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8826 KiB

Open AccessArticle

Poly-ε-caprolactone Coated and Functionalized Porous Titanium and Magnesium Implants for Enhancing Angiogenesis in Critically Sized Bone Defects

by Laura Roland, Michael Grau, Julia Matena, Michael Teske, Matthias Gieseke, Andreas Kampmann, Martin Beyerbach, Hugo Murua Escobar, Heinz Haferkamp, Nils-Claudius Gellrich and Ingo Nolte

Int. J. Mol. Sci. 2016, 17(1), 1; https://doi.org/10.3390/ijms17010001 - 22 Dec 2015

Cited by 11 | Viewed by 7186

Abstract

For healing of critically sized bone defects, biocompatible and angiogenesis supporting implants are favorable. Murine osteoblasts showed equal proliferation behavior on the polymers poly-ε-caprolactone (PCL) and poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate) (P(3HB)/P(4HB)). As vitality was significantly better for PCL, it was chosen as a suitable coating material [...] Read more.

For healing of critically sized bone defects, biocompatible and angiogenesis supporting implants are favorable. Murine osteoblasts showed equal proliferation behavior on the polymers poly-ε-caprolactone (PCL) and poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate) (P(3HB)/P(4HB)). As vitality was significantly better for PCL, it was chosen as a suitable coating material for further experiments. Titanium implants with 600 µm pore size were evaluated and found to be a good implant material for bone, as primary osteoblasts showed a vitality and proliferation onto the implants comparable to well bottom (WB). Pure porous titanium implants and PCL coated porous titanium implants were compared using Live Cell Imaging (LCI) with Green fluorescent protein (GFP)-osteoblasts. Cell count and cell covered area did not differ between the implants after seven days. To improve ingrowth of blood vessels into porous implants, proangiogenic factors like Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) were incorporated into PCL coated, porous titanium and magnesium implants. An angiogenesis assay was performed to establish an in vitro method for evaluating the impact of metallic implants on angiogenesis to reduce and refine animal experiments in future. Incorporated concentrations of proangiogenic factors were probably too low, as they did not lead to any effect. Magnesium implants did not yield evaluable results, as they led to pH increase and subsequent cell death. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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3363 KiB

Open AccessArticle

Effects of PMMA and Cross-Linked Dextran Filler for Soft Tissue Augmentation in Rats

by Jung-Bo Huh, Joo-Hyun Kim, Soyun Kim, So-Hyoun Lee, Kyung Mi Shim, Se Eun Kim, Seong Soo Kang and Chang-Mo Jeong

Int. J. Mol. Sci. 2015, 16(12), 28523-28533; https://doi.org/10.3390/ijms161226112 - 1 Dec 2015

Cited by 7 | Viewed by 9086

Abstract

This study was conducted for evaluation of the ability to maintain efficacy and biocompatibility of cross-linked dextran in hydroxypropyl methylcellulose (DiHM) and cross-linked dextran mixed with PMMA in hydroxypropyl methylcellulose (PDiHM), compared with hyaluronic acid (HA) filler. Saline and HA solution was administered [...] Read more.

This study was conducted for evaluation of the ability to maintain efficacy and biocompatibility of cross-linked dextran in hydroxypropyl methylcellulose (DiHM) and cross-linked dextran mixed with PMMA in hydroxypropyl methylcellulose (PDiHM), compared with hyaluronic acid (HA) filler. Saline and HA solution was administered in the negative and positive control groups, and DiHM and PDiHM were administered in the test groups (n = 10 in each group). The site of cranial subcutaneous injection was the mid-point of the interpupillary line, and the site of intraoral submucosal injection was the ridge crest 2 mm below the cervical line of the mandibular left incisor. Before and immediately after filler injection, intraoral photos and lateral cephalometric radiographs were taken for analysis and comparison of the effect of the filler on the injection sites. The filler injected areas were converted into sequential size changes (%) of the baseline. Histomorphologic examination was performed after 12 weeks. The smallest value in the filler injected area was observed during the experimental period in the normal saline group (p < 0.001), which was almost absorbed at 4 weeks (7.19% ± 12.72%). The HA group exhibited a steady decrease in sequential size and showed a lower value than the DiHM and PDiHM groups (saline < HA < DHiM, PDHiM, p < 0.001). DiHM and PDiHM tended to increase for the first 4 weeks and later decreased until 12 weeks. In this study on DiHM and PDiHM, there was no histological abnormality in cranial skin and oral mucosa. DiHM and PDiHM filler materials with injection system provide an excellent alternative surgical method for use in oral and craniofacial fields. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Intraoral photo measurements of the filler area. Values are expressed as mean ± standard deviation of sequential size changes (%) of saline, HA, DiHM, and PDiHM. hyaluronic acid (HA), cross-linked dextran in hydroxypropyl methylcellulose (DiHM), cross-linked dextran mixed with PMMA in hydroxypropyl methylcellulose (PDiHM). Full article ">Figure 2
Lateral cephalometric radiograph measurements of the filler area. Values are expressed as mean ± standard deviation of sequential size changes (%) of saline, HA, DiHM, and PDiHM. hyaluronic acid (HA), cross-linked dextran in hydroxypropyl methylcellulose (DiHM), cross-linked dextran mixed with PMMA in hydroxypropyl methylcellulose (PDiHM). Full article ">Figure 3
Selected representative specimens were histological features induced by physiological saline (A,B); HA (C,D); DiHM (E,F); and PDiHM (G,H) of H & E stain at 12 weeks after injection. Hyaluronic acid (HA), cross-linked dextran in hydroxypropyl methylcellulose (DiHM), and cross-linked dextran mixed with PMMA in hydroxypropyl methylcellulose (PDiHM). Scale bar = 1 mm. Red boxes are the areas of enlarge image in left upward of each figure. Full article ">Figure 4
Selected representative specimens were histological features induced by physiological saline (A,B); HA (C,D); DiHM (E,F); and PDiHM (G,H) of Masson’s trichrome stain at 12 weeks after injection (E,F); Infiltration of macrophages (yellow arrow) around the dextran microsphere (red arrow) in connective tissue (G,H); Formation of fibro-connective tissue (black arrow) around the PMMA microspheres (blue arrow). Hyaluronic acid (HA), cross-linked dextran in hydroxypropyl methylcellulose (DiHM), and cross-linked dextran mixed with PMMA in hydroxypropyl methylcellulose (PDiHM). Scale bar = 50 μm. Full article ">Figure 5
Injection site of cranial skin and in oral submucosa. (A) Asterisk (*): injection site, L1: interpupillary line, L2: sagittal line; (B) Asterisk (*): injection site, C1: cervical line, C2: 2 mm below the cervical line. Full article ">Figure 6
Method for standardizing the measurements of intraoral submucosal injection. (A) Initial photo taken with a transparent microscopic ruler before filler injection; (B) Photo taken after treatment (circle is the filler injected area); (C) The color of the initial photo A was reversed, and overlapped to photo B (arrow indicates augmentation area induced by filler). Full article ">Figure 7
Method for standardizing measurements of cranial subcutaneous injection. (A) Initial lateral cephalometric radiograph before filler injection; (B) Lateral cephalometric radiograph after treatment (circle is the filler injected area); (C) The color of X-ray A was reversed and overlapped to X-ray B (marked circle indicates the augmentation area induced with filler). Full article ">

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Open AccessArticle

Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study

by Kaile Zhang, Xuran Guo, Weixin Zhao, Guoguang Niu, Xiumei Mo and Qiang Fu

Int. J. Mol. Sci. 2015, 16(11), 27659-27676; https://doi.org/10.3390/ijms161126050 - 19 Nov 2015

Cited by 52 | Viewed by 7362

Abstract

Objective: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001) delivering collagen/poly(l-lactide-co-caprolactone) (P(LLA-CL)) scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM) expression in vitro and in vivo. Methods: ICG-001 (1 mg [...] Read more.

Objective: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001) delivering collagen/poly(l-lactide-co-caprolactone) (P(LLA-CL)) scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM) expression in vitro and in vivo. Methods: ICG-001 (1 mg (2 mM)) was loaded into a (P(LLA-CL)) scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron) and high-performance liquid chromatography (HPLC). Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay) and scanning electron microscopy (SEM) were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-β1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. Results: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL) scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more smooth muscle and thicker epithelium in urethras repaired with ICG-001 delivering scaffolds. Conclusion: After loading with the Wnt signal pathway inhibitor ICG-001, the Collagen/P(LLA-CL) scaffold could facilitate a decrease in the ECM deposition of fibroblasts. The ICG-001 delivering Collagen/P(LLA-CL) nanofibrous scaffold seeded with epithelial cells has the potential to be a promising substitute material for urethroplasty. Longer follow-up study in larger animals is needed in the future. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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9717 KiB

Open AccessArticle

The Effect of Alendronate Loaded Biphasic Calcium Phosphate Scaffolds on Bone Regeneration in a Rat Tibial Defect Model

by Kwang-Won Park, Young-Pil Yun, Sung Eun Kim and Hae-Ryong Song

Int. J. Mol. Sci. 2015, 16(11), 26738-26753; https://doi.org/10.3390/ijms161125982 - 6 Nov 2015

Cited by 35 | Viewed by 8627

Abstract

This study investigated the effect of alendronate (Aln) released from biphasic calcium phosphate (BCP) scaffolds. We evaluated the in vitro osteogenic differentiation of Aln/BCP scaffolds using MG-63 cells and the in vivo bone regenerative capability of Aln/BCP scaffolds using a rat tibial defect [...] Read more.

