GB2512072A - Composition and method of implantable devices for localized delivery of bioactive compounds - Google Patents

Abstract

A composite device for bone comprises a biocompatible microstructured implantable device (ID); a resorbable photopolymerizable biocompatible matrix (BM) loaded on or in the implantable device; and bioactive components (BC) loaded in the biocompatible matrix. Preferably the implantable device is rough with an irregular profile and is porous. The biocompatible matrix may include a photoinitiator which is cytocompatible and is activated by visible or ultraviolet light. The bioactive components may be drugs, peptides, nucleic acids , viral particles or nanoparticles. Also claimed is a pharmaceutical composition wherein the device further comprises pharmaceutically acceptable excipients.

Description

Composition and method of implantable devices for localized delivery of bioactive compounds

BACKGROUND

The integration of implanted devices in bone depends on several factors, such as the capability of the biomaterial to support and promote bone formation around and inside, if possible, the implanted device. Implanted biomaterials in bone can be used to treat a vast array of pathological situations, such as localized bone defects (LBDs) or to support prosthetic devices. LBDs are an important and diffuse health problem. They can be the result of several pathological processes such as malformation, trauma, surgery, atrophy and their therapy usually requires the use of scaffolds that bridge the defect, allowing cells from the neighboring tissues to migrate and fill the gap and create new tissue. Moreover, dental and orthopedic implants are a very common treatment choice to replace missing teeth or bone segments, and they require bone to heal around the implant to be retained and function properly. Although commercially available biomaterials for bone implantation such as calcium phosphate-based bone substitutes or titanium and zirconia for endosseous implants are well tolerated, they still lack the capability to actively stimulate and control bone formation, and the use of available bioactive factors such as Bone Morphogenetic Proteins or Platelet Derived Growth Factor, in the routine practice in both the dental and orthopedic field is still very limited, in part also because of the difficulty to correctly and easily deliver them to the wound site to treat.

The present invention generally relates to the field of hard tissue healing and, more particularly, to the field of microstructured implantable devices loaded with photopolymerizable hydrogels to stimulate the natural process of hard tissue regeneration and bone wound healing.

DETAILED DESCRIPTION OF THE INVENTION

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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The present invention is concerned with the therapeutic use (human and veterinary) of a device aiming at locally delivering bioactive compounds or particles, such device consisting of: a) a biocompatible microstructured implantable device, herein labelled as ID; b) a resorbable photopolymerizable biocompatible matrix loaded on or in ID, herein labelled as BM; c) bioactive components loaded in BM, herein labelled as BC.

Such device can be used in the context of a bone defect or to treat any condition that requires implantation of medical devices in bone, such as tooth loss or orthopedic prosthesis.

Bone defects are a medical condition. In this application "defects" include damage or injury to bone in which a certain amount of bone is lacking, missing, cracked, broken or chipped, due to traumatic injury, disease or atrophy.

Such defects can be localized in the oral and maxillofacial district or in the peripheral skeleton. In the oral district, such defects may be localized in the alveolar bone and be confined to bone tissue or involve teeth. Alveolar bone defects can be a consequence of periodontal disease, atrophy after tooth loss, neoplastic disease, surgery, trauma. Bone defects in the skeleton as a consequence of surgery, trauma, several diseases such as multiple myeloma, arthritis can also involve joints and result in osteochondral defects, because cartilage is damaged as well.

Bone defects can also include fractures. Bone fractures can be classified as closed or open and simple or multi-fragmentary fractures. In closed fractures the skin remains intact, whilst in an open fracture the bone may be exposed through the wound site. Simple fractures occur along a single line, tending to divide the bone in two. Multi-fragmentary fractures spilt the bone into multiple pieces. As used herein, a wound" is any disruption, from whatever cause, of normal anatomy (internal and/or external anatomy) including but not limited to traumatic injuries such as mechanical (i.e. contusion, penetrating), thermal, chemical, electrical, concussive and incisional injuries; elective injuries such as operative surgery and resultant incisional hernias, fistulas, etc.; acute wounds, chronic wounds, infected wounds, and sterile wounds, as well as wounds associated with disease states (i.e. ulcers caused by diabetic neuropathy or ulcers of the gastrointestinal or genitourinary tract). A wound is dynamic and the process of healing is a continuum requiring a series of integrated and interrelated cellular processes that begin at the time of wounding and proceed beyond initial wound closure through arrival at a stable scar.

In most subjects fracture healing occurs naturally and is initiated following injury. Bleeding normally leads to clotting and attraction of white blood cells and fibroblasts, followed by production of collagen fibres. This is followed by bone matrix (calcium hydroxyapatite) deposition (mineralisation) transforming the collagen matrix into bone. Immature re-generated bone is typically weaker than mature bone and over time the immature bone undergoes a process of remodeling to produce mature "lamellar" bone. The complete bone healing process takes considerable time, typically many months. However, most bone defects, especially in the oral and maxillofacial district, although healed, may present with a different anatomy from the pristine morphology.

Bone defects also include pathological porosity, such as that exhibited by subjects with osteoporosis.

Bone defects may benefit from treatment using the described invention to improve not only healing time but to ameliorate the formation of adequate tissue to acquire a tissue morphology similar to the pristine one, which may facilitate subsequent therapy, rehabilitation, and function.

