and are protected by the following embodiments.

The following examples are presented to those skilled in the art to carry out the present methods and to provide a complete disclosure and description of how to use the compositions and compounds disclosed herein. While efforts have been made to ensure accuracy with respect to numerical values (e.g., quantity, temperature, etc.), some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperatures are in degrees Celsius (C), and pressures are atmospheric or near atmospheric. Standard temperature and pressure are defined as 20 ° C and 1 atm.

It should be noted that the ratio, concentration, amount, and other numerical data may be expressed herein in a range format. It should be understood that this range format is used for the sake of brevity for convenience and thus includes not only the numerical values explicitly recited as ranges of limits but also the numerical values and sub- But should also be interpreted in a flexible manner that includes all the individual numerical values or subranges that are present. By way of example, a concentration range of "about 0.1% to about 5%" includes not only the concentrations explicitly stated from about 0.1 wt% to about 5 wt% , 3%, and 4%) and subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%). In one embodiment, the term "about" may include conventional rounding depending on the significant number of numerical values.

Example

Having generally described the embodiments of the present disclosure so far, the following embodiments describe some additional embodiments of the disclosure. The embodiments of the present disclosure are described with reference to the following embodiments and corresponding text and drawings, but are not intended to limit the embodiments of the present disclosure to the above description. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the spirit and scope of the embodiments of the present disclosure.

Example  One

placenta Derived  Extract In vitro Derived In the BIOS CAFOLD  Induction and regulation of angiogenesis

Introduction

This example describes a method for inducing vascularization using a complex human placenta extract (PE). PE is derived from the human placenta and is capable of inducing in vivo angiogenesis in biologically engineered tissue graft as well as in 2D and 3D in vitro models. This example also describes the effective screening of thrombospondin 1 as an angiogenesis inhibiting protein with increased susceptibility compared to current in vitro models using placental extracts. In particular, this model allows for control over the rate and type of angiogenesis (intestinal versus germination) and offers broad application in regenerative medicine and pharmacy as well as many advantages over conventional approaches.

The limitations of mass transfer within tissues are a barrier to the production of effective biomaterials. Even though this disorder can be temporarily overcome by improving cell migration within the human bioscaffold, the creation of effective vasculature remains an important goal to provide nutrition to thick, cellular dense materials over long periods of time. In the adult, the new blood vessels are primarily generated by the physiological processes of angiogenesis, which ultimately forms a nutrient-rich vascular network ^47. This example demonstrates that angiogenesis can be induced in human vein (HUV) vascular grafts and can lead to a long-term nutritional delivery system.

Successful vascularization of the engineered organ through an angiogenic regulator and in vivo restoration of infarct tissue has been a major obstacle to clinically delivering successful antibiotic therapy. Several different approaches have been used to initiate angiogenesis and to induce larger angiogenesis, including direct cell seeding (single and co-culture), stem cells, and a combination of human-derived regulators / growth factors. Until now, little success has been achieved in converting an in vitro approach using typically non-human animal compounds into clinical trials.

An important issue in the field is that the most common / successful approach (Matrigel or basement membrane matrix) is derived from Angel Bres-Holm-Swarmsome sarcoma cells and is thus inappropriate for human therapy. Thus, an approach or mechanism using human-based materials that actively promote angiogenesis in both in vitro and in vivo systems will have a significant impact. This example provides a human placenta extract (hPE) that is capable of inducing angiogenesis in a biologically engineered tissue graft, as well as in 2D and 3D in vitro models. PE is a complex of active human biomolecules and in addition to inducing in vivo and in vivo angiogenesis in human IV vein grafts derived from ex vivo, this model can be used to control the rate and stage of angiogenesis . This example also demonstrates that using PE-administered collagen-based bioscalves improves capillary angiogenesis while reducing fibrosis.

Way

Placenta extract induction . The term placenta was collected from UF Health Shands Hospital (Gainesville, FL) within 12 hours of delivery. The umbilical cord and fetal membranes were removed and the placenta was incised into 2 cm cube and frozen. After gradual freezing to -86 ° C at a rate of 1 ° C / min and after 12 hours, the placenta cube was transferred to a cooling chamber maintained at 4 ° C to complete the rest of the procedure. At 4 DEG C, 100 grams of tissue was mixed with 150 mL cold 3.4 M NaCl buffer (198.5 g NaCl in 1 L distilled water, 12.5 ml 2 M Tris, 1.5 g EDTA, and 0.25 g NEM). The NaCl buffer / tissue mixture was homogenized using a TissueTek Homogenizer at 3200 RPM and centrifuged at 7000 RPM for 15 minutes to separate from the supernatant. The above NaCl washing step was repeated two more times, and the supernatant was discarded every time to remove blood.

The pellet was then homogenized in 100 mL of 4 M urea buffer (240 g urea in 1 L distilled water, 6 g base, and 9 g NaCl), stirred on a magnetic stir plate for 24 hours, centrifuged at 14,000 RPM for 20 minutes The supernatant was removed and 2.5 ml of chloroform for sterilization and 1 L of TBS (6 g of 1 L distilled water in distilled water) was added to the reaction mixture, and the supernatant was removed (Sorvall RC6 + Centrifuge, Thermo Scientific, NC, USA) (Spectrum Laboratories, Inc., Calif., USA) in an 8000 MW dialysis tube (base and 9 g NaCl) .The buffer was replaced with fresh TBS more than 4 times at 2 hour intervals Finally, the contents of the dialysis tube were polymerized by centrifugation at 3000 RPM for 15 minutes (Allegra X-12R Centrifuge, Beckman Coulter, Inc, California, USA) Protein , And collecting the supernatant (pink viscous lysate) was stored at -86 ℃ until use.

Biomolecular composition analysis. A sandwich immunoassay array (Human Cytokine Antibody Array C Series 1000, manufactured by Ray Biotech, Inc., USA) was used to construct a sandwich immunoassay array (Human Cytokine Antibody Array C Series 1000, The chemiluminescence was detected using a Foto / Analyst Luminaryfx Workstation (Fotodyne Incorporated, Wisconsin, USA) and signal intensity was measured using a total The relative abundance ratios of basement membrane biomolecules were measured using the Orbitrap Velos Pro (ThermoFisher, Mass., USA) using TotalLab 100 software (Nonlinear Dynamics, Equipped with a NanoAcquity HPEC (Waters, Milford, Mass., USA) system connected to a &lt; RTI ID = 0.0 & And MS Bioworks (Ann Arbor, Mich.) Using nano LC / MS / MS. Proteins were obtained from a primary sequence database using a Mascot database search engine (Boston, Mass., USA) Respectively.

RT- PCR analysis of cells from the network of hPL- induced angiogenesis . Relative angiogenesis gene expression was measured using a 384 well RT ² Human Angio Genesis RT ² Profiler PCR Array (RT ² Human Angiogenesis RT ² Profiler PCR Arrays) (PAHS-024A, Quijagen, CA). EC was removed from the culture plate using Accutase (Innovative Cell Technologies, San Diego, Calif., USA) and immediately stored in 100 [mu] l of RNA Later (RNA later ). RNA was extracted using an RNeasy Mini Kit (Qiagen: California, USA), and genomic DNA was extracted with an RNase-Free DNase kit (Qiagen: Calif., USA) . After the purified RNA was at 42 ℃ incubated for 15 minutes, then, RT ² first-strand kit of from 95 ℃ incubated for 5 minutes to stop the reaction ^{(RT 2 First Strand Kit) (} SA Bio Cyan System (SA Biosciences: Texas ) Was used to reverse-transcribe the cDNA. The cDNA was then mixed with RT ² SYBR Green Mastermix (SA Biosciences, TX, USA) and loaded in a 384-well Human Angiogenesis PCR Arrays. Stacked array plates using a Bio Rad CFX384 Real-Time System (BioRad, Calif., USA) were incubated at 95 ° C for 10 min, denaturation cycle, 60 ° C for 30 sec annealing / The elongation cycle was run 40 times and finally ramped from 60 占 폚 to 95 占 폚 at a rate of 占 폚 per second to obtain a melting curve. The data ΔΔC _t method and RT ² Profiler PCR Arrays data aeneolrisiseu template (RT ² Profiler PCR Array Data Analysis Template) v4.0 software package, was analyzed by (Qiagen, California, USA).

Human intravenous endothelial cell isolation and myxoblast cell culture . Endothelial cells were obtained from human vein (UF), which was detached from the vessel wall using a 1 mg / ml solution of small type I collagenase in phosphate buffered saline (Gibco, Invitrogen (New York, USA) Resins from Heath Schoenzhafthal (Gainesville, Fla.)). Human primary intravenous endothelial cells (HUVECs) derived from primary were used one to three subcultures for all experiments. For propagation, the cells were cultured using the complete VascuLife Basal media (VascuLife VEGF Medium Complete Kit, Lifeline, Maryland, USA). For angiogenesis experiments, the endothelial cell culture medium was supplemented with 500 mL of a solution containing 25 mL of glutamine, 0.5 mL of hydrocortisone, 0.5 mL of ascorbic acid, 10 mL of FBS, and 1.25 L of bFGF, Life Technologies, Maryland) using a Q-Life VEGF Medium Complete Kit. Human myofibroblast (CRL 2854) was used as a 5- to 10-fold subculture (ATCC, Manassas, Va.) And cultured in low glucose DMEM supplemented with 10% FBS.

Preparation of Placenta Extract - Derived Angiogenesis Assay . Unless otherwise noted, the 32 [mu] l placental extract was thawed and pipetted into each well of a 96 well plate. The extract was evenly coated on the bottom of each well at 30 RPM for 1 minute using a rotary shaker. The coated plates were then incubated at 37 [deg.] C for 30 minutes. The HUVEC was then plated by direct pipetting at 20,000 cells / cm2, 40,000 cells / cm2, or 80,000 cells / cm2. Several times, including days 1, 3, and 5, were examined at each concentration. Trombospondin 1 was tested as an angiogenesis inhibitory drug using final concentrations of 0, 5, 10, 20, and 35 ug / 희 diluted in endothelial cell medium.