This study investigated the effect of alendronate (Aln) released from biphasic calcium phosphate (BCP) scaffolds. We evaluated the in vitro osteogenic differentiation of Aln/BCP scaffolds using MG-63 cells and the in vivo bone regenerative capability of Aln/BCP scaffolds using a rat tibial defect model with radiography, micro-computed tomography (CT), and histological examination. In vitro studies included the surface morphology of BCP and Aln-loaded BCP scaffolds visualized using field-emission scanning electron microscope, release kinetics of Aln from BCP scaffolds, alkaline phosphatase (ALP) activity, calcium deposition, and gene expression. The in vitro studies showed that sustained release of Aln from the BCP scaffolds consisted of porous microstructures, and revealed that MG-63 cells cultured on Aln-loaded BCP scaffolds showed significantly increased ALP activity, calcium deposition, and gene expression compared to cells cultured on BCP scaffolds. The in vivo studies using radiograph and histology examination revealed abundant callus formation and bone maturation at the site in the Aln/BCP groups compared to the control group. However, solid bony bridge formation was not observed at plain radiographs until 8 weeks. Micro-CT analysis revealed that bone mineral density and bone formation volume were increased over time in an Aln concentration-dependent manner. These results suggested that Aln/BCP scaffolds have the potential for controlling the release of Aln and enhance bone formation and mineralization. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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2151 KiB

Open AccessArticle

The Effect of Interferon-γ and Zoledronate Treatment on Alpha-Tricalcium Phosphate/Collagen Sponge-Mediated Bone-Tissue Engineering

by Peiqi Li, Yoshiya Hashimoto, Yoshitomo Honda, Yoshiyuki Arima and Naoyuki Matsumoto

Int. J. Mol. Sci. 2015, 16(10), 25678-25690; https://doi.org/10.3390/ijms161025678 - 26 Oct 2015

Cited by 10 | Viewed by 6481

Abstract

Inflammatory responses are frequently associated with the expression of inflammatory cytokines and severe osteoclastogenesis, which significantly affect the efficacy of biomaterials. Recent findings have suggested that interferon (IFN)-γ and zoledronate (Zol) are effective inhibitors of osteoclastogenesis. However, little is known regarding the utility [...] Read more.

Inflammatory responses are frequently associated with the expression of inflammatory cytokines and severe osteoclastogenesis, which significantly affect the efficacy of biomaterials. Recent findings have suggested that interferon (IFN)-γ and zoledronate (Zol) are effective inhibitors of osteoclastogenesis. However, little is known regarding the utility of IFN-γ and Zol in bone tissue engineering. In this study, we generated rat models by generating critically sized defects in calvarias implanted with an alpha-tricalcium phosphate/collagen sponge (α-TCP/CS). At four weeks post-implantation, the rats were divided into IFN-γ, Zol, and control (no treatment) groups. Compared with the control group, the IFN-γ and Zol groups showed remarkable attenuation of severe osteoclastogenesis, leading to a significant enhancement in bone mass. Histomorphometric data and mRNA expression patterns in IFN-γ and Zol-injected rats reflected high bone-turnover with increased bone formation, a reduction in osteoclast numbers, and tumor necrosis factor-α expression. Our results demonstrated that the administration of IFN-γ and Zol enhanced bone regeneration of α-TCP/CS implants by enhancing bone formation, while hampering excess bone resorption. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Full article ">Figure 1
Alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS) material observations and measurements. (a) Visual image of an α-TCP/CS; (b) SEM image of an α-TCP/CS. The white asterisk is overlaid on a collagen fiber, and the white arrows point to an α-TCP particle; (c) FTIR spectra recordings of an α-TCP powder, an α-TCP/CS; and a CS (d) XRD patterns of α-TCP/CS particles, α-TCP particles, and a CS. Full article ">Figure 2
In vivo models generated with critically sized defects in rat calvarias filled with α-TCP/CS material. (a) After four weeks, rats were administered interferon (IFN)-γ, and bone regeneration was compared with that occurring in no-drug-treated control rats and zoledronate (Zol)-injected rats; (b) Micro-computed tomography and bone-mineral density (BMD) images of rat calvarias defects. Scale bars = 10 mm (long bars) or 2 mm (short bars); (c) Post-operative bone volumes/tissue volumes (BV/TV) and BMDs were measured each week. The data shown represent the mean ± standard deviation (SD; n = 4). * p < 0.05, ** p < 0.01 (Tukey–Kramer method). Full article ">Figure 3
Bone-turnover capacities of material implants following drug treatment. (a) Von Kossa staining. Brown staining represents bone tissue, and white arrows show the α-TCP/CS particles in the bone defect. Broken squares represent the magnified areas; (b) Alkaline phosphatase (ALP) staining. Black staining represents ALP-positive tissue; (c) Alp mRNA expression in bone defects. Data show the mean ± SD (n = 4). # Control vs. IFN-γ; * Control vs. Zol; *,# p < 0.05; **,## p < 0.01 (Tukey–Kramer method); (d) Fluorescence labeling analysis. Calcein (blue staining: new bone growth at 4–6 weeks post-implantation) and tetracycline (green: new bone growth at 6–8 weeks) labeling of regenerative bone tissue in calvarial defects. Scale bars: von Kossa = 1.8 mm and 120 μm (magnified areas), ALP staining = 120 μm, fluorescence labeling = 100 μm; (e) Quantification of labeling fluorescence. The data show the mean ± SD (n = 4). * p < 0.05, ** p < 0.01 (Tukey–Kramer method). Full article ">Figure 4
Effect of Zol and IFN-γ administration on osteoclastogenesis and immune responses in defects treated with α-TCP/CS. (a) Tartrate-resistant acid phosphatase (TRAP) staining representing the presence of osteoclasts (TRAP-positive cells) in tissue sections; (b,c) Tumor necrosis factor alpha (TNF-α; purple) and receptor activator of nuclear factor-kappa B ligand (RANKL) expression (brown). Scale bars: 120 μm; (d) Expression of genes closely related to osteoclast differentiation and bone resorption (Rankl, Tnf-α, Il-1β, and M-csf). The data show the mean ± SD (n = 4). # Control vs. IFN-γ, * Control vs. Zol; *,# p < 0.05, **,## p < 0.01 (Tukey–Kramer method). Full article ">

5971 KiB

Open AccessArticle

Evaluating Osteogenic Potential of Ligamentum Flavum Cells Cultivated in Photoresponsive Hydrogel that Incorporates Bone Morphogenetic Protein-2 for Spinal Fusion

by Chih-Wei Chiang, Wei-Chuan Chen, Hsia-Wei Liu, I-Chun Wang and Chih-Hwa Chen

Int. J. Mol. Sci. 2015, 16(10), 23318-23336; https://doi.org/10.3390/ijms161023318 - 28 Sep 2015

Cited by 7 | Viewed by 6616

Abstract

Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated [...] Read more.

Regenerative medicine is increasingly important in clinical practice. Ligamentum flava (LF) are typically removed during spine-related surgeries. LF may be a source of cells for spinal fusion that is conducted using tissue engineering techniques. In this investigation, LF cells of rabbits were isolated and then characterized by flow cytometry, morphological observation, and immunofluorescence staining. The LF cells were also cultivated in polyethylene (glycol) diacrylate (PEGDA) hydrogels that incorporated bone morphogenetic protein-2 (BMP-2) growth factor, to evaluate their proliferation and secretion of ECM and differentiation in vitro. The experimental results thus obtained that the proliferation, ECM secretion, and differentiation of the PEGDA-BMP-2 group exceeded those of the PEGDA group during the period of cultivation. The mineralization and histological staining results differed similarly. A nude mice model was utilized to prove that LF cells on hydrogels could undergo osteogenic differentiation in vivo. These experimental results also revealed that the PEGDA-BMP-2 group had better osteogenic effects than the PEGDA group following a 12 weeks after transplantation. According to all of these experimental results, LF cells are a source of cells for spinal fusion and PEGDA-BMP-2 hydrogel is a candidate biomaterial for spinal fusion by tissue engineering. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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13382 KiB

Open AccessArticle

Selenium-Substituted Hydroxyapatite/Biodegradable Polymer/Pamidronate Combined Scaffold for the Therapy of Bone Tumour

by Ewa Oledzka, Marcin Sobczak, Joanna Kolmas and Grzegorz Nalecz-Jawecki

Int. J. Mol. Sci. 2015, 16(9), 22205-22222; https://doi.org/10.3390/ijms160922205 - 14 Sep 2015

Cited by 15 | Viewed by 5861

Abstract

The present study evaluated a new concept of combined scaffolds as a promising bone replacement material for patients with a bone tumour or bone metastasis. The scaffolds were composed of hydroxyapatite doped with selenium ions and a biodegradable polymer (linear or branched), and [...] Read more.