The subject to be treated may be any animal or human. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including cells from any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle, horse, non-human primate). The subject may be male or female. The subject may be a patient.

The microstrutured ID device is composed of a biocompatible material, or biomaterials.

Biomaterials are non-toxic materials which are compatible, i.e. they can be used in a tissue without eliciting adverse responses such as inflammation, immune responses.

Biomaterials should also possess a functional compatibility, that is fit in the tissue mechanical or physiological function. An example of mechanical compatibility of a biomaterial is titanium used for implantable devices in load-bearing bones.

The term "Microstructured device" as used in the present application refers to devices possessing a surface that presents with topographic features in the micron range. The terms "topographic features" or "topography", as used herein, refer to the characteristics of a surface shape. Topographic features can be the presence of a pre-determined texture, roughness, the presence of irregularities or the presence of pores.

Several studies have shown that osteoblastic cells can sense the presence of surface features and respond accordingly (Fasseri, Cacchioli et al. ; Galli, Guizzardi et al. 2005). It has been repeatedly shown that bone cells growing on microrough titanium surfaces express higher levels of differentiation markers, and rough devices in vivo induce a better tissue integration, also known as osseointegration (Le Guehennec, Soueidan et al. 2007; Wennerberg and Albrektsson 2010). The degree of roughness and the topographic features of a biomaterial are commonly measured using profilometers and are expressed by 2D and 3D parameters. One of the most common and best known 2D parameters is average roughness Ra. This parameter is used to indicate the average distance between the highest points, or peaks, and the lowest points, or valleys, of the topographic features.

Most implant surfaces have Ra between 1 and 10 1dm, although a growing body of literature and current consensus indicate that an average surface roughness between 1 and 3 pm is associated to best in vitro and in vivo success outcomes for bone implants.

Several methods and techniques are routinely used in the experimental and industrial practice to create surface features on a biomaterial or a device such as a dental or orthopedic implant, such as plasma spray, grit blasting, acid etching, syntherization, laser treatment, milling, lithographic techniques. These are known to those skilled in the art, and can be considered within the scope of the present invention.

In a preferred embodiment of the invention, the microstructured ID is porous. A porous ID device is preferably permeable to nutrients and growth factors required for bone growth.

During wound healing oxygen and nutrients supply depends exclusively on blood vessels in the tissues surrounding the defect until new vessels have been formed in the newly formed tissue. Therefore the survival of cells in the center of the defect is limited by how easily oxygen and nutrients can permeate the defect and the biomaterials that have been placed in it. A porous structure makes it easier for oxygen and nutrients to diffuse and are also more easily resorbed because a greater surface is exposed to bone cells, such as osteoblasts and osteoclasts. Porous scaffolding materials, according to some embodiments, can comprise pores having diameters ranging from about 1 pm to about 1 mm. In one embodiment, a scaffolding material comprises macropores having diameters ranging from about 100 pm to about 1 mm. In another embodiment, a scaffolding material comprises mesopores having diameters ranging from about 10 pm to about 100 pm. In a further embodiment, a scaffolding material comprises micropores having diameters less than about 10 pm. Embodiments of the present invention contemplate scaffolding materials comprising macropores, mesopores, and micropores or any combination thereof.

The microstructured ID device can have any shape and dimension, according to bone substitutes and implantable devices commonly used in the dental or orthopedic practice.

It can be in the form of a single device or blocks or granules, or a powder, irrespective of the granule size, with a minimum of at least 1 micron.

The porous ID device can be porous by nature, if naturally derived, or porosity may be induced during or after manufacture, using commercially available techniques known to those skilled in the art.

Suitable biomaterials for the microstructured ID can be provided by granules or blocks of biocompatible materials such as but not limited to autologous or homologous bone, natural apatite, hydroxyapatite, fluorapatite, coralline apatite, natural or synthetic calcium carbonate, calcium sulfate, or a combination thereof. Calcium phosphate-based ceramics comprise a vast range of compounds containing calcium, phosphate, and possibly other inorganic elements, such as but not limited to magnesium or strontium. The chemical composition can affect the crystal structure and affect the material behavior in vivo.

Calcium phosphate ceramics can be of synthetic or natural origin, if obtain by processing natural tissues or organisms. In this latter case they are often referred to as "natural apatite". Non-limiting examples of calcium phosphates suitable for use as bone scaffolding materials comprise amorphous calcium phosphate, monocalcium phosphate monohyd rate (MCPM), monocalcium phosphate anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), octacalcium phosphate (OCP), a-tricalcium phosphate, -tricalcium phosphate, hydroxyapatite (OHAp), poorly crystalline hyd roxapatite, tetracalcium phosphate (TTCP), heptacalcium decaphosphate, calcium metaphosphate, calcium pyrophosphate dihyd rate, calcium pyrophosphate, carbonated calcium phosphate, or mixtures thereof.

Other non-limiting examples of biomaterials for ID are calcium sulfate, calcium carbonate, bio-glasses, poly lactic acid, polyglycolic acid or titanium. An example of commercially available natural apatite material of bovine origin is BioOss. An example of commercially available coralline apatite is Algipore®.