Morphological characterization of angiogenic networks. The endothelial cell culture medium was stained with Calgain AM (Invitrogen-Life Technologies, New York, USA) at a concentration of 2 μg / mL and the network formation was analyzed. In the dark room, the stained cells were incubated for 30 min at 37 ° C, 5% CO ₂ and then incubated with a Zeiss Axiovert 200 Inverted Fluorescence Microscope (Zeiss, USA Thornwood, NY). Images were analyzed using image J (ImageJ) 1.45s (NIH, Bethesda, Md., USA) to determine tubule length, tubing width, bifurcation, and other meshwork characterization. The branch point was manually designated as the position where all branches where the branch meets or the germination of the tubule are present, and the length of the tubule is measured by measuring the curve length from the branch point to the connected branch point. The tubing width was measured in two different zones per customs tube, 10 ㎛ from the beginning and end, and one zone in the middle of the curve length. The reach area (%) was measured by processing the image using the image J function "binary >> convert to mask" and then measuring the "average". In the TSP-1 experiment, the final value was normalized for the un-dosed sample, calculated as a ratio of the value of "1" relative to the total coefficient of the pixel value, and presented as the "area of arrival (%).

For the analysis of differential scanning microscopy of the form, microvessel networks grown on glass slide were fixed in 2.5% glutaraldehyde, washed in PBS, fixed in 1% osmium tetroxide solution, 25%, 50% 75%, 85%, 95%, and 3x100% ethanol. The sample was then dried at the critical point, coated with gold / palladium, and imaged using a Hitachi S-4000 FE-SEM (Hitachi S-4000 FE-SEM).

Human intravenous scaffold induction and placenta extract incubation . The placenta was collected from UF HealthShangs Hasselfalter (Gainesville, Fla., USA) and the HUV was incised using an automated method as described previously. ³² incised HUV samples were removed in 1% SDS (Thermo Scientific, Rockford, IL, USA) with a solvent / tissue mass of 20: 1 (w: v). The samples were removed on a rotary shaker plate at 100 RPM for 24 hours and then washed with PBS and incubated overnight at 37 ° C with 70 U / mL DNase I solution (Sigma-Aldrich, Sigma-Aldrich, : St. Louis, Missouri, USA). Samples were finally sterilized with 0.2% peracetic acid / 4% ethanol (Sigma-Aldrich, St. Louis, Mo.) solution for 2 hours and finally pH equilibrated with PBS (7.4). After removal of the cells, the scaffold was cut into 1.5 cm x 1.5 cm x 0.075 cm sheets and pre-frozen at -85 deg. C, followed by centrifugation using a Millrock benchtop manifold freeze dryer (Kingston, NY, USA) And lyophilized at -85 캜 for 24 hours under 10 mT vacuum. Immediately prior to cell seeding, the scaffold was soaked in hPE, Matrigel, or PBS (control) for 2 hours and seeded.

Studies on vascularization of animal grafts . Male Sprague-Dawley rats (6 months, 200 g) were purchased from Charles River Laboratories (Wilmington, Mass., USA) and all procedures were performed at the University of Florida ) IACUC (UF # 201207728). In the biological hood, the final sterilized HUV scaffold was incubated in 5 mL of hPE, Matrigel, or PBS (control) for 2 hours each. Animals were anesthetized with isoflurane inhalation, and subcutaneous pouches were made in the dorsal and right side by dulling with scissors. A scaffold was inserted into each subcutaneous pocket and the skin was sutured using a 4-0 suture (Coviden, Mansfield, Mass., USA). Five days after transplantation, animals were euthanized and samples were removed for analysis.

To analyze the capillary network formation, immediately after removal from the animal, the fibro-capsules were incised with a scalpel and the HUV samples were placed on a glass slide. A top-down image of a translucent scaffold sheet was imaged using an Imager M2 optical microscope (Zeiss: Ober-Cohen, Germany) equipped with an Axiocam HRm digital camera (Zeiss, To quantify cell migration and scaffold remodeling, tissue samples were mounted on Neg-50 frozen section media and cut into 7 쨉 m sections (Microm HM550 cryostat, Thermosynthetic: Waltham, Mass., USA) , Standard hematoxylin and eosin (H & E) staining (Richard-Alan Scientific, Kalamazoo, MI).

Statistics . Results are reported as mean ± standard deviation. Linear regression was performed using SPSS (IBM: Somers, NY). Rt-PCR data is RT ² Profiler PCR Arrays data aeneolrisiseu v3.2 software (Bio SA Cyan System (SABiosciences: Valencia, California)) were analyzed by.

Perfusion bioreactor culture and HUV BIOS caching induced angiogenesis in the fold. Cell-seeded tubular structures were cultured in a dual perfusion bioreactor (FIG. 7) at a flow rate of 4 mL / min at a rate of 60 pulses / min for 5 days. The shear stress on the vessel wall was calculated using the Haagen Poisseuille equation, assuming that the medium flow was steady and the laminar flow and vessels were inelastic, cylindrical, and straight: ¹³⁴

Where Q is the average volume flow rate, and μ is the fluid viscosity of water at 37 占 폚 (0.000692 kg / (m * s)). ^{The 134} shear stress was cycled from 0 dyne / cm2 to 0.04 dyne / cm2 cycles for each pulse. The environment was maintained under standard cell culture conditions of 37 ° C and 5% CO ₂ . The pressure in the system remained negligible (<2 mmHg) in both the lumenal and luminal flow circulation, and there was no pressure gradient across the scaffold. The culture medium was replenished once every two days in the bioreactor. Five days after perfusion culture, a 10 cm long tubular scaffold was incised into a ringlet for histological analysis.

result

Induction and Characterization of Human Placenta Extract

After initial mechanical homogenization and centrifugation, the hPE induction technique linearized and solubilized the molecules using a urea stage. Subsequently, the urea was removed by dialysis and separation so that the biomolecule was refolded in its original three-dimensional form (Fig. 1A). All steps of induction were performed in a 4 ° C cold room. The final solution of PE was translucent, viscous and consisted of biomolecules between 8 kD and 868 kD.

hPE could be immersed in biomaterials, or could be made into thin films for tissue culture assays. The reproducibility of hPE was assessed by analyzing the standard deviation of the total protein content in n¼3 hPE batches (each batch was generated using the same mass of tissue from three individual donors), which was similar to Martrigel &&Lt; / RTI &gt; Differential scanning microscopy images show that the surface morphology of hPE (Fig. 1B-1C) and HUVEC were seeded at 4 x 10 ⁴ cells / cm 2 on the hPE membrane and formation of the angiogenic network when cultured for 3 days (Fig. 1d- 1e).

The angiogenic potential of human placenta extract (hPE) was initially characterized by seeding primary human endogenous endothelial cells (HUVEC) on tissue culture plates coated with hPE. Early stage cell coding and germination were visually observed (not shown) within 1 hour after cell seeding and the angiogenic network remained mature continuously until the end of the 3 day (d) experiment (Figure 1f-1g ). The length of individual cell cords (multicellular) was significantly increased between the first day after seeding (Fig. 1C) to the third day (Fig. 1I). Three days after incubation, cells formed extensive angiogenic networks compared to control samples (Fig. 1j, 1k).

Biomolecular Characterization of Placenta Extract

Among the 120 cytokines evaluated, 54 angiogenesis-related cytokines were detected in placental lysates (Fig. 2a). The most common angiogenic chemokine was angiogenin, a potent stimulant of new angiogenesis ¹⁶ . But are not limited to, hepatocyte growth factor (HGF), fibroblast growth factor-4 (FGF4), leptin (LEP), ICAM-1, ICAM-2 and TIMP-2 Important angiogenic chemokines were detected. Immunologically related proteins, including annexin (ANXA1, ANXA2, ANXA4, and ANXA5), neutrophil dyspensin (DEFA1), interleukin enhancer binding factors (ILF2 and ILF3), IL27, ITBG1 and MRC1 (Fig. 2B). LAMB1, LAMB2, LAMB3, and LAMC1), fibronectin (FN1), heparin sulfate (HSPG2) and type 4 collagen (COL4A1, COL4A2, and COL4A3) 2c) were also detected (Fig. 2c), which have been shown to play an important role in angiogenesis, respectively. ^17-20

hPE  In the network of induced angiogenesis Endothelial cell  Gene expression

In addition to chemokine analysis, HUVEC gene analysis further confirmed the angiogenic character of placenta extract. RT-PCR analysis revealed that endothelial cells seeded with hPE for 3 days (d) expressed a wide range of essential angiogenic genes including hepatocyte growth factor, epidermal growth factor, and placental growth factor (Fig. 2d). Additional up-regulated genes include MMP2 and MMP9, which are not only endothelial cells, but proteases that aid in the degradation of the surrounding extracellular matrix to promote the migration of other cells associated with ECM remodeling ²¹ . In the maturation of the microvasculature, type IV collagen associated with basement membrane formation was also upregulated [ ^22] .

In vitro  Capillary blood vessel development and morphological control

So far, in vitro methods have been unable to control the rate and stage of angiogenesis with little or no control. ⁹ This data shows that in vitro hPE-based angiogenesis assays can be modified to control the maturation and morphology of angiogenic network formation by altering the initial cell seeding density. After one day, the HUVEC seeded at a density of 40,000 cells / cm2 formed more distinct tubules with contours when compared to those seeded at a density of 80,000 cells / cm2, but cells seeded at both densities up to day 5 Had clear-cut tubules (Fig. 3A). These results show that the maturation step of network formation can be controlled when the culture is exposed to hPE by varying the cell seeding density. For example, quantitative image analysis results of capillary network (average tubule length [mm], tubing density [# / mm 2], junction [# / mm 2], mesh [# / mm 2], and average tubule width [mm] (FIG. 3c), the higher the seeding density, the slower the network maturation rate (the longer the period to reach the longest average tubing length and the maximum number of mesh / mm2) This allows for more detailed analysis. Similarly, lower cell densities have been associated with a faster maturation rate, which may be more helpful in a rapid screening approach (e.g., testing the efficacy of angiogenesis blockers for cancer therapy).

The Martrigel-induced angiogenic network as an excellent standard for in vitro angiogenesis assays was compared to the hPE inductive network (Fig. 3d). First, the cell culture was exposed to Matrigel or hPE, and the morphology of the endothelial capillary network was analyzed using Calcine AM to determine the viability and network structure. On the first day after seeding, HUVEC in the matrigel coated plate formed a well-defined angiogenic tubule network, but after 3 d (d), the network structure collapsed into spherical balls of apoptotic cells (Fig. 3a, 3d ). Some apoptosis was observed in the hPE inducible network, but no apoptotic ball formation was observed after an extended period of 5 days (d).

In the late stage of angiogenesis, EC mobilizes smooth muscle cells (SMC) to stabilize blood vessels as the capillary network matures. Thus, the effects of hPE and matrigel on smooth muscle cell morphology were evaluated. Interestingly, in the absence of HUVEC, the SMC seeded in Matrigel formed tubules after 1 d (Fig. 3b.i), while maintaining a typical 'hill and valley' formation in hPE coated plates 3b), indicating that there is a significant difference in the molecular signaling pathway between cell types. SMC is not known to form tubules in the early stages of microvascular formation, and thus the results may suggest that hPE-based angiogenesis may more accurately represent normal physiology.