The present study evaluated a new concept of combined scaffolds as a promising bone replacement material for patients with a bone tumour or bone metastasis. The scaffolds were composed of hydroxyapatite doped with selenium ions and a biodegradable polymer (linear or branched), and contained an active substance—bisphosphonate. For this purpose, a series of biodegradable polyesters were synthesized through a ring-opening polymerization of ε-caprolactone or d,l-lactide in the presence of 2-hydroxyethyl methacrylate (HEMA) or hyperbranched 2,2-bis(hydroxymethyl)propionic acid polyester-16-hydroxyl (bis-MPA) initiators, substances often used in the synthesis of medical materials. The polymers were obtained with a high yield and a number-average molecular weight up to 45,300 (g/mol). The combined scaffolds were then manufactured by a direct compression of pre-synthesized hydroxyapatite doped with selenite or selenate ions, obtained polymer and pamidronate as a model drug. It was found that the kinetic release of the drug from the scaffolds tested in vitro under physiological conditions is strongly dependent on the physicochemical properties and average molecular weight of the polymers. Furthermore, there was good correlation with the hydrolytic biodegradation results of the scaffolds fabricated without drug. The preliminary findings suggest that the fabricated combined scaffolds could be effectively used for the sustained delivery of bioactive molecules at bone defect sites. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Full article ">Figure 1
1H NMR spectra of HEMA-PCL100. Full article ">Figure 2
1H NMR spectra of bis-MPA-PCL200. Full article ">Figure 3
Release profile of pamidronate (PAM) from the manufactured combined scaffolds (pH 7.4 ± 0.05). Full article ">Figure 4
SEM micrographs of HA-HEMA-PCL100-PAM: before immersion in PBS (a); after immersion in PBS for nine days (b) and 30 days (c) and HA-bis-MPA-PCL200-PAM: before immersion in PBS (d); after immersion in PBS for nine days (e) and 30 days (f). Full article ">Figure 5
Effect of hydrolytic degradation time on the weight loss (WL) of the manufactured combined scaffolds. Full article ">Figure 6
SEM micrographs of HA-bis-MPA-PCL200: before hydrolytic degradation (a); after hydrolytic degradation for (b) nine days and (c) 30 days. Full article ">Figure 7
Synthesis of linear and branched polyesters using HEMA and bis-MPA as initiators. Full article ">

1981 KiB

Open AccessArticle

Influence of Pre-Freezing Temperature on the Corneal Endothelial Cytocompatibility and Cell Delivery Performance of Porous Hyaluronic Acid Hydrogel Carriers

by Jui-Yang Lai

Int. J. Mol. Sci. 2015, 16(8), 18796-18811; https://doi.org/10.3390/ijms160818796 - 11 Aug 2015

Cited by 13 | Viewed by 5648

Abstract

The development of porous hyaluronic acid (HA) hydrogels for corneal endothelial tissue engineering is attractive because they can be used as functional cell delivery carriers to help in the reconstruction of damaged areas. The purpose of this study was to investigate the corneal [...] Read more.

The development of porous hyaluronic acid (HA) hydrogels for corneal endothelial tissue engineering is attractive because they can be used as functional cell delivery carriers to help in the reconstruction of damaged areas. The purpose of this study was to investigate the corneal endothelial cytocompatibility and cell delivery performance of porous HA hydrogel biomaterials fabricated at different pre-freezing temperatures. As compared to their counterparts prepared at −80 °C, the HA samples fabricated at higher pre-freezing temperature (i.e., 0 °C) exhibited a larger pore size and higher porosity, thereby leading to lower resistance to glucose permeation. Live/dead assays and gene expression analyses showed that the restricted porous structure of HA carriers decreases the viability and ionic pump function of cultured corneal endothelial cells (CECs). The results also indicated that the porous hydrogel biomaterials fabricated at high pre-freezing temperature seem to be more compatible with rabbit CECs. In an animal model of corneal endothelial dysfunction, the wounded rabbit corneas receiving bioengineered CEC sheets and restricted porous-structured HA carriers demonstrated poor tissue reconstruction. The therapeutic efficacy of cell sheet transplants can be improved by using carrier materials prepared at high pre-freezing temperature. Our findings suggest that the cryogenic operation temperature-mediated pore microstructure of HA carriers plays an important role in corneal endothelial cytocompatibility and cell delivery performance. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Full article ">Figure 1
Scanning electron microscopic images of various hyaluronic acid (HA) carriers. (a) High pre-freezing temperature (HFT) and (b) low pre-freezing temperature (LFT) groups. CS: cross-section; S: surface. Scale bars: 200 μm. Full article ">Figure 2
Pore size of various HA carriers. An asterisk indicates statistically-significant differences (* p < 0.05; n = 4) vs. HFT (compared only within the CS or S groups). Full article ">Figure 3
Porosity of various HA carriers. An asterisk indicates statistically-significant differences (* p < 0.05; n = 4) as compared to the HFT groups. Full article ">Figure 4
Concentration of glucose permeated through various HA carriers at 34 °C. An asterisk indicates statistically-significant differences (* p < 0.05; n = 6) as compared to the HFT groups. Full article ">Figure 5
Cell viability of rabbit corneal endothelial cell (CEC) cultures was determined by staining with the Live/Dead Viability/Cytotoxicity Kit in which the live cells fluoresce green and dead cells fluoresce red. (a) Fluorescence images of cells in controls (without test materials) after 8 h of direct contact with different types of HA samples. Scale bars: 50 μm; (b) Quantitative results are expressed as the percentage of control groups (viability of cells cultured in the absence of test materials). An asterisk indicates statistically-significant differences (* p < 0.05; n = 3) as compared to the control groups. Full article ">Figure 6
Gene expression level of ATP1A1 in rabbit CECs after 8 h of direct contact with various HA carriers, measured by real-time reverse transcription polymerase chain reaction. Normalization was done by using GAPDH. Data in the experimental groups are percentages relative to that of control groups (cells cultured in the absence of HA materials). An asterisk indicates statistically-significant differences (* p < 0.05; n = 4) as compared to the control groups. Full article ">Figure 7
(a) Representative slit-lamp biomicroscopic images of rabbit eyes four weeks after surgical treatment of corneal endothelial dysfunction. Control group: cornea denuded of endothelium; HFT group: endothelial scrape-wounded cornea implanted with HFT carriers and bioengineered CEC sheet; LFT group: endothelial scrape-wounded cornea implanted with LFT carriers and bioengineered CEC sheet. Scale bars: 5 mm; (b) Slit-lamp examination scores of rabbit eyes four weeks after surgical treatment of corneal endothelial dysfunction. An asterisk indicates statistically-significant differences (* p < 0.05; n = 6) as compared to the control groups. Full article ">Figure 8
Representative specular microscopic images of rabbit eyes four weeks after surgical treatment of corneal endothelial dysfunction. (a) Control group: cornea denuded of endothelium; (b) HFT group: endothelial scrape-wounded cornea implanted with HFT carriers and bioengineered CEC sheet; and (c) LFT group: endothelial scrape-wounded cornea implanted with LFT carriers and bioengineered CEC sheet. Full article ">

4472 KiB

Open AccessArticle

Differentiation Effects of Platelet-Rich Plasma Concentrations on Synovial Fluid Mesenchymal Stem Cells from Pigs Cultivated in Alginate Complex Hydrogel

by Hao-Che Tang, Wei-Chuan Chen, Chih-Wei Chiang, Lei-Yen Chen, Yu-Ching Chang and Chih-Hwa Chen

Int. J. Mol. Sci. 2015, 16(8), 18507-18521; https://doi.org/10.3390/ijms160818507 - 7 Aug 2015

Cited by 20 | Viewed by 6792

Abstract

This article studied the effects of platelet-rich plasma (PRP) on the potential of synovial fluid mesenchymal stem cells (SF-MSCs) to differentiate. The PRP and SF-MSCs were obtained from the blood and knees of pigs, respectively. The identification of SF-MSCs and their ability to [...] Read more.

This article studied the effects of platelet-rich plasma (PRP) on the potential of synovial fluid mesenchymal stem cells (SF-MSCs) to differentiate. The PRP and SF-MSCs were obtained from the blood and knees of pigs, respectively. The identification of SF-MSCs and their ability to differentiate were studied by histological and surface epitopes, respectively. The SF-MSCs can undergo trilineage mesenchymal differentiation under osteogenic, chondrogenic, and adipocyte induction. The effects of various PRP concentrations (0%, 20% and 50% PRP) on differentiation were evaluated using the SF-MSCs-alginate system, such as gene expression and DNA proliferation. A 50% PRP concentration yielded better differentiation than the 20% PRP concentration. PRP favored the chondrogenesis of SF-MSCs over their osteogenesis in a manner that depended on the ratios of type II collagen/type I collagen and aggrecan/osteopontin. Eventually, PRP promoted the proliferation of SF-MSCs and induced chondrogenic differentiation of SF-MSCs in vitro. Both PRP and SF-MSCs could be feasibly used in regenerative medicine and orthopedic surgeries. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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12495 KiB

Open AccessArticle

Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

by Jung-Bo Huh, June-Jip Yang, Kyung-Hee Choi, Ji Hyeon Bae, Jeong-Yeol Lee, Sung-Eun Kim and Sang-Wan Shin

Int. J. Mol. Sci. 2015, 16(7), 16034-16052; https://doi.org/10.3390/ijms160716034 - 14 Jul 2015

Cited by 25 | Viewed by 8205

Abstract

Anorganic bovine bone matrix (Bio-Oss^®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss^®. [...] Read more.

Anorganic bovine bone matrix (Bio-Oss^®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss^®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss^® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss^® or BMP-2/Bio-Oss^®. The Bio-Oss^® and BMP-2/Bio-Oss^® groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss^® group (16.50 ± 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss^® group (9.43 ± 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss^® markedly enhances bone regeneration. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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3172 KiB

Open AccessArticle

3D-Printed ABS and PLA Scaffolds for Cartilage and Nucleus Pulposus Tissue Regeneration

by Derek H. Rosenzweig, Eric Carelli, Thomas Steffen, Peter Jarzem and Lisbet Haglund

Int. J. Mol. Sci. 2015, 16(7), 15118-15135; https://doi.org/10.3390/ijms160715118 - 3 Jul 2015

Cited by 276 | Viewed by 18854

Abstract

Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds [...] Read more.

Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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ABS and PLA scaffold design. Construct design was downloaded from the freeware website <a href="http://www.thingiverse.com/thing:45228/#files" target="_blank">http://www.thingiverse.com/thing:45228/#files</a>. (A) Representative .stl images of scaffold structure; (B) representative images of printed ABS and PLA scaffolds. Horizontal scale bar: 0.5 cm; vertical scale bar: 0.3 cm; (C) Quantification of scaffold weight; (D) quantified porosity of the ABS and PLA scaffolds used in the studies. Error bars represent ± SD, n = 6. Full article ">Figure 2
Mechanical testing of ABS and PLA scaffolds. (A) Young’s modulus was calculated between 5%–10% compressive strain/strain curves for freshly printed (day 0) and culture medium bathed (day 21) ABS and PLA scaffolds. For each set, n = 5. Error bars represent ± SD; (B) Cartoon representation of mechanical test data for printed scaffolds placed in context of known mechanical bulk properties of ABS and PLA plastics, as well as human NP tissue, articular cartilage, and bone. Horizontal arrows indicate a range of stiffness, while downward arrows indicate exact stiffness. Full article ">Figure 3
Cell ingrowth on ABS and PLA scaffolds. Representative phase contrast images of cell-seeded scaffolds at 4× and 10× magnification after 21 days of culture (n = 4). Black arrows indicate cell growth and neo-tissue deposition. White scale bar represents 1 mm; grey scale bar represents 500 µm. Full article ">Figure 4
Cell viability on ABS and PLA scaffolds. Live/dead assay was performed on chondrocytes and NP cell-seeded ABS and PLA scaffolds after 21 days’ culture. Live cells are shown in green (calcein AM) while dead cells are shown in red (ethidium homodimer). Representative images (n = 4) show that the number of dead cells on either scaffold is negligible. Inset panels are enlarged 3× from indicated regions, and show individual live and dead cells. Scale bar: 1.0 mm. Full article ">Figure 5
Proteoglycan analysis of scaffolds. (A) Fixed acellular, chondrocyte-, and NP-seeded ABS and PLA scaffolds were stained with safranin-O/fast green after 21 days’ culture. Representative images (n = 4) showing that both chondrocyte- and NP-seeded ABS and PLA scaffolds retained intense safranin-O staining. Ruler ticks represent millimeters; (B) DMMB assay for quantifying sulfated GAG in total extract per scaffold (in 1 mL); (C) DNA content was quantified by HOECHST 33258 assay per scaffold; (D) The sGAG/DNA ratio was determined. Error bars represent ± SD. * indicates p < 0.05. Full article ">Figure 6
Collagen type II analysis of cell-seeded scaffolds. (A) Representative Western blot probing extracts for newly synthesized, non-crosslinked collagen type II; (B) densitometry quantification for collagen type II deposition on scaffolds; (C) extractable collagen type II band density/sGAG was determined; (D) extractable collagen type II band density/DNA was determined. Error bars represent ± SD. # indicates p = 0.0601. Full article ">Figure 7
Immunofluorescence analysis of cell-seeded scaffolds. Acellular, chondrocyte-, and NP-seeded ABS and PLA scaffolds were probed by immunofluorescence for collagen type II (red) and aggrecan core protein (green) after 21 days’ culture. Acellular scaffolds show some autofluorescent background. Cell-seeded scaffolds produce fluorescent signals well above background, specifically in regions within the pores, where only cells are growing. Scale bar: 1.0 mm. Full article ">

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Open AccessArticle

Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability

by Yoshika Suzawa, Norihiko Kubo, Soichi Iwai, Yoshiaki Yura, Hajime Ohgushi and Mitsuru Akashi

Int. J. Mol. Sci. 2015, 16(6), 14245-14258; https://doi.org/10.3390/ijms160614245 - 23 Jun 2015

Cited by 24 | Viewed by 6904

Abstract

Hydroxyapatite (HA) or calcium carbonate (CaCO₃) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells [...] Read more.

Hydroxyapatite (HA) or calcium carbonate (CaCO₃) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO₃-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO₃-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO₃-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO₃-formed agarose at an early stage, and both HA- and CaCO₃-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO₃-formed agarose could potentially be employed for the repair of critical bone defects. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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(a) Fluorescence microscope images of mesenchymal stem cells (MSCs) cultured for 16 days on agarose, hydroxyapatite (HA)/agarose, and CaCO3/agarose gels with live/dead stain. Left panels: Agarose gel; middle panels: HA/agarose; right panels: CaCO3/agarose gel. The lower panels show high-power magnification (200×) images of the upper panels (40×). Disk shaped gels with 1.0-mm thickness and 10-mm diameters; (b) Fluorescence microscope images of MSCs cultured for 21 days on the HA/agarose gel with live/dead stain. The cultured gel was cut perpendicular to the round surface of the gel at its center. The cross-section was observed under the microscope by focusing surface to bottom layer. Disk shaped gels with 1.0-mm thickness and 10-mm diameters. Full article ">Figure 2
SEM images of MSCs cultured on HA/agarose and CaCO3/agarose gels for 21 days. (A) HA/agarose gel; (B) CaCO3/agarose gel. Full article ">Figure 3
Adenosine triphosphate (ATP) content of MSCs cultured in the presence (Dex (+)) or absence (Dex (−)) of dexamethasone (Dex) on agarose, HA/agarose, and CaCO3/agarose gels. The data represent means ± SD (n = 6). * p < 0.05, ** p < 0.01. Full article ">Figure 4
Alkaline phosphatase (ALP) staining of MSCs cultured in the presence (Dex (+)) or absence (Dex (−)) of dexamethasone on agarose, HA/agarose, and CaCO3/agarose gels. Cultures were performed for 14, 21, and 28 days. Full article ">Figure 5
ALP activity of MSCs cultured in the presence (Dex (+)) or absence (Dex (−)) of dexamethasone on agarose, HA/agarose, and CaCO3/agarose gels. Cultures were performed for 4 to 28 days. The data represent means ± SD (n = 6). * p < 0.05, ** p < 0.01. Full article ">Figure 6
Osteocalcin content of MSCs cultured in the presence (Dex (+)) or absence (Dex (−)) of dexamethasone on agarose, HA/agarose, and CaCO3/agarose gels. Cultures were performed for 4 to 28 days. The data represent means ± SD (n = 6). * p < 0.05, ** p < 0.01. Full article ">

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Open AccessArticle

Engraftment of Prevascularized, Tissue Engineered Constructs in a Novel Rabbit Segmental Bone Defect Model

by Alexandre Kaempfen, Atanas Todorov, Sinan Güven, René D. Largo, Claude Jaquiéry, Arnaud Scherberich, Ivan Martin and Dirk J. Schaefer

Int. J. Mol. Sci. 2015, 16(6), 12616-12630; https://doi.org/10.3390/ijms160612616 - 4 Jun 2015

Cited by 31 | Viewed by 7427

Abstract

The gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to [...] Read more.