The microstructured ID device can also be constituted by polymeric materials.

Suitable polymer materials include, but are not limited to, biodegradable/bioresorbable polymers which may be chosen from the group of: polycaprolactone, poly(DL-lactide-co-caprolactone), polyp lactide-co-caprolactone-co-glycolide), polyglycolide, polylactide, polyhydroxyalcanoates, co-polymers thereof. Non-limiting examples of non-biodegradable polymers are cellulose acetate, cellulose butyrate, alginate, agarose, polysulfone, polyurethane, polyacrylonitrile, su Ifonated polysulfone, polyamide, polyacrylonitrile, polymethylmethacrylate or co-polymers thereof.

In one preferred embodiment of the invention ID comprises, consists of or essentially consists of an implantable device for dental or orthopedic use, such as a titanium or zirconia endosseous implant, herein simply referred to as "endosseous implant".

Titanium and zirconia implants are common devices used to support different kind of prosthetic devices, such as dental prosthesis. They are generally constituted by a cylindrical or conical (root-form") threaded device, which can be inserted and retained in bone tissue after healing. Depending on the manufacturing techniques used endosseous implants can have a microstructured, roughened or porous structure (Le Guehennec, Soueidan et al. 2007). An example of commercially available rough implants are SLA implants by Straumann AG. Examples of commercially available porous endosseous implants are titanium-tantalium Trabecular Metal implants or syntherized titanium Tixos® implants.

In one embodiment of the invention ID can be processed by CAD/CAM techniques, according to commonly used techniques in the art, to obtain the desired shape to match a tissue defect or to perform a certain therapy.

In one embodiment of the invention the polymeric microstructured scaffold can be formed using computer-controlled prototyping machinery, according to commonly used techniques in the art.

The invention further comprises a photopolymerizable biocompatible matrix (BM).

Several biomaterials can be utilized as BM, such as polyethylene glycole derivatives, polyvinyl alcohol derivatives, dextran derivatives, chitosan derivatives, hyalu ronic acid derivatives, collagen derivatives, alginate derivatives, polyurethane derivatives, or a combination thereof. Derivatives of further compounds can be used, such as but not limited to derivatives of polylactic acid (PLA); polyglycolid acid (PGA); copolymers of lactic acid and g lycolic acid (PLGA); polycaprolactone; polyphosphoester; polyorthoester; poly(hydroxy butyrate); poly(diaxanone); poly(hydroxy valerate); poly(hyd roxy butyrate-co-valerate); poly(g lycolide-co-trimethylene carbonate); polyanhydrides; polyphosphoester; poly(ester-amide); polyphosphoeser; polyphosphazene; poly(phosphoester-urethane); poly(amino acids); polycyanoacrylates; derivatives of biopolymeric molecules such as fibrin; fibrinogen; cellulose; starch; or a mixture or a copolymer thereof.

In a preferred embodiment, BM comprises, consists of or essentially consists of a hydrogel composed of polethyleng lycole diacrylate.

The term "photopolymerizable" indicates that is the application of light, either in the visible or UV spectrum, turns the material from a sol phase into solid phase.

Some types of matrices can be photopolymerized in vivo and in vitro in the presence of photoinitiators using visible or ultraviolet (UV) light. Non limiting examples of water-soluble photopolymerizable polymers are PEG acrylate derivatives, PEG methacrylate derivatives, polyvinyl alcohol (PVA) derivatives, modified polysaccharides such as but not limited to hyaluronic acid derivatives and dextran methacrylate and a photoinitiator, as described by K.T. Nguyen and J.L. West (Nguyen and West 2002).

Photopolymerizable materials can also be co-polymers, that is they are formed by a mixture of different monomers.

Visible or UV light can interact with light-sensitive compounds called photoinitiators to create free radicals that can initiate polymerization to form crosslinked hydrogels.

Non-limiting example photoinitiators include 1 -[4-(2-hyd roxyethoxy)-phenyl]-2-hydroxy-2-methyl-i-propane-i-one, acetophenone, benzophenone, and the benzoin ethers. Other non-limiting examples of photoinitiators are 2,2-dimethyoxy-2-phenylacetophenone, benzoin methyl ether, as described by H. Singer in Patent U54620954. An example of commercially available UV photoinitiator is lrgacureTM 2959. Non-limiting examples of visible light photoinitiators are eosin Y, or triethanolamine. N-vinyl pyrrolidinone (NVP) can be used as a monomer and as an accelerator for photocrosslinking. Other non-limiting example accelerators include N1N dimethyl toluidine or tetramethyl-ethylenediamine.

Preferably, the photoinitiators and accelerators are cytocompatible.

BM contains bioactive components (BC). BC include cytokines such as IL-i, IL-2, IL-6, IL-7, IL-B, IL-b, IL-12, IL-i3, IL, i6, IL-i7, IL-23, IL-34, IL-35, Tumor necrosis factor (TNF), Interferon, chemokines, such as, but not limited to CCL.1, CCL2, CCL3, CCL5, CCL6, CCL7, CCLB, CCL9,CCL11, CCL12, CCL13, CCL14, CCL1, CCL16, CCL17, CCL18, CCL19, CCL2O, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, Osteoprotegerin, RANKL, Sclerostin, DKK, sFRP, immunoglobulins, growth factors such as but not limited to Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMP5), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF), Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor (TGF), Vascular endothelial growth factor (VEGE), Wnt family growth factors, Placental growth factor (PIGE), hormones, such as but not limited to Parathyroid hormone (PTH), Growth Hormone (GH), Insulin, Calcitriol, Estrogen, Progesterone, Prolactin, Testosterone.