For drug screening applications hPE  Analysis of inducible angiogenesis

In addition to his role in regenerative medicine, human placenta extract (hPE) induced angiogenesis was tested for its ability to screen angiogenesis-related drugs in vitro. Matrigel was used as a control. Matrigel and hPE-based angiogenesis assays were screened against thrombospondin 1 (TSP-1), a glycoprotein with potent inhibitory activity against neovascularization. TSP-1 represents a model drug for the anti-angiogenic treatment of solid tumors ²³ . After 1 d of incubation, the control HUVEC seeded directly onto the tissue culture plate was not affected by TSP-1 and the cells formed a typical pebble morphology. In contrast, HUVEC cultured in hPE treated culture plates significantly reduced angiogenesis network formation (Fig. 4A). The results indicate that total tubule length and bifurcation decrease linearly as a function of TSP-1 concentration (FIG. 4B). Significantly, these studies indicate that hPE-based assays are more susceptible to drug concentrations than matrigel-based assays. This is expressed in a higher correlation between the TSP-1 concentration and the% reduction in the area of reach of the angiogenic network with R ² values of 0.97 and 0.36, respectively (FIG. 4C). Inhibition of network formation further demonstrates actual angiogenesis besides unrecognized stress response. Additionally, since the hPE is derived from human, inaccuracy based on the species that is caused by screening using a non-human system avoids ^13,14.

3D BIOS CABLE  Within In vitro  Network formation of angiogenesis

Because successful vascularization in engineered organs has been a major impediment to the development of successful regenerative medicine therapies ²⁴ , the potential for hPE to induce angiogenesis in in vitro derived bioscaffolds has been analyzed. The present study showed that hPE induced the formation of an angiogenic network in engineered (cell removed) human vein (HUV) bioscapes (Fig. 5A). Consistent with assays on 2D culture plates, endothelial cells (ECs) seeded on the biosecolds developed into a rectangular shape connected by multicellular cords to form a complex network of interconnected cells.

In vivo angiogenesis occurs by a variety of mechanisms, most commonly by germination or intestinal overgrowth. This data shows that when incubated with hPE, germinal vesicle superimposed angiogenesis can be regulated by varying cell densities in vitro. The network form in the hPE incubated scaffold at lower cell density (2 x 10 ⁴ cells / cm 2) showed germination (Fig. 5c.i, 5C.ii), medium density ( ⁴ x 10 ⁴ cells / The morphology showed a combination of germination and intestinal angiogenesis (Fig. 5c.iii, 5C.iv) and at a higher density (6x10 ⁴ cells / cm 2) the network morphology showed a similarity to that of long-overlapping angiogenesis (Fig. 5c.v, 5C.vi). Since germination occurs at an early stage of angiogenesis in low cell density areas, which are generally capillary free, the correlation between cell density and the specific mechanism of angiogenesis is currently understood for vascular capillary and network formation Lt; / RTI &gt; On the other hand, Chapter overlap occurs at a higher cell density and area for capillary endothelial cells is already present ^25,26.

In vivo  Induction of angiogenesis

Using the subcutaneous rat model (Fig. 6A), angiogenic responses to the administered scaffolds (Matrigel and hPE) were assessed on day 5 post-implantation. Both the control and Matrigel incubated scaffolds showed significant fibrosis around the scaffold, whereas there was no discernable fibrosis around the graft in the hPE administered scaffold (Fig. 6b.i-6b.iii.). Anti-fibrosis in the hPE sample was detected in the presence of anti-inflammatory annexin (ANXA1, ANXA2, ANXA4, and ANXA5) ²⁷ , antimicrobial dipentin peptides such as DEFA1- ²⁸ , and MRC1 Which are known to bind to a potential pathogen, including, but not limited to, viruses and bacteria.

As shown by bright field microscopy, both matrigel and hPE incubated scaffolds showed a significant increase in neovascularization when compared to the control (Fig. 6b.iv-6b.vi). While total vascularization was similar, hPE treatment showed formation of mature capillary vessels, while matrigel samples appeared to be less structured without evidence of mature capillary vasculature. The H & E stained slices showed that the cells migrated from hPE to incubated scaffolds and throughout, while the Matrige incubated samples showed decreased cell invasion and the cells in the control samples were localized around the scaffold (Fig. 6b.vii.-6b.ix.). Improved cell migration in both matrigel and hPE incubated samples was the result of chemotactic and growth factor signals adsorbed to the scaffold structure. Despite improved cell migration in both the hPE and matrigel-administered scaffolds compared to the control group, cell remodeling varied among the sample groups. In the Matrigel incubated sample, the cell density area was defined as the original, well-structured, generally more amorphous structure, as compared to an hPE incubated scaffold that appeared to be almost completely remodeled, thereby exhibiting more organized fiber and cell structure Of HUV fibers (Fig. 6b.vii.-6b.ix.).

Argument

Tissue regeneration, infarct tissue and ischemic wound restoration are three clinical areas that have a clinically significant impact on improved strategies for wound healing or tissue replacement. The use of amniotic membrane and chorionic villi has greatly increased in various applications over the last five years, with an increasing number of evidence suggesting that the post-natal tissue has a significant clinical ^outlook29-32 .

The results herein focus on the physiological ratios of the human-derived potent stimulating factors of angiogenesis and tissue remodeling, and describe in detail a novel approach to delivering it. This data demonstrates the ability to initiate capillary angiogenesis (in vitro and in vivo), to regulate the EC phenotype during angiogenesis with the ability to regulate growth or matured dynamic properties, and to provide improved Cell activity.

The ability to control the rate of in vitro maturation of capillary network formation and to control the occurrence of germination and the network form of intestinal neovascularization is a useful platform for understanding the control pathways in wound healing and tissue regeneration . Based on a comparison with Martrigel, the mechanism by which hPE stimulates cells appears fundamentally different. SMCs incubated with Matrigel initiated capillary similarity formation, but SMCs exposed to hPE retained their typical heel and valley morphology, and thus human-derived hPE is a model more representative of physiological angiogenesis in more complex models Can be provided.

Many of the current methods are based on the human-derived (recombinant) modulators that rely on a single angiogenic regulators or a combination of the individual ^33. Although individual combinations control mutations and reduce the complexity inherent in the various approaches, they limit the screening process and can be used to test a broad set of human in vivo agents that can be important in testing the potential of antiangiogenic, Molecular interaction.

The complexity inherent in the hPE-based model can lead to advances in the pharmaceutical industry by providing a more effective screening approach for tumor suppressor drugs. Compared to current technology, exposure of human EC to hPE appeared to increase susceptibility to an angiogenesis inhibitory drug concentration (TSP-1) with a lower detection limit.

hPE-based models induce angiogenesis using a broad set of human-derived molecules at close physiological proportions. Since the in vivo blood vessel formation requires the induction of various metabolic pathways, it is considered that only the adjustment of only the selected path to molecules limited the attempts to develop a new anti-angiogenic drugs ^34,35. Thus, drugs can regulate angiogenesis through interaction with any of these many pathways, but if the local environment is not complex, the effect of inducing sufficient angiogenesis may be scarcely visible. The in vivo analysis results in this example further provide evidence that the composite PE can affect many biochemical pathways and result in a wide range of effects. According to the data, hPE not only improves angiogenic properties, but also has immunosuppressive properties, as shown by reduced fibrosis in hPE-administered bioscalves. Considering the complex interconnection between angiogenesis and immunologic molecular pathways, the molecular composition of hPE ³⁶ is an appropriate basis for developing clinically applicable techniques for inducing capillary angiogenesis without significant immunological inflammatory response to provide.

The positive results of the studies described above seem to be related not only to the presence of important growth factors (GF) and gene regulatory factors in PE but also to their presence in physiological proportions. For example, VEGF was up-regulated in hPE-inducible EC, but hPE solution did not contain detectable levels. This is in contrast to Matrigel (BMM), which contains VEGF as the active concentration in both the standard version and the GFR version (growth factor reduced). The results of this study showed that more mature capillary vessels were formed, the presence of VEGF is only one of many contributing factors, and the presence of other regulatory factors may possibly play an important role in angiogenesis. Similarly, compared with the human recombinant protein, which is used to initiate angiogenesis, these highly concentrated (typically) depends on a single GF ^{37 -39.} It has been reported that a single, highly reactive GF is clinically applied and has undesirable effects ^40-42 .

hPE angiogenesis models have been demonstrated in 2D and 3D in vitro models as well as in vivo in vivo manipulated tissue implants and can be easily adapted for a variety of clinical or pharmaceutical applications. The fact that this model is derived from a physiologically sound human blood vessel in combination with its angiogenesis and immunosuppressive properties makes it unique among current angiogenesis models. The data presented here demonstrate that hPE plays a central role in many important clinical issues where the need for an alternative, more successful approach is clinically preferred.

Example References to 1

Example  2

Regulated delivery of placenta extract for angiogenic stimulation

Introduction

Tissue manipulation aims at building tissue and organs from scratch in vitro to transplant into sick patients. However, this innovative alternative to grafting is subordinated to the failure to form an appropriate vasculature to supply oxygen and nutrition to the seeded cells in the grafted graft. Therefore, there is a desperate need for an effective method for inducing angiogenesis in tissue engineered structures.

To date, all methods attempted to promote vasculization in engineered products have produced unsatisfactory results. The present disclosure provides a protocol for obtaining angiogenesis-induced extracts from the human placenta that has been shown to induce and modulate the early stages of angiogenesis, as described in the above examples.

This example describes a 3D in vitro neovascular assay that promotes the formation of a capillary network and persists it over time. For this purpose, the angiogenic potential and its bioactivity of the placenta extract over time were analyzed. These studies have shown that frequent administration of extracts promotes the formation of mature, long-lasting capillary networks. Therefore, biodegradable gelatin microparticles for the introduction and controlled release of the extracts were prepared and their degradation kinetic properties were studied. Finally, a 3D in vitro angiogenesis assay was developed. The placenta-loaded microparticles were embedded in HUVEC-seeded type I collagen hydrogel scaffold and evidence for the early stages of microvascular formation in the matrices was demonstrated.