The gold standard treatment of large segmental bone defects is autologous bone transfer, which suffers from low availability and additional morbidity. Tissue engineered bone able to engraft orthotopically and a suitable animal model for pre-clinical testing are direly needed. This study aimed to evaluate engraftment of tissue-engineered bone with different prevascularization strategies in a novel segmental defect model in the rabbit humerus. Decellularized bone matrix (Tutobone) seeded with bone marrow mesenchymal stromal cells was used directly orthotopically or combined with a vessel and inserted immediately (1-step) or only after six weeks of subcutaneous “incubation” (2-step). After 12 weeks, histological and radiological assessment was performed. Variable callus formation was observed. No bone formation or remodeling of the graft through TRAP positive osteoclasts could be detected. Instead, a variable amount of necrotic tissue formed. Although necrotic area correlated significantly with amount of vessels and the 2-step strategy had significantly more vessels than the 1-step strategy, no significant reduction of necrotic area was found. In conclusion, the animal model developed here represents a highly challenging situation, for which a suitable engineered bone graft with better prevascularization, better resorbability and higher osteogenicity has yet to be developed. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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(A) Amount of nucleated cells extracted per bone marrow biopsy; each dot represents one donor; (B) Percentage of colony forming units per nucleated cells extracted from bone marrow biopsy; (C) Silicon mold with scaffold during seeding; (D) Tetrazolium (MTT) staining showing the distribution of cells along the periphery after seeding: the top image is a midline section, and the bottom is the outside surface. Black bar represents 1 mm. Full article ">Figure 2
(A) Experimental set-up of the different groups; (B) Representative intraoperative images of the orthotopic defect. Full article ">Figure 3
(A) Follow-up radiological image showing scaffold in orthotopical defect after 10 weeks. Callus formation is visible on the opposite side of the plate. Arrows indicate proximal and distal edges of defect, white bar represents 5 mm; (B) Radiological quantification of callus as seen in (A), represented as average mm of radioopaque mass at the proximal and distal edges of the defect. Dotted line represents normal bone diameter at the same location; (C) Macroscopical appearance after plate removal. Arrows indicate the edges of the defect; (D) Post-explantation microtomography. Red bar represents 1 mm. None of the groups displayed macroscopic or microradiographic evidence of fracture consolidation during the experimental period. Full article ">Figure 3 Cont.
(A) Follow-up radiological image showing scaffold in orthotopical defect after 10 weeks. Callus formation is visible on the opposite side of the plate. Arrows indicate proximal and distal edges of defect, white bar represents 5 mm; (B) Radiological quantification of callus as seen in (A), represented as average mm of radioopaque mass at the proximal and distal edges of the defect. Dotted line represents normal bone diameter at the same location; (C) Macroscopical appearance after plate removal. Arrows indicate the edges of the defect; (D) Post-explantation microtomography. Red bar represents 1 mm. None of the groups displayed macroscopic or microradiographic evidence of fracture consolidation during the experimental period. Full article ">Figure 4
(A) Analysis of necrotic core formation based on hematoxylin and eosin (H&E) staining. Blue rectangle indicates the total area considered during quantification. Black dotted outline is an example of necrotic area. Black bar represents 1 mm; (B) Necrotic area observed in the experimental groups, represented as fold of the average necrotic area in the pedicle 2-step group; (C) Representative Goldner Trichrome staining with vessels appearing red in high magnification. Black bar represents 50 mm; (D) Average vessel number inside the scaffold (blue rectangle in A), counted by Goldner Tichrome staining. Significant difference is marked by *; (E) Correlation of vessel number and necrotic area for each sample. Black line represents linear regression fit and dotted lines represent 95% confidence intervals. Full article ">Figure 4 Cont.
(A) Analysis of necrotic core formation based on hematoxylin and eosin (H&E) staining. Blue rectangle indicates the total area considered during quantification. Black dotted outline is an example of necrotic area. Black bar represents 1 mm; (B) Necrotic area observed in the experimental groups, represented as fold of the average necrotic area in the pedicle 2-step group; (C) Representative Goldner Trichrome staining with vessels appearing red in high magnification. Black bar represents 50 mm; (D) Average vessel number inside the scaffold (blue rectangle in A), counted by Goldner Tichrome staining. Significant difference is marked by *; (E) Correlation of vessel number and necrotic area for each sample. Black line represents linear regression fit and dotted lines represent 95% confidence intervals. Full article ">Figure 5
Representative tartrate resistant acid phosphatase (TRAP) stainings. Black bar represents 50 mm in each image. (A) Schematical display of TRAP staining location; (B) Normal bone has closely associated TRAP positive osteoclasts; (C) Periost around fracture displays increased presence of TRAP positive cells; (D) Tutobone scaffold with invading granulation tissue, however no closely associated TRAP positive cells. Full article ">

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Open AccessArticle

A Solid-State NMR Study of Selenium Substitution into Nanocrystalline Hydroxyapatite

by Joanna Kolmas, Marzena Kuras, Ewa Oledzka and Marcin Sobczak

Int. J. Mol. Sci. 2015, 16(5), 11452-11464; https://doi.org/10.3390/ijms160511452 - 19 May 2015

Cited by 22 | Viewed by 6479

Abstract

The substitution of selenium oxyanions in the hydroxyapatite structure was examined using multinuclear solid-state resonance spectroscopy (ssNMR). The study was supported by powder X-ray diffractometry (PXRD) and wavelength dispersion X-ray fluorescence (WD-XRF). Samples of pure hydroxyapatite (HA₃₀₀) and selenate (HA₃₀₀ [...] Read more.

The substitution of selenium oxyanions in the hydroxyapatite structure was examined using multinuclear solid-state resonance spectroscopy (ssNMR). The study was supported by powder X-ray diffractometry (PXRD) and wavelength dispersion X-ray fluorescence (WD-XRF). Samples of pure hydroxyapatite (HA₃₀₀) and selenate (HA₃₀₀-1.2SeO₄) or selenite (HA₃₀₀-1.2SeO₃) substituted hydroxyapatites were synthesized using the standard wet method and heated at 300 °C to remove loosely bonded water. PXRD data showed that all samples are single-phase, nanocrystalline hydroxyapatite. The incorporation of selenite and selenate ions affected the lattice constants. In selenium-containing samples the concentration of Se was very similar and amounted to 9.55% and 9.64%, for HA₃₀₀-1.2SeO₄ and HA₃₀₀-1.2SeO₃, respectively. PXRD and ssNMR data showed that the selenite doping significantly decreases the crystallite size and crystallinity degree. ³¹P and ¹H NMR experiments demonstrated the developed surface hydrated layer in all samples, especially in HA₃₀₀-1.2SeO₃. ¹H NMR studies showed the dehydroxylation of HA during the selenium oxyanions substitution and the existence of hydrogen bonding in structural hydroxyl group channels. ¹H→⁷⁷Se cross polarization NMR experiments indicated that selenites and selenates are located in the crystal lattice and on the crystal surface. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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PXRD patterns of the analysed samples: HA300, HA300-1.2SeO3 and HA300-1.2SeO4. Full article ">Figure 2
31P BD (A) and CP (B) MAS NMR spectra of the analysed samples. Full article ">Figure 3
Representative peak fittings of the 31P CP NMR spectrum for the HA300-1.2SeO3 sample. Full article ">Figure 4
The 1H BD NMR spectra of HA300, HA300-1.2SeO3 and HA300-1.2SeO4. Full article ">Figure 5
Structural hydroxyl groups content in reference to stoichiometric hydroxyapatite (%). Full article ">Figure 6
The 1H→77Se CP NMR spectra for the selenium containing samples. Samples of HA100-1.2SeO3 and HA100-1.2SeO4 were dried at 100 °C. Full article ">

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Open AccessArticle

Cell Adhesion and in Vivo Osseointegration of Sandblasted/Acid Etched/Anodized Dental Implants

by Mu-Hyon Kim, Kyeongsoon Park, Kyung-Hee Choi, Soo-Hong Kim, Se Eun Kim, Chang-Mo Jeong and Jung-Bo Huh

Int. J. Mol. Sci. 2015, 16(5), 10324-10336; https://doi.org/10.3390/ijms160510324 - 6 May 2015

Cited by 59 | Viewed by 9991

Abstract

The authors describe a new type of titanium (Ti) implant as a Modi-anodized (ANO) Ti implant, the surface of which was treated by sandblasting, acid etching (SLA), and anodized techniques. The aim of the present study was to evaluate the adhesion of MG-63 [...] Read more.

The authors describe a new type of titanium (Ti) implant as a Modi-anodized (ANO) Ti implant, the surface of which was treated by sandblasting, acid etching (SLA), and anodized techniques. The aim of the present study was to evaluate the adhesion of MG-63 cells to Modi-ANO surface treated Ti in vitro and to investigate its osseointegration characteristics in vivo. Four different types of Ti implants were examined, that is, machined Ti (control), SLA, anodized, and Modi-ANO Ti. In the cell adhesion study, Modi-ANO Ti showed higher initial MG-63 cell adhesion and induced greater filopodia growth than other groups. In vivo study in a beagle model revealed the bone-to-implant contact (BIC) of Modi-ANO Ti (74.20% ± 10.89%) was much greater than those of machined (33.58% ± 8.63%), SLA (58.47% ± 12.89), or ANO Ti (59.62% ± 18.30%). In conclusion, this study demonstrates that Modi-ANO Ti implants produced by sandblasting, acid etching, and anodizing improve cell adhesion and bone ongrowth as compared with machined, SLA, or ANO Ti implants. These findings suggest that the application of Modi-ANO surface treatment could improve the osseointegration of dental implant. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Scanning electron microscopy (SEM) images of surface-modified titanium substrates. SLA: sandblasted and acid etched Ti, ANO: anodized Ti, Modi-ANO: sandblasted/acid etched and anodized Ti. Full article ">Figure 2
Cell adhesion of MG-63 cells cultured on titanium substrates. Crystal violet assays were performed after incubating MG-63 cells on each Ti surface for 3 h. * p < 0.05 and ** p < 0.01 indicate statistical significant between groups. Full article ">Figure 3
SEM images of the morphologies of MG-63 cells grown on Ti control, SLA, ANO, and Modi-ANO titanium substrates for 24 h. Full article ">Figure 4
(A) Histological evaluations of (a) Control; (b) SLA; (c) ANO; and (d) Modi-ANO. (Magnification: Central, ×12.5; Right and Left, ×40); (B) Histological images of (a) Control; (b) SLA; (c) ANO; and (d) Modi-ANO (Original magnification ×100). Full article ">Figure 5
Implant placement. Implants were placed 4 mm apart using a ruler. Releasing incision was made for tension free structure of the flap. Full article ">

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Open AccessArticle

Area-Specific Cell Stimulation via Surface-Mediated Gene Transfer Using Apatite-Based Composite Layers

by Yushin Yazaki, Ayako Oyane, Yu Sogo, Atsuo Ito, Atsushi Yamazaki and Hideo Tsurushima

Int. J. Mol. Sci. 2015, 16(4), 8294-8309; https://doi.org/10.3390/ijms16048294 - 14 Apr 2015

Cited by 3 | Viewed by 8321

Abstract

Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP)-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to [...] Read more.

Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP)-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA–fibronectin–apatite composite (DF-Ap) layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in theunderlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Schematic depiction of the sample setting used in the dual reporter gene transfer study. Full article ">Figure 2
Scanning electron microscopy (SEM) images of the surfaces of the DNA–fibronectin–apatite composite layers (DF-Ap layers) immobilizing firefly luciferase (FL) gene (Sample DF-FL) and Renilla luciferase (RL) gene (Sample DF-RL). Full article ">Figure 3
Thin-film X-ray diffraction (TF-XRD) patterns of the surfaces of the DNA–fibronectin–apatite composite layers (DF-Ap layers) immobilizing firefly luciferase (FL) gene (Sample DF-FL) and Renilla luciferase (RL) gene (Sample DF-RL). Full article ">Figure 4
Contents of (a) calcium and phosphorus; and (b) DNA and fibronectin of the DNA–fibronectin–apatite composite layers (DF-Ap layers) immobilizing firefly luciferase (FL) gene (Sample DF-FL) and Renilla luciferase (RL) gene (Sample DF-RL). The results shown are the mean and standard deviation of three independent experiments. Full article ">Figure 5
Luciferase (firefly luciferase (FL) and Renilla luciferase (RL)) activity in CHO-K1 cells cultured on the DNA–fibronectin–apatite composite layers (DF-Ap layers) immobilizing FL gene (Sample FL) and RL gene (Sample DF-RL). Both Samples FL and RL were adjacently placed in one well of a four-well chamber slide. The results shown are the mean and standard deviation of five independent experiments. Full article ">Figure 6
Optical microscopy image of CHO-K1 cells cultured on the DNA–fibronectin–apatite composite layer (DF-Ap layer) immobilizing firefly luciferase (FL) gene (Sample DF-FL). The scale bar indicates 200 μm. Full article ">

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Open AccessArticle

Synthesis of Spongy-Like Mesoporous Hydroxyapatite from Raw Waste Eggshells for Enhanced Dissolution of Ibuprofen Loaded via Supercritical CO₂

by Abdul-Rauf Ibrahim, Xiangyun Li, Yulan Zhou, Yan Huang, Wenwen Chen, Hongtao Wang and Jun Li

Int. J. Mol. Sci. 2015, 16(4), 7960-7975; https://doi.org/10.3390/ijms16047960 - 9 Apr 2015

Cited by 50 | Viewed by 7952

Abstract

The use of cheaper and recyclable biomaterials (like eggshells) to synthesize high purity hydroxyapatite (HAp) with better properties (small particle size, large surface area and pore volume) for applications (in environmental remediation, bone augmentation and replacement, and drug delivery systems) is vital since [...] Read more.

The use of cheaper and recyclable biomaterials (like eggshells) to synthesize high purity hydroxyapatite (HAp) with better properties (small particle size, large surface area and pore volume) for applications (in environmental remediation, bone augmentation and replacement, and drug delivery systems) is vital since high-purity synthetic calcium sources are expensive. In this work, pure and mesoporous HAp nanopowder with large pore volume (1.4 cm³/g) and surface area (284.1 m²/g) was produced from raw eggshells at room temperature using a simple two-step procedure. The control of precursor droplets could stabilize the pH value of the reaction solution, because of the size of the needle (of the syringe pump used for precursor additions) leading to production of HAp with high surface area and pore size. The as-produced HAp revealed high ibuprofen (as a model drug) loading (1.38 g/g HAp), enhanced dissolution and controllable release of the drug via solute-saturated supercritical carbon dioxide. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
XRD Patterns of (a) calcite from waste eggshell; hydroxyapatite (Hap) produced using (b) Na2HPO4 and (c) H3PO4 (HAp284PA); and the calcined HAp284PA at (d) 700 °C and (e) 950 °C; bars: HAp reference (ICSD-PDF2: 01-084-1998). Full article ">Figure 2
SEM images of HAp produced using (a) Na2HPO4 and (b) H3PO4 (HAp284PA); and the calcined HAp284PA at (c) 700 °C and (d) 950 °C. Full article ">Figure 3
XRD patterns for (a) pure ibuprofen; (b) as-HAP (HAp284PA); and (c) ibuprofen-HAp mixture samples. Full article ">Figure 4
TG curves for the as-produced HAp, pure ibuprofen and ibuprofen loaded samples. Full article ">Figure 5
Dissolution profiles for ibuprofen raw material and ibuprofen-HAp samples in a simulated gastric fluid. Full article ">Figure 6
Illustration for the preparation of HAp. Full article ">Figure 7
Schematic diagram of the solute-saturated supercritical loading process; (1) CO2 cylinder; (2) dryer; (3) back pressure valve; (4) compressor; (5,5’) valves; (6) filter; (7) water bath; (8) loading vessel; (9) tube ; (10) ibuprofen loaded; (11) cotton; (P) pressure gauge. Full article ">

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Open AccessArticle

Multi-Layer Electrospun Membrane Mimicking Tendon Sheath for Prevention of Tendon Adhesions

by Shichao Jiang, Hede Yan, Dapeng Fan, Jialin Song and Cunyi Fan

Int. J. Mol. Sci. 2015, 16(4), 6932-6944; https://doi.org/10.3390/ijms16046932 - 26 Mar 2015

Cited by 42 | Viewed by 7334

Abstract

Defect of the tendon sheath after tendon injury is a main reason for tendon adhesions, but it is a daunting challenge for the biomimetic substitute of the tendon sheath after injury due to its multi-layer membrane-like structure and complex biologic functions. In this [...] Read more.

Defect of the tendon sheath after tendon injury is a main reason for tendon adhesions, but it is a daunting challenge for the biomimetic substitute of the tendon sheath after injury due to its multi-layer membrane-like structure and complex biologic functions. In this study, a multi-layer membrane with celecoxib-loaded poly(l-lactic acid)-polyethylene glycol (PELA) electrospun fibrous membrane as the outer layer, hyaluronic acid (HA) gel as middle layer, and PELA electrospun fibrous membrane as the inner layer was designed. The anti-adhesion efficacy of this multi-layer membrane was compared with a single-layer use in rabbit flexor digitorum profundus tendon model. The surface morphology showed that both PELA fibers and celecoxib-loaded PELA fibers in multi-layer membrane were uniform in size, randomly arrayed, very porous, and smooth without beads. Multi-layer membrane group had fewer peritendinous adhesions and better gliding than the PELA membrane group and control group in gross and histological observation. The similar mechanical characteristic and collagen expression of tendon repair site in the three groups indicated that the multi-layer membrane did not impair tendon healing. Taken together, our results demonstrated that such a biomimetic multi-layer sheath could be used as a potential strategy in clinics for promoting tendon gliding and preventing adhesion without poor tendon healing. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Figure 1
Scanning electron microscopy (SEM) observation for cross-sectional features of the multi-layer membrane (A) and surface morphological features of the poly(l-lactic acid)-polyethylene glycol (PELA) electrospun fibers (B) and celecoxib-loaded PELA electrospun fibers (C). Full article ">Figure 2
(A) Gross evaluation of the rabbit flexor digitorum profundus (FDP) tendon model in the untreated control group, PELA membrane group, and multi-layer membrane group. Tendon (T) is indicated in the figures and adhesion tissue is pointed to using black arrows. Tendon repair and peritendinous adhesions are evaluated by determining macroscopic evaluation of tendon adhesions (B), maximum tensile strength (C), and work of flexion (D). * p < 0.05 compared with control group; † p < 0.05 compared with PELA membrane group. Data are expressed as mean ± SEM for six tendons/group. Full article ">Figure 3
HE (A) and Masson (B) staining of untreated repair site, repair sites wrapped with unloaded PELA fibrous membrane and wrapped with a multi-layer fibrous membrane. White arrowheads indicate the interface without peritendinous adhesions, while black arrowheads indicate peritendinous adhesions between the membrane (M) and tendon (T) (Scale bar = 1 mm). Peritendinous adhesions and tendon repair are evaluated by determining histological adhesion grade (C) and histological healing grade (D). * p < 0.05 compared with control group; † p < 0.05 compared with PELA membrane group. Data are expressed as mean ± SEM for six tendons/groups. Full article ">Figure 4
Western blotting assay for collagen I and collagen III expression in repair sites with the untreated group, PELA fibrous membrane group, and multi-layer fibrous membrane group for three weeks (A). Densitometry of collagen I (B) and collagen III (C) from Western blot experiment. Data are expressed as mean ± SEM for six tendons/group. Full article ">Figure 5
Schematic illustration of multi-layer membrane as physical barrier for preventing tendon adhesions after tendon injury. Full article ">

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Open AccessArticle

In Vitro Corrosion and Cytocompatibility Properties of Nano-Whisker Hydroxyapatite Coating on Magnesium Alloy for Bone Tissue Engineering Applications

by Huawei Yang, Xueyu Yan, Min Ling, Zuquan Xiong, Caiwen Ou and Wei Lu

Int. J. Mol. Sci. 2015, 16(3), 6113-6123; https://doi.org/10.3390/ijms16036113 - 17 Mar 2015

Cited by 24 | Viewed by 6804

Abstract

We report here the successful fabrication of nano-whisker hydroxyapatite (nHA) coatings on Mg alloy by using a simple one-step hydrothermal process in aqueous solution. The nHA coating shows uniform structure and high crystallinity. Results indicate that nHA coating is promising for improving the [...] Read more.