In an embodiment of the invention, the hydrogel matrix further comprises extracellular matrix components, such as Collagen, Laminin, Elastin, Fibronectin, Vitronectin, F-spondin, Periostin, Trombospondin, proteoglycans such as Heparan Sulfate, Chondroitin Sulfate, Cheratan Sulfate.

The peptides useful in the present invention can be natural or recombinant. The natural peptides useful in the present invention can be obtained for example from human tissues as described by U.S. Pat. N. 4388234, U.S. Pat. N. 20060241016, U.S. Pat. N. 7547761 or from other mammalian sources as easily recognized by those skilled in the art.

The recombinant peptides useful in the present invention can be prepared by standard recombinant technology using both prokaryotic and eukaryotic expression systems in a conventional manner. In this respect, a reference is made for instance to E.U. Patent N. 0 433 225 Al, U.S. Patent N. 7749731, U.S. Patent N. 6242219, U.S. Patent N. 7951559.

In an embodiment of the invention, BC are nucleic acids, selected from the group consisting of plasmids, shRNA or siRNA. Plasmids are defined as small circular, double-stranded DNA molecules, usually found in bacteria. Plasmid size can vary considerably, from a few kbp to several hundreds kbp and contain one or more genes of interest, including regulatory sequences such as promoters. "Promoter" refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA.

In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native sequence, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a nucleotide sequence in different tissues or cell types and/or at different stages of development and/or in response to different environmental or physiological conditions.

Promoters that cause a nucleotide sequence to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a nucleotide sequence to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a nucleotide sequence to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a nucleotide sequence to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that, because in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleotide sequences of different lengths may have identical promoter activity.

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.

Physiologically, double stranded RNAs (dsRNAs), such as those found in viruses, initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves dsRNAs to produce double-stranded fragments of 20-25 base pairs with a 2-nucleotide overhang at the 3' end. These short double-stranded fragments are called small interfering RNAs (siRNAs) (Gavrilov and Saltzman 2012; Nimesh 2012). These siRNAs are then separated into single strands and integrated into an active RISC complex. After integration into the RISC, siRNAs base-pair to their target mRNA and induce cleavage of the mRNA, thereby preventing it from being used as a translation template and functionally silence the genes these mRNA were transcribed from. Introducing a custom made 5iRNA is a well known method to those skilled in the art to silence a gene of interest in a cell by targeting a known mRNA. An alternative approach to obtain a prolonged silencing of a gene of interest is by short hairpin RNA (5hRNA) (Lambeth and Smith 2013). A shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. At first, polymerase Ill promoters such as U6 and Hi were used; however, these promoters lack spatial and temporal control. As such, there has been a shift to using polymerase II promoters to regulate expression of shRNA.

shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. Once the vector has been introduced into a cell and has integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase Ill, depending on the promoter choice. This product is processed by the enzyme Drosha. The resulting pre-shRNA is exported from the nucleus by Exportin 5. This product is then processed by Dicer and loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand is degraded. The antisense (guide) strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarity, RISC cleaves the mRNA. In the case of imperfect complementarity, RISC represses translation of the mRNA. In both of these cases, the shRNA leads to target gene silencing.

BC such as the above mentioned examples can be mixed to the BM monomer or can be chemically bound to it. If BC is chemically bound, the bond can be covalent. Covalent bonds between the bioactive compounds and BM can vary according to the chemical nature of the bioactive compounds and the presence of functional groups on the hydrogel monomer or polymer.

In one preferred embodiment BM is composed of polyethylenglycole diacrylate (FEGDA).

The presence of acrylic groups can create bonds between the monomers, but can also be used for Michael's addition-type reaction and bind certain functional groups such as aminic or thiol groups present on several bioactive compounds, especially if these are proteins.

The Michael reaction or Michael addition is the nucleophilic addition of a carbanion or another nucleophile to an a,-unsaturated carbonyl compound. It belongs to the larger class of conjugate additions.

Addition of a protein to PEGDA results in the spontaneous formation of covalent bonds between the two compounds, so that the bioactive compound is retained in the hydrogel structure.

A crosslinker can be utilized to bind BC to BM. Crosslinking is the process of chemically joining two or more molecules by a covalent bond. The term "crosslinker" (or crosslinking reagent) as used herein refers to is used in the present text to indicate molecules that contain two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules, in this case BC and BM of the present invention. The chemical nature of the crosslinker depends on the functional groups present on the components. Crosslinking is conducted using techniques known to those skilled in the art, which depend on the nature of the present functional groups. A non-limiting example of crosslinking between a thiol-functionalized BM and a amine-containing BC can be performed as follows: BC is dissolved in Phosphate buffered saline (pH 7.2) at 0.1 mM. The crosslinker, such as but not limited to Succinimidyl-([N-maleimidopropionamido]-ethylenglycoQester, is added to the dissolved BC at 1 mM final concentration. The reaction mixture is incubated for 30 minutes at room temperature or 2 hours at 4°C. The excess crosslinker is removed using a desalting column equilibrated with Phosphate buffered saline. The thiol-BM and desalted BC-crosslinker are combined and mixed in a 1:1 molar ratio or in a molar ratio corresponding to that desired for the final conjugate and consistent with the relative number of sulfhydryl and activated amines that exist on the two molecules. The reaction mixture is incubated at room temperature for 30 minutes or 2 hours at 4°C.