In modern society, loss or impairment of an institution or organization is one of the most serious and costly human health problems. Many approaches have been attempted to address these problems, including surgical reconstruction, pharmacotherapy, prosthetics, mechanical devices, and implants. In the long term, this approach does not completely replace the function of the missing organs or tissues. ² , In the case of grafts, the number of donors is often high and the number of donors is often not enough.

One solution would be to develop a manipulated organ or tissue. However, the current organizational manipulation approach has achieved limited success due in part to the nutritional barriers that result from passing through thick bioscapes. A major obstacle to operating thick (> 200 [mu] m) tissue structures is that oxygen diffusion is typically confined to distances of 150 to 200 [mu] m in the tissue manipulated scaffold, thereby damaging cell growth and viability in 3D tissue structures It is ^{6 , 7} . Oxygen is an important nutrient for cell survival. Without oxygen, cells in the scaffold die. Further, when the transportation conditions are poor, the fibrous capsules secreted by the cells are usually formed. The encapsulation lacks nutrient transport in the scaffold, resulting in low cell density. Thus, manipulating long-lived sustainable tissue will benefit from a method of promoting delivery of oxygen and nutrients to seeded cells in 3D tissue structures. The placenta extracts and methods of this disclosure provide an approach to overcome oxygen and malnutrition, including inducing rapid nutrient-rich capillary blood vessels in the scaffold. An implementation method that can supply the essential nutrients to the center as well as the limb of the structure will help prevent the formation of fibrous capsules.

Angiogenesis is important for tissue development and maintenance, and when the angiogenesis is successfully regulated, the established vascular network of the implanted graft is facilitated to be regulated. Several biological factors and molecular pathways are involved in regulating such complex processes, which are still not yet partly known. To facilitate a better understanding of the molecular mechanisms that initiate and regulate vessel growth, studies have shown that vessels, which are cell culture systems that regenerate the final elements of angiogenesis in vitro or in vivo under conditions that are streamlined, We have been focusing on developing new assays. Although a variety of different approaches have been used to promote angiogenesis in vitro, to date there has been little success in interpreting him as a clinical trial. The limitations of most angiogenic models depend entirely on the use of animal-derived (e.g., matrigel-based) or live animals (such as chick villi and rabbit corneal micropocket). In addition, there are no various cytokines and chemical gradients present in the natural living body . As a result, the results of the assay were generally disappointing because there was no long-lasting angiogenesis. Thus, an in vitro model of authentic human origin will be useful for mechanistic research and for screening human angiogenic drugs.

Considering that the placenta is an easily available tissue and is an abundant source of vascular tissue and angiogenesis-related growth factors, this study demonstrated that, as described above, and as described in Example 1, (HPE or hPM), which is a viscous protein compound rich in cytokines and angiogenesis-related growth factors. hPE has been shown to induce and regulate the early stages of angiogenesis in vitro for a limited period of time. In addition, hPE has also been found to significantly reduce fibrosis.

This embodiment provides a method of continuously delivering hPE so that growth factors in the hPE can remain functional for a sufficiently long period of time to allow the formation of a mature capillary network. For this purpose, microparticles have been developed as a delivery system due to its ability to efficiently encapsulate and release the polypeptide at a constant rate over a long period of time, and due to its versatility ²² .

Fine particle size is in the range of 1 to 1,000 ㎛, surface to a near solid particles in a volume large, nearly spherical ratio is ^23. This may be done using a number of different materials, both natural (e.g., starch, gum) and synthetic (e.g., polylactic acid and polyglycolic acid) materials using different techniques, such as hot melt extrusion, spray drying or solvent removal ²⁴ can be prepared. The release rate of the fine particles can be controlled by varying their size: the smaller the particle size, the faster the particle is dissolved than the larger particles as the surface to volume ratio increases. For this reason, the size can change the transfer rate by combining the different particles ^25. Drug or protein release from microparticles usually occurs by simple matrix bioerosion. This process involves corrosion of the particle surface, followed by bulk corrosion and the introduction of the release medium into the particle voids ^{26 , 27} .

In this example, biodegradable gelatin microparticles were prepared and used for 3D angiogenesis assay in vitro. Angiogenesis assay is a cell culture system that is simplified, and only reproduces the final element of angiogenesis in vitro or in vivo under conditions which are modulated ^28. Which may be two-dimensional or three-dimensional. 3D In vitro In the assay, EC is then present on the substrate surface and generating a tubular structure which enters the space surrounding matrix usually consists of bio-gel ^13.

This example demonstrates that in a 3D in vitro assay that promotes the formation of a long-lasting vascular network within an implanted graft An embodiment of an angiogenesis assay is provided. In this embodiment, the assay is prepared by embedding human hematopoietic endothelial cells (HUVEC) with microparticles loaded with PE in a collagen type I matrix. The angiogenic response of HUVEC was then analyzed at other incubation times.

Experimental Method

Endothelial cell isolation and culture . As described by Jaffe et al. (Culture of Human Endothelial Cells Derived from Umbilical Veins. 52 , 2745-2756; 1973), which is incorporated herein by reference in the context of isolation and cultivation of HUVECS) Pre-isolated, pre-cultured human intravenous endothelial cells (HUVEC) from the vein were removed from the flask using an accelerator (Fisher Scientific) and centrifuged at 1,000 rpm for 5 minutes. The cell pellet was resuspended in medium (25 ml glutamine, 0.5 ml hydrocortisone, 0.5 ml ascorbic acid, 10 ml FBS, 1.25 l VEGF, and 1.25 l bFGF in 500 ml Basque Life Life Basal medium) And HUVEC was counted using a hemocytometer.

HPM according to time function Angiogenesis analysis . To evaluate how hPE incubation time affects HUVEC angiogenesis, cells were plated on hPE coated tissue culture plates and incubated with hPE for 1, 3 or 5 days. Briefly, the placental matrix was prepared using the isolation method described in the above examples. The vial containing the extract was thawed and pipetted (100 μl / cm 2) into the wells of a 96-well plate. HUVECS was prepared by directly pipetting and plating the cell solution at 20,000 cells / cm2. Was added to each plate sample prepared angiogenesis and medium (200 ㎕ / ㎠), placed on the wetting of the latter in the incubator 37 ℃ 5% CO _2.

To determine how hPE maintains bioactivity over time, incubation was carried out at 37 &lt; 0 &gt; C and 5% CO ₂ In an incubator). The film hPE from each time point was then coated onto a 96 well plate for tissue culture as described above and seeded with HUVEC. Cells were cultured for 3 days and the angiogenic network was qualitatively characterized.

HPE according to inoculation frequency function Angiogenesis analysis . The response of HUVEC to the number of hPE inoculations was also analyzed. On day 1, the cells were first seeded onto hPE and hPE was mixed with the culture medium for hPE administration after day 1. Cells were cultured for 5 days, with some sample groups receiving a single hPE inoculation on day 1 (day of seeding), the other sample group being inoculated twice on days 1 and 3, the other sample groups being 1, 3, and On the 4th day, she was inoculated three times.

Manufacture of gelatin microparticles . (Hereby incorporated by reference herein in relation to the manufacture of gelatin microparticles) (Tabata), etc. to prepare a gelatin microparticles using the method described by ^33. All reagents used were obtained from Fisher Scientific. Briefly, a 10% wt B-type gelatin aqueous solution was prepared by adding 1 g of gelatin to 9 mL of deionized water. The temperature was raised to 45 &lt; 0 &gt; C and the solution was added dropwise to 375 ml warm (45 &lt; 0 &gt; C) olive oil through a syringe and 21 G needle with constant stirring at 400 rpm. After 10 min, the emulsion temperature was lowered to 15 캜 and stirring was continued for 30 min to induce gelation. 100 mL of cold acetone was added, and the emulsion was stirred for 1 hour. The fine particles were removed by vacuum filtration, washed with acetone and dried. Once dried, it was placed in an aqueous solution containing 0.1% wt Tween 80 and 0.5% wt glutaraldehyde. The solution was stirred constantly at 125 rpm for 15 hours at 4 &lt; 0 &gt; C to promote cross-linking of the fine particles. The crosslinked microparticles were collected by vacuum filtration, washed in deionized water, and then stirred in 100 mL of 10 mM glycine aqueous solution to block any unreacted glutaraldehyde. After 1 hour, the fine particles were again collected by filtration, washed in deionized water, and lyophilized. 100 [mu] l of pure hPM per 1 mg of fine particles was incubated to load cross-linked lyophilized gelatin microparticles. The mixture was vortexed at full speed and allowed to adsorb by overnight incubation at 4 &lt; 0 &gt; C.

In vitro Release kinetic properties of hPE loaded microparticles . To evaluate the decomposition kinetic properties of the gelatin microparticles, non-crosslinked blank microparticles of 10 mg (dry weight) at 37 占 폚 were incubated in 1 ml of phosphate buffered saline (PBS) (pH 7.4) , And protein release kinetic properties were compared using a kit for protein assay from crosslinked microparticles loaded with hPE and crosslinked microparticles loaded with hPE. Briefly, supernatants from each sample were periodically collected (1, 2, 3, and 6 hours, and then daily) and replaced with fresh PBS. (BioTech Sinergy 2 plate reader, Bio Teck Instrument, Inc.) using a Micro BCA Assay Kit (Thermo Scientific, Waltham, Mass., USA) Protein in supernatant was quantitated by absorbance measured at 562 nm using a fluorescence microscope. Protein concentrations were compared to freshly prepared bovine serum albumin standards with concentrations ranging from 200 to 0.5 [mu] g / mL. The final value is presented as the total protein release from the start of the experiment and is normalized as a function of the non-crosslinked blank microparticle dry weight or as a function of the wet weight estimate before the start of the experiment. The absorbance value is normalized to the value of phosphate buffered saline (pH 7.4). The linear range of action was 2-40 ㎍ / mL and the detection limit was 0.1 ㎍ / mL. Each experiment was repeated three times.

3D in vitro angiogenesis assay . HUVEC and PE-loaded microparticles were embedded in non-planar 3D in vitro Angiogenesis assay was prepared. 8 mL of cold Vitrogen collagen, 1 mL of sterile PBS and 1.166 mL of 0.1 M NaOH solution were mixed to prepare a collagen hydrogel matrix. The color transition of the solution from red to violet showed a pH change. The pH of the solution was checked with pH paper, and a few drops of 0.1 M NaOH or 0.1 M HCl solution was added dropwise to adjust the pH to 7.4. As previously described ¹⁷ , before gelling, 3 mL of HUVEC in solution was added at a concentration of 40,000 cells / mL and 2 mg of loaded microparticles were added. The collagen / cell / microparticle solution was then pipetted to mix until homogenized and 500 [mu] l was pipetted into each well of a 48 well plate. To induce collagen gelation, the culture plates were incubated in a 37 &lt; 0 &gt; C oven for 30 min. The final thickness of the collagen hydrogel was 6.6 mm. Finally, 100 ㎕ / ㎠ vascular a new medium of (the cells being seeded and gelation, the fine particles containing the embedded hydrogel) was added to each well, wetting the plates at 37 ℃ Chemistry 5% CO ₂ incubator .