We report here the successful fabrication of nano-whisker hydroxyapatite (nHA) coatings on Mg alloy by using a simple one-step hydrothermal process in aqueous solution. The nHA coating shows uniform structure and high crystallinity. Results indicate that nHA coating is promising for improving the in vitro corrosion and cytocompatibility properties of Mg-based implants and devices for bone tissue engineering. In addition, the simple hydrothermal deposition method used in the current study is also applicable to substrates with complex shapes or surface geometries. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Figure 1
X-ray diffraction (XRD) patterns of uncoated and nano-whisker hydroxyapatite (nHA) coated ZK60 samples (a.u and deg. are the abbreviation of arbitrary unit and degree, respectively). Full article ">Figure 2
Schematic illustration of the reaction mechanism of calcium phosphate coatings on ZK60 substrate. (1) Anode reaction; (2) Cathode reaction; (3) Transformation of H2PO4− to PO43−; and (4) HA phase producing process. Full article ">Figure 3
SEM images with different magnifications of nHA coatings on ZK60 substrate. (a) Integral morphology of the HA coatings; (b) Middle magnification SEM image; (c) High magnification SEM image; and (d) Bilayer structure of HA. Full article ">Figure 4
Potentiodynamic polarization curves of uncoated and nHA coated ZK60 samples. Full article ">Figure 5
Variation of pH value of immersion solution at different immersion time of uncoated and nHA coated ZK60 samples. Full article ">Figure 6
Surface morphologies of uncoated and nHA coated ZK60 samples after immersion test for 7 days. (a) Uncoated ZK60; and (b) nHA coated sample. Full article ">Figure 7
Cell viability cultured in individual extraction mediums of uncoated and nHA coated ZK60 samples. Full article ">

1680 KiB

Open AccessArticle

Biomechanical Evaluation of Ti-Nb-Sn Alloy Implants with a Low Young’s Modulus

by Kenta Takahashi, Naru Shiraishi, Risa Ishiko-Uzuka, Takahisa Anada, Osamu Suzuki, Hiroshi Masumoto and Keiichi Sasaki

Int. J. Mol. Sci. 2015, 16(3), 5779-5788; https://doi.org/10.3390/ijms16035779 - 12 Mar 2015

Cited by 19 | Viewed by 5841

Abstract

Dental implants are widely used and are a predictable treatment in various edentulous cases. Occlusal overload may be causally related to implant bone loss and a loss of integration. Stress concentrations may be diminished using a mechanobiologically integrated implant with bone tissue. The [...] Read more.

Dental implants are widely used and are a predictable treatment in various edentulous cases. Occlusal overload may be causally related to implant bone loss and a loss of integration. Stress concentrations may be diminished using a mechanobiologically integrated implant with bone tissue. The purpose of this study was to investigate the biomechanical behavior, biocompatibility and bioactivity of a Ti-Nb-Sn alloy as a dental implant material. It was compared with cpTi. Cell proliferation and alkaline phosphatase (ALP) activity were quantified. To assess the degree of osseointegration, a push-in test was carried out. Cell proliferation and ALP activity in the cells grown on prepared surfaces were similar for the Ti-Nb-Sn alloy and for cpTi in all the experiments. A comparison between the Ti-Nb-Sn alloy implant and the cpTi implant revealed that no significant difference was apparent for the push-in test values. These results suggest that implants fabricated using Ti-Nb-Sn have a similar biological potential as cpTi and are capable of excellent osseointegration. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Surface characteristics of the specimens. (a) 3-D digital images; (b) SEM micrographs; and (c) EDX spectra. Full article ">Figure 2
Hydrophilicity of cpTi and Ti-Nb-Sn alloy disks. (a) Mean contact angle of 1 μL H2O onto the cpTi and Ti-Nb-Sn alloy disks. Data are the mean ± SD (n = 8); (b) Photographic images of H2O droplets pipetted onto the cpTi and Ti-Nb-Sn alloy disks. Full article ">Figure 3
Cell proliferation assay. Data are the mean ± SD (n = 5). Full article ">Figure 4
ALP activity. Data are the mean ± SD (n = 5). Full article ">Figure 5
Push-in test values for cpTi and Ti-Nb-Sn alloy. Data are the mean ± SD (n = 5). Full article ">Figure 6
SEM micrographs and EDX spectra of excised implant after push-in tests. (a) cpTi; (b) Ti-Nb-Sn alloy are SEM micrographs at healing period week 2; (g–j) show EDX spectra, which are the EDX analyses for (c–f) in (a,b), respectively. Bars = 100 μm. Full article ">

3352 KiB

Open AccessArticle

Novel Cholesterol-Based Cationic Lipids as Transfecting Agents of DNA for Efficient Gene Delivery

by Jia Ju, Meng-Lei Huan, Ning Wan, Hai Qiu, Si-Yuan Zhou and Bang-Le Zhang

Int. J. Mol. Sci. 2015, 16(3), 5666-5681; https://doi.org/10.3390/ijms16035666 - 11 Mar 2015

Cited by 35 | Viewed by 8460

Abstract

The design, synthesis and biological evaluation of the cationic lipid gene delivery vectors based on cholesterol and natural amino acids lysine or histidine are described. Cationic liposomes composed of the newly synthesized cationic lipids 1a or 1b and neutral lipid DOPE (1,2-dioleoyl-l-α-glycero-3-phosphatidyl-ethanolamine) exhibited [...] Read more.

The design, synthesis and biological evaluation of the cationic lipid gene delivery vectors based on cholesterol and natural amino acids lysine or histidine are described. Cationic liposomes composed of the newly synthesized cationic lipids 1a or 1b and neutral lipid DOPE (1,2-dioleoyl-l-α-glycero-3-phosphatidyl-ethanolamine) exhibited good transfection efficiency. pEGFP-N₁ plasmid DNA was transferred into 293T cells by cationic liposomes formed from cationic lipids 1a and 1b, and the transfection activity of the cationic lipids was superior (1a) or parallel (1b) to that of the commercially available 3β-[N-(N',N'-dimethylaminoethyl)-carbamoyl] cholesterol (DC-Chol) derived from the same cholesterol backbone with different head groups. Combined with the results of agarose gel electrophoresis, transfection experiments with various molar ratios of the cationic lipids and DOPE and N/P (+/−) molar charge ratios, a more effective formulation was formed, which could lead to relatively high transfection efficiency. Cationic lipid 1a represents a potential agent for the liposome used in gene delivery due to low cytotoxicity and impressive gene transfection activity. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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2122 KiB

Open AccessArticle

Effect of Platelet-Rich Plasma and Bioactive Glass Powder for the Improvement of Rotator Cuff Tendon-to-Bone Healing in a Rabbit Model

by Yang Wu, Yu Dong, Shiyi Chen and Yunxia Li

Int. J. Mol. Sci. 2014, 15(12), 21980-21991; https://doi.org/10.3390/ijms151221980 - 28 Nov 2014

Cited by 10 | Viewed by 6700

Abstract

To test the hypothesis that a platelet-rich plasma (PRP) plus bioactive glass (BG) mixture could shorten the tendon-bone healing process in rotator cuff tendon repair, thirty mature male New Zealand white rabbits were randomly divided into three groups, Control, PRP, and PRP + [...] Read more.

To test the hypothesis that a platelet-rich plasma (PRP) plus bioactive glass (BG) mixture could shorten the tendon-bone healing process in rotator cuff tendon repair, thirty mature male New Zealand white rabbits were randomly divided into three groups, Control, PRP, and PRP + BG. All groups underwent a surgical procedure to establish a rotator cuff tendon healing model. Mechanical examinations and histological assays were taken to verify the adhesion of the tendon-bone. Real-time PCR was adopted to analyze Bone Morphogenetic Protein-2 (BMP-2). The maximum load-to-failure value in mechanical examinations was significantly higher in the PRP + BG group than that in the control group after six weeks (Control 38.73 ± 8.58, PRP 54.49 ± 8.72, PRP + BG 79.15 ± 7.62, p < 0.001), but it was not significantly different at 12 weeks (PRP 74.27 ± 7.74, PRP + BG 82.57 ± 6.63, p = 0.145). In histological assays, H&E (hematoxylin-eosin) staining showed that the interface between the tendon-bone integration was much sturdier in the PRP + BG group compared to the other two groups at each time point, and more ordered arranged tendon fibers can be seen at 12 weeks. At six weeks, the mRNA expression levels of BMP-2 in the PRP + BG group were higher than those in the other groups (PRP + BG 0.65 ± 0.11, PRP 2.284 ± 0.07, Control 0.12 ± 0.05, p < 0.05). However, there was no significant difference in the mRNA expression levels of BMP-2 among the three groups at 12 weeks (p = 0.922, 0.067, 0.056). BMP-2 levels in PRP and PRP+BG groups were significantly lower at 12 weeks compared to six weeks (p = 0.006, <0.001).We found that the PRP + BG mixture could enhance tendon-bone healing in rotator cuff tendon repair. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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5532 KiB

Open AccessArticle

Mechanical Reinforcement of Diopside Bone Scaffolds with Carbon Nanotubes

by Cijun Shuai, Tingting Liu, Chengde Gao, Pei Feng and Shuping Peng

Int. J. Mol. Sci. 2014, 15(10), 19319-19329; https://doi.org/10.3390/ijms151019319 - 23 Oct 2014

Cited by 15 | Viewed by 6574

Abstract

Carbon nanotubes are ideal candidates for the mechanical reinforcement of ceramic due to their excellent mechanical properties, high aspect ratio and nanometer scale diameter. In this study, the effects of multi-walled carbon nanotubes (MWCNTs) on the mechanical properties of diopside (Di) scaffolds fabricated [...] Read more.