Useful crosslinker reagents can also be obtained via commercial sources, such as Molecular Biosciences Inc. (Boulder, CD, USA), or Thermo Pierce (Rockford, IL, USA) synthesized in accordance with procedures described in Toki et al (Toki, Cerveny et al. 2002); U.S. Pat. N. 6214345; WO 02/088172; U.S. Pat. N. 2003130189; U.S. Pat. N. 2003096743; WO 03/026577; WO 03/043583; and WO 04/032828.

In one embodiment of the invention BM contains a thiol group or is functionalized with a thiol group, or a thiol group is created by reducing a disulfide bond, such as thiolated hyaluronic acid. A crosslinker carrying a functional group or groups capable to bind thiol groups such as but not limited to maleimide or acrylate groups may be used. Non-limiting examples of crosslinkers for thiol groups are Bismaleimidoethane, Bismaleimidobutane, Bismaleimidohexane, Tris(2-maleimidoethyl)amine, N-alpha-Maleimidoacet- oxysuccinimide ester, N-beta-Maleimidopropyl-oxysuccinimide ester, N-gamma-Maleimidobutyryl-oxysuccinimide ester, m-Maleimidobenzoyl-N-hydroxysuccinimide ester, Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, N-epsilon-Malemidocaproyl-oxysuccinimide ester, Succinimidyl 4-(p-maleimidophenybutyrate, Succinimidyl 6-[(beta-maleimidopropionamido)hexanoate], Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate), N-kappa-Maleimidoundecanoyl-oxysu Ifosuccini mide ester can be used.

In one preferred embodiment BM contains and aminic group or is functionalized with an aminic group. A crosslinker carrying a functional group or groups capable to bind aminic groups such as but not limited to acrylate, epoxy groups or Succinimidyl ester groups may be used. Non-limiting examples of crosslinkers for aminic groups are Dimethyl adipimidate-2HCI, Dimethyl pimelimidate-2HCI, Dimethyl suberimidate-2HCI, Dimethyl 3,3-dithiobispropionimidate-2HCI, 1,5-Difluoro-2,4-dinitrobenzene, Bis(succinimidyl) penta(ethylene glycol), Bis(sulfosuccinimidyl) suberate, Bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone, Disuccinimidyl glutarate, Dithiobis(succinimidylpropionate), Disuccinimidyl suberate, Disuccinimidyl tartrate, 3,3'-Dithiobis(sulfosuccinimidylpropionate), Ethylene glycol bis(succinimidylsuccinate), Ethylene glycol bis(sulfosuccinimidylsuccinate), Tris-succinimidyl aminotriacetate, DicyclohexylcarbodUmide, 1 -Ethyl-3-(3-dimethylaminopropycarbodUmide hydrochloride, N-Hydroxysuccinimide, Sulfosuccinimidyl (4-iodoacetyaminobenzoate, Succinimidyl (4-iodoacetyaminobenzoate, Succinimidyl 3-(bromoacetamido)propionate, Succinimidyl iodoacetate, 2-Pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide, 2- Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide, Su Ifosuccinimidyl 6-[3'-(2-pyridyldithio)propionamido]hexanoate, Succinimidyl 6-[3(2-pyridyldithio) propionamido] hexanoate, Succinimidyl 3-(2-pyridyldithio)propionate, 4-Succini midyloxycarbonyl-alpha- methyl-alpha(2-pyridyldithio)toluene, Sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate, Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate, Succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxy-(6-amidocaproate), N-epsilon-Malemidocaproyl- oxysuccinimide ester, N-gamma-Maleimidobutyryl-oxysulfosuccinimide ester, N-gamma- Maleimidobutyryl-oxysuccinimide ester, N-kappa-Maleimidoundecanoyl-oxysulfosuccinimide ester, m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, Succinimidyl 4-(p-maleimidophenybutyrate, N-alpha-Maleimidoacet-oxysuccinimide ester, N-beta-Maleimidopropyl-oxysuccinimide ester, Succinimidyl 6-[(beta-maleimidopropionamido)hexanoate].

In on embodiment of the invention, crosslinkers can comprise, consist of or essentially consist of a peptide, thus comprising one or more amino acid units. Peptide linker reagents may be prepared by solid phase or liquid phase synthesis methods (Bodanszky 1984) that are well known in the field of peptide chemistry, including t-BOC chemistry (Geiser 1988) and Fmoc/HBTU chemistry (Fields and Noble 1990), on an automated synthesizer such as the Rainin Symphony Peptide Synthesizer (Protein Technologies, Inc., Tucson, Ariz.), or Model 433 (Applied Biosystems, Foster City, CA, US).