Analysis method

Fluorescent staining with Calcine AM . Calcine AM staining was performed using Live-Dead Assay (Invitrogen-Life Technologies, New York, USA). Briefly, Calcine AM was directly pipetted into the medium present in the culture well at a final concentration of 2 [mu] g / ml. The stained cells were incubated at 37 [deg.] C for 30 min, and then observed using an inverted fluorescence microscope (Zeiss ASSOCOAT 200 inverted fluorescence microscope).

Qualitative and quantitative network formation analysis. In previous studies, cell response analyzes were performed on experimental conditions in an in vitro model of angiogenesis with several semi-quantitative and quantitative methods (each of which is incorporated herein by reference) ^34-37 . Both the morphological parameters (mean tubulin length) and the local anatomical parameters (bifurcation number and mesh count) were considered because they enable the characterization of the spatial organization of the EC in the capillary-like network.

After cell staining, the selected field of view was photographed for each sample. The acquired images were analyzed. Images were analyzed using images J 1.45s (Wayne, Rasband - National Institutes of Health, USA).

The branch point (BP), a node where the branch met or from which germination of the tubule occurred, was identified and counted. In addition, the mesh of the cell network was identified as a blood vessel area surrounded by hexagonally arranged blood vessels ³⁸ , which was manually counted. Finally, the tubule length was evaluated by drawing a line along each tubule (white dotted line in the zoom image of FIG. 8), and the measured length of the line was automatically calculated by software.

Scanning Electron Microscopy ( SEM ). The fine particle surface structure was investigated by differential scanning microscopy (S-4000 FE-SEM, Hitachi High Technologies, Texas, USA). The lyophilized sample was mounted on an aluminum stub using double-sided graphite tape and coated with gold and palladium using a spreader (desk V, Denton Vacuum). Samples were taken using a Hitachi S-4000 FE-SEM (Texas, USA).

Statistical analysis . The experiment was repeated in triplicate. The graph data and the data in the table were all shown as mean ± standard deviation. Data analysis was performed using SPSS (IBM: Somers, NY, USA). The significance test was calculated using independent, bilateral and Student's t test with this variance. The significance level was set to p &lt; 0.05.

result

The angiogenic potential of PE . The collected data indicate that the degree of angiogenesis response of HUVEC to PE is greatly influenced by the incubation time and the number of PE doses provided.

For incubation time, HUVEC incubated with PE formed an angiogenic neural network as a function of incubation time, and its morphology was different (Figs. 9a-b). On the first day after seeding, HUVEC did not yet form a well-defined network (Fig. 9A, day 1). Mesh was large (102.67 ± 3.52), small, and short tubules were observed (average tubule length: 78.85 ± 3.24 μm). In addition, although the number of bifurcations (BP) was large (105 ± 9.85), it was difficult to confirm in the presence of some cell clusters. On the third day of culture (day 3 in FIG. 9A), a well-formed network emerged. This is also confirmed by the presence of fewer BPs (71 +/- 6.25), longer tubules (average length: 136.43 +/- 15.23 mu m), and wider, fewer number of meshes (61.33 +/- 8.02). On the fifth day of culture (Fig. 9A, day 5), the network was still visible but began to degrade: the length of tubules was longer (170.56 +/- 16.51 mu m), but the number was smaller and thinner. The mesh was difficult to identify and its number decreased (36.33 ± 6.02). For BP, the number decreased slightly (63 ± 4). The network degradation may be due to the cells already depleting the angiogenic molecules contained in the PE. No tubular formation was observed in the control plate (upper right corner in each image), suggesting that changes in cell morphology are not due to stress or other external causes.

A semi-quantitative analysis of the spatial organization of the HUVEC is shown in Figure 9b as a histogram. The bar represents the average tubular length, BP number, and mesh number. It can be noted that statistical differences were found between day 1 and day 5 in all three parameters considered.

In this study, capillary-like network formation was affected by the number of PE inoculations (Fig. 10a-b). Mean tubular length increased from cells inoculated to cells inoculated three times, while BP number and mesh count decreased (Figs. 10a and 10b). There was no well-defined network formation after one round of PE: mesh was high (76.67 ± 19.74), but not diffuse as also confirmed by mean tubular length (129.71 ± 12.88 μm) (Figure 10a, panel). In addition, several BPs (110.78 +/- 15.40) and cell clusters were present, thus suggesting that the cells were not well connected. The cells inoculated twice, but some clusters still existed (Figure 10a, second panel). This could be suggesting that the network was not fully mature. However, while the average tubular length was increased (139.91 ± 8.93 μm), the number of meshes and number of bifurcations decreased (52.67 ± 19.74 and 84.33 ± 11.86, respectively), thus indicating that the network is likely to become more prominent . The triplicate cells were wider, but had fewer meshes (43.89 +/- 6.52), and a reduced number of BPs (80.67 +/- 11.92) (Figure 10a, third panel). The length of the tubing increased (153.89 ± 8.54 ㎛), suggesting that the network improved structure by connecting the tubing together. In this case as well, no tubular formation was observed in the control plates (upper right corner in each panel).

The bar graph in Figure 10B shows a semi-quantitative analysis of the spatial structure of the HUVEC. The average tubule length, number of BPs, and number of meshes are divided into three groups (1, 2, or 3 groups) according to the number of inoculations with PE. The data indicate that the mean tubulin length was increased from the first inoculated cells (129.71 ± 12.88 μm) to the third inoculated cells (153.89 ± 8.54 μm), suggesting that the cells were organized into a more mature network. These observations are also supported by observations that the number of meshes (from 76.67 ± 19.74 to 43.89 ± 6.52) and the number of BPs (from 110.78 ± 15.40 to 80.67 ± 11.92), both decreased. A statistical analysis performed revealed differences in all three parameters between the first and third inoculated cells.

With respect to the bioactivity maintenance of the extract, it maintains its angiogenic potential until 15 days of storage in a 5% CO ₂ incubator humidified at 37 ° C (FIG. 11). No significant differences were observed between days 1 and 7 of storage. Considering all of these characteristics of PE, it was considered to be constant over time and suitable for sustained release.

Characterization of gelatin microparticles . Analysis of the size distribution of the gelatin microparticles of the various preparations revealed that the size range was 20.886 ± 3.53 to 124.083 ± 13.01 μm. Its nearly 80% diameter ranged from 20-80 μm (FIG. 12B). When the fine particles were examined by SEM, there was a difference in surface morphology between the blank fine particles and the loaded fine particles as shown in FIG. 12A. Blank fine particles (upper line) showed smooth surface and regular shape. After loading (bottom row), the particle size was larger and had an irregular surface. This suggests the two terms of (i) adhering to the surface of the PE (adsorption), and (ii) infiltration (absorption) of the particles inside the PE ^27.

Fig. 13 shows the decomposition profile of the non-crosslinked fine particles. This experiment was performed using only blank microparticles to evaluate the degradative kinetic properties of gelatin in PBS. An initial burst was observed: after 6 hours, the cumulative release rate (%) was 6.45% ± 0.12 (Figure 13, illustration). Subsequently, slowed-down release was achieved after the burst. After 20 days, the cumulative cumulative release rate was 18.65% ± 0.09 (FIG. 13, main graph).

14A shows in vitro dissolution (black PE, no PE) and in vitro release (PE PE) of the microparticles loaded with PE. In both cases, a small initial burst was observed. After 48 hours, the cumulative release (%) from the loaded microparticles was 1.03% ± 0.08, while for blank microparticles it was 0.76% ± 0.05, in both cases thereafter the release occurred almost constant . After 22 days, the cumulative release (%) from loaded microparticles and blank microparticles was approximately the same, 4.65% ± 0.07 and 4.65% ± 0.11, respectively, as also highlighted in the graph. On day 23, the emissions from the blank microparticles were slightly larger (5.18 +/- 0.05 and 4.92 +/- 0.09, respectively) than those from the loaded particles. Based on the evidence that most of the PE was released, this experiment was terminated.

Fig. 14B shows the difference in the in vitro release rate between the loaded fine particles and the blank fine particles. Two peaks, the first peak from the first hour to the fifth day, and the second peak from the fifth day to the 22nd day, can be observed. The first peak indicates that the PE bound to the particle surface was released after the first to a short time. Possibly the second peak, which may be due to bulk corrosion of the particles, lasts longer and the overall cumulative release rate is greater. This indicates that PE is gradually released from the pores or channels in the microparticles formed by the release medium.

Despite the presence of an initial "burst &quot;, the release kinetic properties exhibited by the fine particles obtained via filtration are close to the zero order profile, meaning that the release occurs almost constantly. It is believed by the present inventors that this can happen because particles of different sizes coexist in the same sample. The release rate is influenced by the particle size, which is precisely as a result of the increase of the surface area to volume ratio, the smaller the microparticles are released more largely. This phenomenon has been already used to control the drug release or proteins in order to achieve close control type in almost zero-order release profile ^{of 25.}

Blank microparticles and loaded microparticles, total accumulated emission amount emitted from the both is usually 20% greater than that of slightly lower than the values reported in the literature ^39,40. This can be attributed to the high degree of crosslinking.

3D in vitro angiogenesis assay . After determining the optimal cell density and PE volume, angiogenesis assays were prepared as described in the methods above. After 3 days of incubation, there was no sign of tube formation, HUVEC was seeded, and no difference was observed between the microparticles loaded with PE and the control (results not shown). After 5 days of culture, cell shape changes and germination were observed, although no capillary-like network was formed yet (Fig. 15). Germination is that after 5 days thus the bar and the early stages of angiogenesis, culture, germination is present to demonstrate the angiogenic response ⁴¹ from HUVEC. Since no network was formed after 3 days of seeding, the release of PE by the particles may not be sufficient to promote angiogenesis during the observed period. However, germination suggests that PE was released and an angiogenic response was initiated. The release rate of the fine particles can be optimized to promote tubule formation in a shorter period of time.