Carbon nanotubes are ideal candidates for the mechanical reinforcement of ceramic due to their excellent mechanical properties, high aspect ratio and nanometer scale diameter. In this study, the effects of multi-walled carbon nanotubes (MWCNTs) on the mechanical properties of diopside (Di) scaffolds fabricated by selective laser sintering were investigated. Results showed that compressive strength and fracture toughness improved significantly with increasing MWCNTs from 0.5 to 2 wt %, and then declined with increasing MWCNTs to 5 wt %. Compressive strength and fracture toughness were enhanced by 106% and 21%, respectively. The reinforcing mechanisms were identified as crack deflection, MWCNTs crack bridging and pull-out. Further, the scaffolds exhibited good apatite-formation ability and supported adhesion and proliferation of cells in vitro. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Morphology of the raw powders (a) Di powders; and (b) MWCNTs powders; (c) Di scaffold prepared by SLS; (d) Raman spectrum of the prepared scaffold, D: D band of CNTs; G: G band of CNTs. Full article ">Figure 2
Compressive strength and fracture toughness of the scaffolds with different MWCNTs content. Full article ">Figure 3
The surface morphology of (a) Di scaffold with 2 wt % MWCNTs; and (b) Di scaffold with 5 wt % MWCNTs. Full article ">Figure 4
SEM of (a) indentation crack propagation of Di scaffold with 2 wt % MWCNTs; (b) MWCNTs crack bridging; (c) crack deflection; (d) MWCNTs pull-out at the fracture surface of the scaffold. Full article ">Figure 5
Bone-like apatite precipitated on the Di scaffolds for different periods of immersion (a) 7 days; (b) 14 days; (c) 21 days; and EDS spectra of (d) the globular bone-like apatite in (c). Full article ">Figure 6
MG-63 cells cultured on Di scaffolds for (a) 3 days; and (b) 7 days. Full article ">

Review

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891 KiB

Open AccessReview

Hydrogels for Engineering of Perfusable Vascular Networks

by Juan Liu, Huaiyuan Zheng, Patrina S. P. Poh, Hans-Günther Machens and Arndt F. Schilling

Int. J. Mol. Sci. 2015, 16(7), 15997-16016; https://doi.org/10.3390/ijms160715997 - 14 Jul 2015

Cited by 234 | Viewed by 12808

Abstract

Hydrogels are commonly used biomaterials for tissue engineering. With their high-water content, good biocompatibility and biodegradability they resemble the natural extracellular environment and have been widely used as scaffolds for 3D cell culture and studies of cell biology. The possible size of such [...] Read more.

Hydrogels are commonly used biomaterials for tissue engineering. With their high-water content, good biocompatibility and biodegradability they resemble the natural extracellular environment and have been widely used as scaffolds for 3D cell culture and studies of cell biology. The possible size of such hydrogel constructs with embedded cells is limited by the cellular demand for oxygen and nutrients. For the fabrication of large and complex tissue constructs, vascular structures become necessary within the hydrogels to supply the encapsulated cells. In this review, we discuss the types of hydrogels that are currently used for the fabrication of constructs with embedded vascular networks, the key properties of hydrogels needed for this purpose and current techniques to engineer perfusable vascular structures into these hydrogels. We then discuss directions for future research aimed at engineering of vascularized tissue for implantation. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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Graphical abstract
Full article ">Figure 1
Young’s modulus of natural soft tissues and organs in kPa. Full article ">Figure 2
Molding approach for fabrication of microfluidic constructs. Microneedles and a Nylon fiber are fixed inside a chamber. Then hydrogel solutions are poured into the chamber and gelled. Then open channels are formed by gently removing the fibers. Medium with ECs can be perfused through the channel. Full article ">Figure 3
Soft lithographic approach for fabrication of microfluidic constructs. Agarose solution embedded with hepatic cells was gelled on a SU-8 patterned silicon wafer and microfluidic channels were formed by heating and sealing of the surface of two molded agarose hydrogels. Full article ">Figure 4
Photopatterning approach for fabrication of microfluidic constructs. A thin film of a photosensitive polymer is exposed to UV light through a predesigned mask. There are transparent regions in the mask that allow the UV light to pass through. The areas where the light can reach through the transparent regions generate crosslinking of the polymer while the dark areas under the mask that cannot receive the light will maintain liquid and can be easily removed to form tubular structures. Full article ">Figure 5
Bioprinting for fabrication of multi-level tubular constructs. With a layer-by-layer printing approach, the collagen precursor was printed and polymerized. Then gelatin/cell mixture was printed in a straight pattern to take the position of the channel. After gelation, more collagen layers were printed to cover the gelatin pattern. The vascular structure was formed after gelatin was liquefied. Full article ">Figure 6
Modular method for fabricating multi-level tubular constructs. (A) The Mask with multi-level tubular structure for a photopatterning approach; (B) The microtubes created after UV-light photocuring; (C) Microtubes are assembled into a larger and longer tubular construct with multi-level interconnected lumens. Full article ">

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Open AccessReview

Biomedical Application of Low Molecular Weight Heparin/Protamine Nano/Micro Particles as Cell- and Growth Factor-Carriers and Coating Matrix

by Masayuki Ishihara, Satoko Kishimoto, Makoto Takikawa, Hidemi Hattori, Shingo Nakamura and Masafumi Shimizu

Int. J. Mol. Sci. 2015, 16(5), 11785-11803; https://doi.org/10.3390/ijms160511785 - 22 May 2015

Cited by 17 | Viewed by 8386

Abstract

Low molecular weight heparin (LMWH)/protamine (P) nano/micro particles (N/MPs) (LMWH/P N/MPs) were applied as carriers for heparin-binding growth factors (GFs) and for adhesive cells including adipose-derived stromal cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs). A mixture of LMWH and P yields [...] Read more.

Low molecular weight heparin (LMWH)/protamine (P) nano/micro particles (N/MPs) (LMWH/P N/MPs) were applied as carriers for heparin-binding growth factors (GFs) and for adhesive cells including adipose-derived stromal cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs). A mixture of LMWH and P yields a dispersion of N/MPs (100 nm–3 μm in diameter). LMWH/P N/MPs can be immobilized onto cell surfaces or extracellular matrix, control the release, activate GFs and protect various GFs. Furthermore, LMWH/P N/MPs can also bind to adhesive cell surfaces, inducing cells and LMWH/P N/MPs-aggregate formation. Those aggregates substantially promoted cellular viability, and induced vascularization and fibrous tissue formation in vivo. The LMWH/P N/MPs, in combination with ADSCs or BMSCs, are effective cell-carriers and are potential promising novel therapeutic agents for inducing vascularization and fibrous tissue formation in ischemic disease by transplantation of the ADSCs and LMWH/P N/MPs-aggregates. LMWH/P N/MPs can also bind to tissue culture plates and adsorb exogenous GFs or GFs from those cells. The LMWH/P N/MPs-coated matrix in the presence of GFs may provide novel biomaterials that can control cellular activity such as growth and differentiation. Furthermore, three-dimensional (3D) cultures of cells including ADSCs and BMSCs using plasma-medium gel with LMWH/P N/MPs exhibited efficient cell proliferation. Thus, LMWH/P N/MPs are an adequate carrier both for GFs and for stromal cells such as ADSCs and BMSCs, and are a functional coating matrix for their cultures. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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1118 KiB

Open AccessReview

Three-Dimensional Cell Culture: A Breakthrough in Vivo

by Delphine Antoni, Hélène Burckel, Elodie Josset and Georges Noel

Int. J. Mol. Sci. 2015, 16(3), 5517-5527; https://doi.org/10.3390/ijms16035517 - 11 Mar 2015

Cited by 724 | Viewed by 28632

Abstract

Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the [...] Read more.

Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D) cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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725 KiB

Open AccessReview

Methods for Biomimetic Remineralization of Human Dentine: A Systematic Review

by Chris Ying Cao, May Lei Mei, Quan-Li Li, Edward Chin Man Lo and Chun Hung Chu

Int. J. Mol. Sci. 2015, 16(3), 4615-4627; https://doi.org/10.3390/ijms16034615 - 2 Mar 2015

Cited by 107 | Viewed by 11603

Abstract

This study aimed to review the laboratory methods on biomimetic remineralization of demineralized human dentine. A systematic search of the publications in the PubMed, TRIP, and Web of Science databases was performed. Titles and abstracts of initially identified publications were screened. Clinical trials, [...] Read more.

This study aimed to review the laboratory methods on biomimetic remineralization of demineralized human dentine. A systematic search of the publications in the PubMed, TRIP, and Web of Science databases was performed. Titles and abstracts of initially identified publications were screened. Clinical trials, reviews, non-English articles, resin-dentine interface studies, hybrid layer studies, hybrid scaffolds studies, and irrelevant studies were excluded. The remaining papers were retrieved with full texts. Manual screening was conducted on the bibliographies of remaining papers to identify relevant articles. A total of 716 studies were found, and 690 were excluded after initial screening. Two articles were identified from the bibliographies of the remaining papers. After retrieving the full text, 23 were included in this systematic review. Sixteen studies used analogues to mimic the functions of non-collagenous proteins in biomineralization of dentine, and four studies used bioactive materials to induce apatite formation on demineralized dentine surface. One study used zinc as a bioactive element, one study used polydopamine, and another study constructed an agarose hydrogel system for biomimetic mineralization of dentine. Many studies reported success in biomimetic mineralization of dentine, including the use of non-collagenous protein analogues, bioactive materials, or elements and agarose hydrogel system. Full article

(This article belongs to the Special Issue Biomaterials for Tissue Engineering)

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