Crosslinkers can include enzyme cleavable sequences for the release of the therapeutic agent. Enzyme cleavable sequences comprise amino acid sequences recognized and cleaved by membrane bound and/or cell-secreted peptidases, which are peptide-cleaving enzymes well known in the art to recognize particular amino acid sequences and to cleave said sequences between specific amino acids. Such enzymes include, for example and without limitation, matrix metalloproteinases or "MMP's" (also referred to herein as matrixins), e.g., MMP-2, MMP-9, MMP-14, serine proteases, cysteine proteases, elastase, stromelysins, human collagenases, cathepsins, granzymes, dipeptidyl peptidases, plasmins, plasminogen activators, lysozymes and e.g., aminopeptidase P, aminopeptidase A, and aminopeptidase N. Peptides with suitable MMP substrate selectivity include, for example and without limitation, those having the amino acid sequences reported in U.S. Pat. N. 6844318 or (Hatakeyama, Akita et al. ; Dettin, Muncan et al. 2011; Fonseca, Bidarra et al. 2011; Jang, Kim et al. 2011; van Duijnhoven, Robillard et al. 2011).

Crosslinkers can further comprise acid labile bonds, photolabile bonds, peptidase labile bonds and a combination thereof.

In one embodiment of the invention the crossinker may be a dendritic type linker for covalent attachment of more than one drug moiety through a branching, multifunctional linker moiety to BM (King, Dubowchik et al. 2002; Sun, Wirsching et al. 2002; Sun, Wirsching et al. 2003; Paleos, Isiourvas et al. 2007). Dendritic linkers can increase the molar ratio of BC to BM.

In one embodiment BC comprises, consists of or essentially consists of viral particles. Viral particles that can be used include but are not limited to retrovirus, lentivirus, adeno- associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirus vectors, as well as any combination thereof.

Such viruses can be engineered using the techniques known to those skilled in the art and can carry vectors that include useful genes for tissue regeneration. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions (e.g., promoters, enhancers, termination sequences, etc.), and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

In one embodiment BC comprises, consists of or essentially consists of nanoparticles, that is particles smaller than 1 micron in size. The term "nanoparticle" includes a wide range of structures and constructs such as polymeric micelles, dendrimers, polymeric, metal and ceramic nanoparticles, protein cage architectures, viral-derived capsid nanoparticles, DNA nanoparticles, polyplexes, solid lipid particles and liposomes (Kesharwani, Gajbhiye et al. 2012).

In one embodiment BC comprises, consists of or essentially consists of liposomes. As used herein, the term liposome" refers to a self-assembling structure comprising one or more lipid bilayers, each of which comprises two monolayers containing oppositely oriented amphipathic lipid molecules. Amphipathic lipids comprise a polar (hydrophilic) headgroup covalently linked to one or two or more non-polar (hydrophobic) acyl or alkyl chains. Energetically unfavorable contacts between the hydrophobic acyl chains and a surrounding aqueous medium induce amphipathic lipid molecules to arrange themselves such that polar headgroups are oriented towards the bilayer's surface and acyl chains are oriented towards the interior of the bilayer, effectively shielding the acyl chains from contact with the aqueous environment.

Liposomes useful in connection with the methods and compositions described herein can have a single lipid bilayer (unilamellar liposomes) or multiple lipid bilayers (multilamellar liposomes) surrounding or encapsulating an aqueous compartment. Various types of liposomes are described, e.g., in (Maurer, Fenske et al. 2001; Heneweer, Gendy et al. 2012; Parhi and Suresh 2012; Urban, Valle-Delgado et al. 2012).

Amphipathic lipids typically comprise the primary structural element of liposomal lipid vesicles. Hydrophilic characteristics of lipids derive from the presence of phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, and other like polar groups. Hydrophobicity can be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, which may be substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s). Non-limiting examples of preferred amphipathic compounds are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phoasphatidylglycerol, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DRAC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine (DSFC), dilinoleoylphosphatidylcholine and egg sphingomyelin.

Other lipids such as sphingolipids and glycosphingolipids, are also useful in methods and compositions provided herein. Additionally, the amphipathic lipids described above may be mixed with other lipids, such as triacylglycerols and sterols.

The nanoparticles used in the present invention may be formed with any technologies among those known to those skilled in the art to which they pertain. Nanoparticles can be functionalized with peptides, proteins, nucleic acids or other biologically active compounds.

BM can further comprise extracellular matrix (ECM) components or fragments thereof in addition to BCs. ECM is mainly composed of proteins, proteoglycan and, in the case of bone, mineral hydroxyapatite crystals (Gillies and Lieber 2011; Lockhart, Wirrig et al. 2011; Shekaran and Garcia 2011). ECM components that can be included in the present invention are selected from the groups including molecules such as but not limited to Collagen family, Laminin, Elastin, Fibronectin, Vitronectin, F-spondin, Fibrillin, Osteocalcin, proteoglycans such as Heparan Sulfate, Chondroitin Sulfate, Cheratan Sulfate, hydroxyapatite crystals.