Discussion & Conclusion

This example demonstrates that the angiogenic response of HUVEC is affected not only by the incubation time, but also by the number of PE doses received. In particular, in the early experiments, the capillary network began to form on day 1 after seeding, became clear on day 3, and began to degrade after 5 days. HUVEC also appeared to form a more mature capillary network when it received more than one PE. In addition, in this embodiment, the network was not degraded after 5 days as in the experiment discussed above. Finally, the extract retains its angiogenic potential until 15 days of storage. When considering all of the above characteristics of PE, it is constant over time and may be considered suitable for sustained release. Accordingly, there is a need for a method for multiple protein mixtures that uses biodegradable gelatin microparticles to further preserve bioactivity and to regulate and prolong the release of hPE, with the goal of improving the ability of the regulatable PE to induce angiogenesis Drug delivery methods have been developed.

For this purpose, biodegradable gelatin microparticles were prepared as delivery systems for PE. Absorption and adsorption of hPE to gelatin particles was evaluated by SEM examination and in vitro release kinetic property analysis. SEM analysis showed that there was a size increase and morphological change between the loaded particles and the blank particles, which is the result of adhesion (adsorption) of hPE to the surface and / or penetration (absorption) into the particles. The release kinetic properties showed two peaks, the first being considered to be the result of protein release bound to the microparticle surface, which was smaller than the second and occurred over a shorter period of time. The second peak was considered to be the result of bulk erosion because of the higher overall cumulative release over a longer period of time. Despite the initial "burst", the release kinetic properties were close to zero order profiles after day 1. This analysis showed that the particles were suitable for use as a vehicle for sustained release of the extract from the 3D in vivo angiogenesis assay.

This study also showed that hPE-loaded gelatin microparticles, when introduced into the 3D I collagen matrix, induced the cells to a rectangular shape five days after incubation and induced early stages of tubule formation with some cell germination (Fig. 15, right). Since germination is an early stage of angiogenesis, germination suggests an angiogenic response from HUVEC. The total cumulative release rate released from both the blank microparticles and the loaded microparticles, observed after 23 days, was nearly 5%, which is the value at which a single protein was released from the gelatin microparticles, Which is significantly lower than the value of 30%. Despite the low cumulative release rate, the hPE concentration released by the particles was still high enough to cause an angiogenic response in the collagen hydrogel. Thus, hPE loaded gelatin microparticles embedded in collagen hydrogels can be used as an in vivo angiogenesis assay, including use ranging from anti-angiogenic drug screen to mechanistic studies on the formation of tubular and angiogenic networks . &Lt; / RTI &gt;

Overall, this example demonstrates that biodegradable gelatin microparticles can be used as a vehicle for sustained release of multiprotein hPE in 3D angiogenesis assays. Cell germination was observed after 5 days of incubation when hPE loaded gelatin microparticles were used, but no interconnected capillary network was formed. Increased hPM emissions or prolonged incubation times emitted by the particles can promote the formation of more interconnected capillary networks.

Example References to 2

Example  3

To control angiogenesis PLGA  Emitting placenta extract from microparticles

Introduction

As described in Example 1 above, a single administration of the human placenta extract of this disclosure has been found to induce and regulate early stages of in vitro and in vivo angiogenesis. It has been shown that multiple administrations of hPM over time promote the development of additional capillary blood vessels and thus the hypothesis that regulated delivery, as described in Example 2 above, further stabilizes network formation over an extended period of time . In this example, hPE was encapsulated in poly (lactic-co-glycolic acid) (PLGA) microparticles to extend the release period. The fine particle formulation was optimized for hPE loading, characterizing morphological characteristics (size, encapsulation efficiency, porosity), and profiling of protein release.

In order to overcome the identified problems in inducing angiogenesis in the case of grafted tissue or tissue structures, the method of the present disclosure uses a human placenta to induce capillary network formation, as described in the above example. Human placenta extracts, referred to herein as human placenta matrix (hPM), which contain angiogenic and immunomodulatory proteins, can be obtained. Endothelial cells (HUVEC) seeded on hPM in vitro have been shown to form an angiogenic network with upregulation of angiogenesis genes in the above, and the matrix in vivo has been shown to inhibit tissue fibrosis, Induced angiogenesis. In addition, endothelial cells subjected to hPM inoculation at multiple time points (day 1, day 3 and day 5 of culture), when compared to cells that received only one inoculation whose network begins to degrade after 5 days, More stable, longer lasting angiogenic networks. Thus, an approach to delivering the matrix in a controlled manner over time has been studied so that a longer lasting, more stable capillary network can be formed.

The complex, heterogeneous nature of h PM can complicate the mechanism of regulated release. For example, due to counteraction interactions and interactions with materials used in controlled release, different charge and chain properties of the protein may affect the loading rate. Both natural and synthetic microparticulate materials, both of which have potential to encapsulate proteins, have been investigated. Natural materials such as collagen, chitosan, and alginate provide biocompatible and non-aggressive encapsulation techniques, and their rate of degradation is about 7 to 10 days, which allows for some sustained release, The use of sustained release of proteins may be limited ^{11 , 12, 13} . Chemical or photochemical cross-linking can slow the degradation of the fine particles ^{9 , 14} .

PLA- copolymers have been used to encapsulate proteins in vivo medical applications, which is a biocompatible synthetic materials that are approved by the FDA in the ^{15, 16 and 17.} The polymer may be released protein in the long run continuous adjustment method may be used to create a complex and multi-layered fine particles ^{15 and 18.} Among these materials, or a PLGA poly (lactic-co-glycolic acid) it is used for the controlled release of the type-specific growth factors ^{(e.g., BMP, VEGF, bFGF) 19} , 20, 21.

Through a number of studies were evaluated PLGA encapsulation of a single protein, several studies have not examined came against the co-encapsulated ^{22 and 23} bar in a review for the encapsulation of complex heterogeneous mixture of proteins in any study of multiple proteins. This example illustrates the composition and encapsulation technique for hPM using PLGA microparticles and evaluates the effect of controlled release of the mixture in a 3D culture of endothelial cells.

Considering that hPM is a heterogeneous composition, this embodiment described in this example optimizes the PLGA synthesis protocol to adapt the multiprotein hPM release for application in cell cultures. Considerations included fine particle size optimization (suitable for regenerative medicine applications), high loading and encapsulation efficiency, low initial burst, and controlled release profile. In addition, it was confirmed that hPM was released from the microparticles at an appropriate concentration to induce angiogenesis using endothelial cells. After microparticle synthesis and loading, an assay was performed to evaluate whether the encapsulation process was selective for the hPM protein. The effect of regulated release of hPM on angiogenesis induction was evaluated using a 3D culture system containing hPM loaded microparticles formatted with alginate based hydrogel seeded with HUVEC. At 28 days, cell behavior was assessed to assess the response between cells receiving a single direct inoculation of hPM on day 1 as compared to cells that had received regulated hPM from PLGA microparticles throughout the incubation period.

Way

Multi bolus Effect of hPM inoculation . An effect analysis of the hPM dosage profile during angiogenesis network formation was confirmed using HUVEC in vitro by pipetting and evenly coating the wells of a 96 well tissue culture plate with a rotary shaker at 30 rpm for 1 minute. The plate was then incubated at 37 DEG C for 30 minutes to warm the hPM. HUVEC was suspended in angiogenesis medium, pipetted on top of hPM, and placed in a humidified 6% CO ₂ incubator at 37 ° C. For the control group, HUVECs were cultured in angiogenesis medium at 20,000 cells / cm2. Three different profiles of hPM inoculation: 1) inoculated hPM only on day 1 (seeding day), 2) inoculated on days 1 and 3 with hPM, and 3) 1) hPM on days 1, 3, and 5 . The cells were cultured for 7 days, and the medium was changed for 3 and 5 days. As a control, cells were seeded directly to the bottom of the plate at the same density, and the medium was changed every 2 days. Cells were stained for 7 days, imaged by fluorescence microscopy, and angiogenesis quantified by imaging as described in the section "Angiogenesis Quantification &quot;.

Manufacture of PLGA microparticles . PLGA (poly (DL-lactide-co-glycolide)) microparticles were prepared using a water-in-oil heavy water emulsion (Protocol 1) (Durect (Cupertino, CA)). An aqueous solution (W1) was prepared using the hPM protein mixture as described previously in Example 1 and the other aqueous solution (W2) was added to 100 mL of DI water with 2 g of polyvinyl alcohol (wt 30,000-70,000 , 87-90% hydrolysis, Sigma-Aldrich: St. Louis, Mo.). The oil solution (O) was obtained by dissolving 90 mg of PLGA in 3 mL of chloroform until the solution appeared transparent. W1 was added to O and homogenized at 20,000 rpm for 1 minute. While stirring at 300 rpm, the primary emulsion obtained by using a micropipette was added dropwise to W2. When all the primary emulsions were added, the resulting secondary emulsion was loosely covered with aluminum foil and stirred overnight (300 rpm) in a fume hood to evaporate the solvent. The secondary emulsion was then centrifuged at 1,000 rpm for 10 minutes, the supernatant was removed, and the microparticles were washed twice more with DI water. The cured microparticles were suspended in DI water, freeze-dried for 48 hours and stored at 4 &lt; 0 &gt; C until needed. In the control group, W1 was replaced with bovine serum albumin (bSA) instead of hPM (Sigma-Aldrich: St. Louis, Mo.) to produce PLGA microparticles loaded with a single protein.

Due to the heterogeneous nature of the hPM mixture, encapsulation represents a challenging element of controlled release from PLGA microparticles. Therefore, we analyzed the effect of protocol modification on size and emission. First, the effect of prolonged homogenization on the first stage was evaluated (protocol 2: 2 minutes instead of 1 minute). Then, the effect of additional homogenization steps introduced after the formation of the secondary water-in-oil emulsion and before the removal of the solvent was investigated (protocol 3: additional homogenization in 1 minute; protocol 4: additional homogenisation in 20 seconds). After each modification, the microparticle morphological characteristics and associated release rates were evaluated and compared before proceeding to the optimization process.

Morphological characterization of PLGA microparticles . Microscopic examination was used to evaluate the morphological characteristics of the PLGA microparticles. The mean size of the microparticles was evaluated using an inverted optical Leica microscope (Leica DM IL LED, Leica Microsystems Inc., IL, USA) with a color digital camera. The images were analyzed using the free software image J 1.45s (Wayne, Rabbend - US National Institutes of Health (USA) - http://imagej.nih.gov/ij/). After manually tracking the particles, the diameter of each particle was measured by automatic measurement using Leica LAS software (Wetzlar, Germany).

The surface morphology and porosity of microparticles were characterized using differential scanning microscopy (SEM) (S-4000 FE-SEM, Hitachi High Technologies, Texas, USA). For shape and surface analysis, the lyophilized sample was mounted on an aluminum stub using double-sided graphite tape and coated with gold and palladium using a spreader (Desk V, Denton Batch). The coated samples were then exposed at an accelerating voltage of 2 kV, photographed using a low magnification to evaluate the average size and shape, and photographed using a high magnification to evaluate porosity and morphological feature changes.

Load factor . After the microparticle dissolution, the encapsulated protein was directly measured to determine the hPM loading rate in the PLGA microparticles. Ravi., Et al., Development and characterization of polymeric microspheres for controlled release protein loaded drug delivery system . 70, (2008)] and Igartua, M. et al., Stability of BSA encapsulated into PLGA microspheres using PAGE and capillary electrophoresis. International Journal of Pharmaceutics 169, 45-54 (1998)), both of which are incorporated herein by reference in their entirety for hydrolysis techniques. Briefly, 15 mg of lyophilized microspheres were digested with 5 mL of 5% w / v SDS containing 0.1 M NaOH and hydrolyzed on a shaker for 15 h at room temperature until a clear solution was obtained. The resulting clear solution was then neutralized to pH 7 by the addition of 1 M HCl and centrifuged at 5,000 rpm for 10 min. The protein concentration in the supernatant was then analyzed in triplicate at 562 nm using a UV-Visible Spectrophotometer along with the Pierce BCA standard protein assay. The encapsulation efficiency is expressed as the ratio of the actual protein content to the theoretical protein content.

In vitro hPM emission characterization. To characterize the release of hPM from PLGA microparticles, 10 mg of the loaded microparticles were suspended in 1 mL of phosphate buffered saline (PBS). The tubes were incubated in a shaking cuvette garter and three samples were evaluated for each protocol. The tubes were centrifuged at 5,000 rpm for 5 minutes every two days, the supernatant was collected, and an equal volume of fresh PBS was added to the tubes. The protein concentration was quantified by measuring the absorbance at 562 nm using a spectrophotometer. By comparing the results from the standard Pierce BSA assay with the results from the microprotein BSA assay, we were able to evaluate the release of the released protein at all time points in a more reliable way. Measurements were collected in triplicate. Protein concentrations were compared to freshly prepared standards ranging from 200 to 0.5 [mu] g / mL. The average absorbance of the standard PBS was subtracted from all measurements. The amount of protein in each sample was added to the amount obtained at each previous time point to create a cumulative release curve.

Since hPM is a complex protein mixture comprising growth factors, extracellular matrix proteins, and biomolecules characterized by various charges and properties, this study analyzed protein release from microparticles loaded with hPM, Were measured. SDS-PAGE analysis was performed on the supernatant collected after overnight stirring. hPM and BSA in PVA (diluted in 10 mg BSA and 5 mL DI water in 50 mL DI water, at the same concentration as the concentration used for loading the fine particles, as well as the microparticles loaded with hPM and BSA, 50 [mu] l hPM). Protein standards were used to evaluate the molecular weight of the detected protein. SDS-page was performed using a BIO-RAD electrophoresis system (Hercules, CA, USA). After electrophoresis, the polyacrylamide gel was stained with Coomassie blue for 1 hour while continuously shaking. The gel was then decolorized in a 4: 1: 5 methanol: acetic acid: DI water solution to enhance the protein band. The final gel was washed in DI water and imaged.

Alginate Hydrogel- based 3D culture . Angiogenic assays were generated using the loaded microparticles to evaluate the application for controlled release of hPM. Human hepatic vein endothelial cells ( HUVECs) were seeded and cultured in 3D alginate gel (1.5% alginate) embedded with hPM loaded microparticles. The sample group included cells suspended in alginate containing pure hPM, cells suspended in alginate containing microparticles loaded with embedded hPM, cells suspended in alginate without microparticles, and blank microparticles Lt; RTI ID = 0.0 &gt; alginate &lt; / RTI &gt; For all sample groups, HUVEC was suspended in the medium and gently suspended in an alginate matrix by pipetting a 0.054 M calcium chloride solution prior to polymerization. The alginate cell suspension was kept stationary for 15 minutes to allow polymerization to occur, then the gel was washed with PBS, the culture medium was added, and the plates were incubated in a humidified 6% CO2 incubator at 37 ° C.

To obtain similar results between the conditions when hPM was added directly to the matrix and the conditions where the hPM was released from the microparticles, the concentration of hPM mixed in each gel was such that the protein released from the embedded microparticles after 21 days Concentration (78 [mu] g protein / cm &lt; 2 &gt;). According to the morphological characterization of the cell network in the stained gel using Carlsein AM (Life Technologies, Grand Island, NY, USA), the effect of the sustained controlled release of hPM from the microparticles is 7, 14 , 21 and 28 days post hPM bolus inoculation.

Quantification of angiogenesis . An image J 1.45s (NIH, Bethesda, MD, USA) was used to quantify the angiogenic outcome. Images of 5X magnification photographed using an inverted fluorescence microscope were processed and the number of meshes, number of bifurcations and length of tubule-like structure formed by HUVEC during the time period described in the following parameters: Example 1 were evaluated and quantified.

Statistical analysis . Experiments on both microparticles and 3D cultures were repeated in triplicate. Both graph data and tabulated data are presented as mean ± standard error. Analysis was performed using Excel (Microsoft Office) and Minitab 15 (Minitab: State College, Pennsylvania, USA). When evaluating the condition above 2, the ANOVA test was used to calculate the significance and the post test was used to assess the specific difference. If only two conditions were similar, independent, bilateral, and Student's t test was used with this variance. The significance level was set to * p <0.05.

result

Multi bolus Effect of hPM inoculation . The formation of HUVEC into the angiogenic network was influenced by the number of times hPM was administered over time (Figs. 16a-16d). As shown in Figure 16e, the tubule length increased as a function of the number of inoculations. Although there were no significant differences in the number of meshes and number of bifurcations (Fig. 16f-16g), it was observed that mature meshes as a function of number of inoculations tended to increase and become more numerous. Mature angiogenesis is characterized by long tubuli and bifurcation and reduced mesh counts, which confirms that continuous administration of hPL helps endothelial cells to organize into a stable network.

Initial PLGA microparticle preparation : Protocol 1. The average size of the PLGA microparticles generated from Protocol 1 was 447 +/- 32 micrometers and the normal distribution range of sizes was 100 to 1,000 micrometers. Three fine particle batches were fabricated under the same conditions and a repeatable distribution could be demonstrated. The loading rate of hPM in the PLGA microparticles was 64 +/- 4% (data not shown).

In vitro release analysis showed that initial bursts were low, corresponding to 10% of the total encapsulated protein (21.38 ± 0.83 μg / mL) and after 21 days 51.23 ± 0.19% of the initial encapsulated proteins (134.56 ± 0.50 g / mL) was released from PLGA microparticles (data not shown). The release rate profile was linear over the estimated 21 day period and appeared to be constant, but at the end of the evaluation period, hPM release from the fine particles (as described above) resulted in bolus injection of hPM to induce an angiogenic response The concentration was significantly lower when used (500 / / mL).

The mean size of the PLGA microparticles loaded with the control BSA was 239.60 ± 9.45 μm and the normal distribution of the sizes was 50 to 400 μm, where 70% of the microparticles were in the range of 150 to 350 μm. The loading rate of PLGA microparticles loaded with BSA was 76 ± 3% of the total amount of the initially loaded protein. In vitro release analysis showed that the initial burst was higher when compared to the loaded microparticles with hPM of 20% (51.52 ± 1.76 ug / mL) of the total encapsulated BSA, whereas after 21 days the initial encapsulated 66.7 ± 9.55% (166.74 ± 23.8 μg / mL) of the protein was released from the PLGA microparticles (data not shown). The release rate profile was linear over a period of 21 days evaluated and appeared to be constant. The observed differences between the hPM loaded PLGA microparticles and the BSA loaded PLGA microparticles suggest that the release kinetic properties can be improved through optimization of the fabrication and loading protocols.

Optimization of PLGA fine particle size and characterization of morphology . Size analysis from the microparticles produced using protocol 2 homogenizing the primary emulsion for 2 minutes (further 1 minute more than protocol 1) resulted in an average diameter of 276.78 +/- 13.41 mu m (Figs. 17A, 17D) Respectively. The average size of the microparticles produced using protocol 3, with a one minute homogenization step added to the second emulsion manufacturing step, was 37.79 ± 1.30 μm (FIGS. 17b and 17e), while the second emulsion was subjected to a 20 second homogenization step The average size of the fine particles generated using protocol 4 added was 91.85 +/- 2.92 mu m (Figs. 17C, 17F).

The results from SEM analysis showed no significant difference in surface porosity between the different protocols. The surfaces of the microparticles obtained using the different variants described all showed rounded formation with homogeneous surfaces (Figures 18a-18f). Figures 18a-18c show the fine particle shape and Figures 18d-f show the microparticle surface porosity of the particles prepared by protocols 2, 3 and 4, respectively.

Load factor . The encapsulation efficiency did not appear to have a linear relationship with the size of the fine particles (Fig. 19A). The encapsulation efficiency increased to 73 ± 4% for protocol 2 and to 63 ± 3% for protocol 3 when the size was significantly reduced. For Protocol 4, the highest encapsulation efficiency was achieved, corresponding to 79 ± 9% of the encapsulated protein during the initial total protein loading.

Optimized hPM- loaded hPM from fine particles In vitro release . The rate of release of hPM from the fine particles produced using the optimized protocol was a direct result of the difference in size and loading ratio and the release profile trends obtained with the modification of the homogenization step appeared to be similar. The initial bursts did not change significantly with duration variation of the primary emulsion homogenization step, but were significantly lowered from 8.14 ± 0.31% (21.38 ± 0.83 μg protein / mL) in protocol 1 (data not shown) to 6.42 &Lt; / RTI &gt; ± 2.07% (18.88 ± 6.11 μg protein / mL). When the homogenization of the secondary emulsion was introduced, a significant increase in the burst release was observed: in protocol 3, compared to 27.72 ± 3.73% (106.33 ± 14.33 μg / mL) for protocol 4, 23.75 + 0.40% of the total protein (60.82 +/- 1.03 [mu] g / mL) was released within the first day (Figure 19b).