Further ECM components that can be included in the present invention are matricellular proteins. Matricellular proteins are dynamically expressed non-structural protein that are present in the ECM (Bornstein and Sage 2002), and include thrombospondin-1 (ISP1), SPARC (secreted protein, acidic and rich in cysteine; also known as osteonectin), tenascin-C, TSP2, osteopontin (OFN), Bone Sialoprotein (BSP) , Periostin, the CON (cyr-61, OTGF [connective tissue growth factor], Nov) family of proteins and tenascin X. BM can also be enriched with adhesion peptides. The term "(cell) adhesion"/"cell adhesiveness" is used to describe the way in which cells adhere to another or to a substrate, as a result of the binding of complementary membrane surface molecules. The terms "(cell) adhesion" and "adherence" are often used synonymously. Among the EOM factors which promote cell/ECM adhesion a particular role is played by fibronectins. In its native form, the fibronectin molecule is a dimer composed of identical sub-units with a relative molecular mass M.sub.r of 220 KD in each case, which are linked together via disulphide bridges. One feature that is particularly pronounced in fibronectin, as also on other proteinogenic ECM components, is its composition of short repeating amino acid sequence units. Several of these sequences, which are homologous--though not identical--to one another, for their part form clearly delimited domains in the individual fibronectin chains, and these domains facilitate binding to fibrin, collagen or heparin. Only one of these domains possesses the ability to bind to cells. The section of the sequence responsible for the ability of this fibronectin domain to adhere to cells was identified and sequenced by Pierschbacher et al. (Proc. NatI. Acad. Sci. USA 80 (1983)1224-1227). This section contains the tetrapeptide "RGDS" (Arg-Gly-Asp-Ser). This recognition sequence was characterised by Pierschbacher and Ruoslahti (Nature 309 (1984) 30-33), by testing smaller and smaller synthetic peptides of the cell recognition domains with regard to their ability to promote cells' adhesion capacity. The RGD sequence (Arg-Gly-Asp) is essential in promoting the binding activity, whereas the amino acid serine in position 4 can also be replaced by other amino acids (Ruoslahti and Pierschbacher 1987). The RGDX sequence has been found not only in the fibronectin molecule, but also in numerous other matrix proteins, the X standing for a variable, though not optional, amino acid (Clements, Newham et al. 1994). Further adhesion sequences have been discovered, as described by Estephan (Estephan, Dao et al. 2012) and in patents W01996006114 A, US Patent N. 20120114617, US Patent N. 5880092. The term "adhesion peptide" or "peptide" is used in the context of the present invention for amino acid sequences in general with at least three amino acids linked together via the ordinary peptide bonds (amide bonds). The chain length of these amino acid chains can vary, with preference being given to peptides (or oligopeptides or polypeptides) extended at the amino terminus (i.e. at Aa3) having up to 30, preferably 20, amino acids. The adhesion peptides of the invention are preferably present in linear form, though branched amino acid chains are also conceivable without there being any inevitable reduction in the influencing effect on cell/cell adhesion as a result.

The inventive composition desirably is formulated to be acceptable for pharmaceutical use, such as, for example, administration to a human host in need thereof. As generally used herein the terms "acceptable for phramaceutical use" or "pharmaceutically acceptable" pertain to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. To this purpose, the ID, BM and BC components of the invention can be formulated into a composition including a physiologically acceptable carrier (e.g., excipient or diluent). Physiologically acceptable carriers are well known and are readily available, and include buffering agents, anti-oxidants, bacteriostats, salts, and solutes that render the formulation isotonic with the blood or other bodily fluid or tissue of the human patient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, co-solvents, detergents, solubilizers, thickening agents, stabilizers (e.g., surfactants), and preservatives. The choice of carrier will be determined, at least in part, by the location of the target tissue, and the particular method used to administer the composition. Examples of suitable carriers and excipients for use in drug conjugate formulations are disclosed in, for example, International (PCT) Patent Application Nos. WO 00/02587, WO 02/060955, and WO 02/092127, and (Ghetie, Ghetie et al. 1988).

Where necessary, the composition may be sterilized with any suitable sterilization method, such membrane filtration.

Any suitable pharmaceutically acceptable suspending agent may be used in connection with the inventive composition. Non-limiting examples of preferred suspending agents include sodium carboxymethyl cellulose, xanthan gum, microcrystalline cellulose, carragenan, veegum, tragacanth, bentonite, methylcellulose, and polyethylene glycols.

Any suitable pharmaceutically acceptable preservative may be used in connection with the inventive composition. Non-limiting examples of preferred preservative include phenol, benzyl alcohol, phenylethyl alcohol, chlorhexidine, benzalkonium chloride, benzethonium chloride, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, butyl p-hydroxybenzoate, propyl p-hydroxybenzoate, ethanol, chlorobutanol, thimerosal, sodium dehydroacetate and myristyl-gamma-picolinium chloride, sodium benzoate, potassium benzoate, potassium sorbate.

Any suitable pharmaceutically acceptable buffering agent may be used in connection with the inventive composition. Non-limiting examples of particularly preferred buffering agents include citrate, acetate, succinate, phosphate, histidine, sodium hydrogenphosphate, potassium dihydrogenphosphate, dipotassium phosphate, anhydrous sodium dihydrogenphosphate, crystalline sodium dihydrogenphosphate, boric acid, borax, sodium acetate, citric acid, citric anhydride, sodium citrate, sodium glutamate and creatinine.