Cumulative release after 21 days correlated with microparticle size, the smaller the microparticle, the greater the release of hPM released during the assay period. After 21 days of release, the microparticles from Protocol 2 released 64.09 ± 1.70% (188.43 ± 5 μg / mL) of the total encapsulated protein, whereas MP from Protocol 3 released 98.62 ± 1.40% (252.48 ± 3.6 μg / mL ), And MP from protocol 4 released 87.60 +/- 1.12% (335.98 +/- 4.72 mu g / mL) (Figure 19B). Further analysis comparing the P3 and P4 release profiles over an extended period of time showed that after 30 days the microparticles from protocol 3 were completely degraded and the release of hPM was no longer achieved, Up to 93.12 ± 0.2% (357 ± 4.09 μg / mL) of the total encapsulated protein.

The results from the SDS page are limited due to the low concentration of protein contained in the supernatant collected and analyzed. However, the analysis showed that the encapsulation of hPM in PLGA microparticles occurred uniformly, and there was no evidence of selective encapsulation of specific proteins. Both the BSA loaded microparticles and the hPM loaded microparticles, both of which were collected after microparticle curing, showed that the protein content was similar when the concentration was lower (Figure 20).

Alginate Hydrogel- based 3D culture . Results from angiogenesis assays showed that regulated release of hPM could improve the stability of capillary-like structures in the matrix in which endothelial cells were seeded. Figure 21 shows that after 7 days of incubation, only a few germination and coronary structures were observed in HUVECs cultured in microparticle-embedded alginate hydrogel loaded with hPM (no mature network was observed), whereas pure hPM Angiogenesis in the matrix was comparatively more mature during the same period wherein the average tubulin length was 207.90 ± 15.31 μm, 1.27 ± 0.84 mesh / mm ² , and 9.60 ± 0.70 bifurcation / mm ² . However, after 14 days of incubation, the MP-loaded alginate gel loaded with hPM started to organize primary tubules with an average length of 137.41 ± 9.77 μm and 6.66 ± 0.47 bifurcation / mm 2, whereas the matrix containing pure hPM Showed isolated angiogenic networks and no meshes were observed and only isolated tubules with an average length of 226.58 ± 25.64 ㎛ were present. On day 21, the MP-embedded gel showed immature meshes, average tubule length was 204.07 ± 12.75 μm, and the average number of bifurcations was 11 ± 1.25 bifurcations / ㎟. Overall, after 21 days of incubation, the established angiogenesis network remained stable, with only minor changes in the number of bifurcations / mm2 (Fig. 21). After 28 days of cultivation, the average tubule length was 168.88 ± 10.31 μm, the average number of branching points was 12.93 ± 1.61, and the average number of meshes was 3.17 ± 0.93 cells / mm 2.

Argument

An encapsulated protein using PLGA microparticles is a drug delivery system for the controlled release of growth factors and a commonly used manufacturing technique is the water-in-oil heavy water method. ^{19, 20, 21 In} this study, a complex human-derived multiparticulate mixture (placental matrix or hPM) containing more than 2,600 angiogenic and anti-fibrotic proteins was encapsulated in PLGA microparticles. According to the results, when using hPM-loaded PLGA microparticles, the release of multiprotein fusions containing angiogenic proteins and fibrotic proteins can be sustained and controlled over time. When used with endothelial cells during in vitro culture, hPM loaded PLGA microparticles enable a stable angiogenic network to be formed.

The results of this study showed that hPM affects multiprotein composition in the form of microparticles produced from PLGA loading and complexes. When compared to single protein (BSA) encapsulation, the mean size of hPM loaded microparticles was significantly greater than that loaded with BSA. This may possibly be the result of intricate interactions of the protein with the PLGA polymer phase in hPM and thus can have a wide array of different molecular charge, pH, and size when compared to the microparticles loaded with BSA . SDS-PAGE analysis showed that although the concentration of protein released from the PLGA microparticles loaded with hPM is low, a broad distribution can be observed in a supernatant similar to the composition-diluted hPM. These results suggest that the encapsulation of the protein mixture is not selective for a particular protein, is homogeneous, and the protein composition of hPM is not affected during microparticle preparation.

Both the duration and rate of the homogenization step were found to affect the fine particle size in advance. ^{17 27 In} this example it has been found that there is a correlation between the hPM loaded PLGA microparticle size and the in vitro release rate. Although optimizing the homogenization of the primary emulsion step produced fine particles with the desired controlled release characteristics, the loading rate of hPM in these particles was somewhat lower than other manufacturing protocols being evaluated. By optimizing the homogenization of the primary emulsion step, the encapsulation of the protein solution was increased (up to 78%) and microparticles with a size range of the desired size range were obtained. Interestingly, although the encapsulation efficiency was not correlated with the size of the fine particles, it was influenced by the homogenization period of the dash secondary emulsion, possibly due to the fragmented incomplete particles caused by the interaction between the heterogeneous protein mixture and the polymer Lt; / RTI &gt;

Using the optimized PLGA microparticle synthesis protocol described above (allowing for higher hPM encapsulation efficiency, optimized fine particle size, and linear release over 30 days when compared to the non-optimized protocol), the hPM loaded Microparticles were introduced into alginate-based angiogenesis assay to assess endothelial cell (HUVECS) response to regulated release of hPM over time. Due to their cost-effective processability and overall superior properties as extracellular material, 3D angiogenesis assays were generated by embedding hPM loaded microparticles in selected alginate gels as the basis for assays. ^30, but is lower than ³¹ after the culture 21, although concentration (263 ㎍ / ㎠) that it passes from the previous 2D angiogenesis studies using total (78 ㎍ / ㎠) followed by bolus injection of a protein that is passed from the fine particles, HUVECS formed angiogenic tubules in the gel in regions close to PLGA microparticles loaded with embedded hPM. By using hPM-loaded microparticles, sustained angiogenesis network formation over a period of 28 days could be achieved over a period of 28 days when compared to bolus administration of hPM directly mixed with an alginate matrix. Cells formed an angiogenic network more rapidly in alginate gels directly mixed with hPM, but this network was degraded more rapidly when compared to gels using hPM loaded microparticles. After 28 days, hPM release from the microparticles was almost zero, but neovascularization of the angiogenic meshes was still visible, indicating that controlled release of hPM from the PLGA microparticles improved stability of angiogenic network formation .

Overall, this example describes the optimization of the PLGA microparticle synthesis protocol for applications delivering complex, multiprotein mixtures with low initial burst and high encapsulation efficiency. We then developed a novel angiogenesis assay by applying the method developed here, thereby continuing to form an angiogenic network within the alginate matrix. Significantly, the techniques developed can be applied to any combination of complex serum and protein mixtures, which can be applied in a wide variety of tissue manipulation and medical systems. The technology makes it possible to develop improved in vitro angiogenesis assays and to better understand the role that cells play in sustained serum delivery in a seeded matrix in which serum-loaded PLGA particles are embedded, (E.g., collagen, fibrin, laminin).

conclusion

This example describes an embodiment of the hPM encapsulation method and angiogenesis formation was observed using an alginate based angiogenesis assay. A sustained angiogenic response over a period of 21 days was observed in the 3D hydrogel culture system. This confirms the effectiveness of the controlled release approach of hPM to guide the formation and maintenance of the capillary network.

Example References to 3

Claims (20)

A human placenta extract obtained from a human placenta sample, wherein blood and solids are substantially removed from the extract, the extract comprising placental proteins, including cytokines and growth factors, wherein the placental proteins were present in a placental sample Human placenta extract, and
A composition comprising biodegradable microparticles,
Wherein the human placenta extract is coupled to the microparticles so that the human placenta extract can be released from the microparticles over a period of time after exposure to the cells in vivo or in vitro. The method according to claim 1,
Removing blood from a sample obtained from a human placenta sample to produce a placenta crude extract;
Mixing the placenta crude extract with a protein solubilizing agent to solubilize the protein in the crude extract;
Separating the solid from the solubilized protein-placenta extract mixture; And
And dialyzing the solubilized protein-placenta extract mixture to remove the protein solubilizing agent from the mixture to produce a human placenta extract. 2. The composition of claim 1, wherein the placental extract comprises at least 20 different cytokines. 3. The composition of claim 2, wherein the protein solubilizing agent is urea. The composition of claim 1, wherein the placenta extract is capable of regulating angiogenesis. The composition of claim 1, wherein the biodegradable microparticles comprise poly (DL-lactide- co -glycolide) (PLGA) microparticles. 8. The composition of claim 7, wherein PLGA microparticles are loaded with human placenta extract (hPE), wherein the hPE loaded PLGA microparticles are prepared by an oil-in-water process comprising double homogenization. The composition of claim 1, wherein the biodegradable microparticles comprise gelatin microparticles. The composition of claim 1, wherein the biodegradable microparticles comprise microparticles of different sizes. A method for inducing angiogenesis comprising contacting a cell with a composition for sustained release angiogenesis,
The sustained angiogenesis-regulating composition comprises:
Biodegradable microparticles, and
Wherein the human placenta extract is coupled to the microparticles so that the human placenta extract can be released from the microparticles during a period of time after exposure of the microparticles to the cells in vivo or in vitro, Wherein the blood and solids are substantially removed from the extract, wherein the extract comprises a placental protein, including cytokines and growth factors, wherein the placental protein is present in a blood vessel that is present in the placenta sample, Induction method of new life. 11. The method of claim 10, wherein the cell is an endothelial cell. 11. The method according to claim 10,
Removing blood from a sample obtained from a human placenta sample to produce a placenta crude extract;
Mixing the placenta crude extract with a protein solubilizing agent to solubilize the protein in the crude extract;
Separating the solid from the solubilized protein-placenta extract mixture;
Wherein the protein-placenta extract mixture is dialyzed against the solubilized protein-placenta extract mixture to remove the protein solubilizing agent from the mixture to produce a human placenta extract. 13. The method of inducing angiogenesis according to claim 12, wherein the protein solubilizing agent is urea. 11. The method of claim 10, further comprising coupling a sustained angiogenesis-regulating composition to the biomaterial. 15. The method of claim 14, further comprising the step of inducing vascularization of the biomaterial in vivo by implanting the biomaterial in the subject. 15. The method of claim 14, wherein the biomaterial is an engineered bioscaffold comprising a human-derived substrate material. 15. The method of claim 14, wherein the bioscaffold comprises a cell-free decellularized human umbilical vein scaffold seeded with human endothelial cells. 18. The method of claim 17, wherein the human endothelial cell is a human endothelial cell (HUVEC). 16. The method according to claim 15, wherein the subject is a human. Comprising administering to the subject a composition comprising a human placenta extract obtained from a human placenta sample wherein the blood and solids are substantially removed from the extract and wherein the extract contains placental proteins including cytokines and growth factors Wherein the placental protein is present in a placental sample.

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