The buffering agent may be present in the inventive composition in any suitable concentration, so long as sufficient stability of the composition is achieved under the desired conditions. The buffering agent typically is present in the inventive composition such that the pH is maintained within a desired range, which is established according to the specific composition of the conjugate compound of the invention.

In addition to the buffering agent discussed above, the inventive composition also optionally contains a surfactant. Any suitable surfactant can be used in connection with the invention. Suitable surfactants are well known to those skilled in the art. Although compositions formulated with surfactants are preferred, compositions formulated without surfactants are also within the scope of the invention.

As an additional stabilizing agent, sodium chloride also is added to the inventive composition. In this regard, the inventive composition comprises a suitable amount, preferably a tonicifying amount, of sodium chloride (NaCI). By the phrase a "tonicifying amount of sodium chloride," it is meant that the concentration of NaCI in the composition is such that the tonicity of the composition is the same as the tonicity of the human tissue the compound is administered to (i.e., isotonic). In this regard, the NaCI can be present in the inventive composition in any suitable concentration, so long as sufficient tonicity and stability is achieved in the inventive composition.

Compositions containing peptides are rendered unstable by oxidation. Thus, in another embodiment of the invention, the composition further comprises an antioxidant. Any suitable antioxidant can be used in the inventive composition. Suitable antioxidants are known in the art and include, for example, superoxide dismutase, glutathione peroxidase, tocotrienols, polyphenols, zinc, manganese, selenium, vitamin C, vitamin E, beta carotene, cysteine, and methionine. The antioxidant can be present in the composition in any suitable concentration.

In addition to antioxidants, the inventive composition can further be stabilized by the addition of sucrose. The use of sucrose to stabilize peptide formulations is known to those of skill in the art. Any suitable amount of sucrose can be used in the inventive composition.

Preferred embodiments of this invention are described herein. Of course, variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

In one preferred embodiment of the invention, BC are added, combined or linked to BM, a process that generates a product herein labelled as (BM+BC), before it is added to ID and ID and (BM+BC) are put together before their insertion into a patient's body and before BM polymerization. ID can be combined to (BM+BC) by the final clinical user just before their clinical use, an operative modality often referred to as "chairside".

Thus, the operative sequence is: BM is enriched with BC using any of the methods that have been described in the present application and it is provided to the final clinical user in this (BM+BC) state. The final clinical user adds (BM+BC) to the proper ID, which is chosen depending on the clinical necessity of the patient. BM is then polymerized by being exposed to an adequate visible light or UV source and the enriched device consisting of ID and (BM+BC) is then clinically inserted into the patient. The terms "adequate light source" or "proper light source", as used herein, refer to a visible or UV light source capable to provide a stimulus of such wavelength and intensity and duration to completely polymerize the photopolymerizable component or components of the invention or to polymerize them to a degree which is deemed clinically acceptable according to the clinical needs of the tissue defect to treat. The ID and (BM+BC) combination can be carried out as it best suits the clinical needs of the patient. In one preferred embodiment of the invention ID is inserted into an excess unpolymerized (BM+BC) mass, which, depending on its nature, can have liquid or viscous properties, it is then retrieved and exposed to a proper light source, so that the remaining (BM+BC) on, and possibly inside, the ID is polymerized and then the whole is inserted into the tissue defect. In an alternative embodiment ID is inserted into an excess unpolymerized (BM+BC) mass contained in a proper transparent or otherwise light-conductive vessel. The whole vessel is then exposed to a light source which polymerizes the whole (BM+BC) mass contained in the vessel and ID contained in it. ID is then retrieved from the excess (BM+BC), retaining some (BM+BC) on its microstructured surface and it is implanted into the tissue defect.

In an alternative embodiment ID is combined to unpolymerized (BM+BC), is inserted into the tissue defect and (BM+BC) is then polymerized inside the tissue, by directly applying an adequate light source.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as described.

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Claims (19)

1. CLAIMSWhat is claimed is: 1. Acampasite device for bane, comprising: a) a biocompatible microstructured implantable device, herein labelled as ID; b) a resorbable photopolymerizable biocompatible matrix loaded an or in ID, herein labelled as BM; c) bioactive components loaded in BM, herein labelled as BC.
2. 2. The device of claim 1 wherein ID is composed of biomaterials selected from the group consisting of titanium, zirconia, a non resorbable bone substitute, monocalcium phosphate monohydrate (MCPM), dicalcium phosphate (DCP), tricalcium phosphate (TCP), amorphous calcium phosphate (ACP), hydroxyapatite (HA), tetracalcium phosphate (tetCF), fluorapatite, calcium carbonate, calcium sulphate, bio-glass, autologous bone, homologous bone, demineralized bone matrix, biodegradable polymers, biodegradable copolymers, non bio-degradable polymers, non biodegradable copolymers or a combination thereof.
3. 3. The device of claim 1, wherein the surface of ID is rough, and presents with an irregular profile.
4. 4.The device of claim 3, wherein average roughness Ra is comprised between 0.1 and 10 km.
5. 5.The device of claim 1, wherein ID is porous and contains macropores, mesopores or microporoes or a combination thereof and at least a portion of the plurality of pores are interconnected and connect the inner surface of the device body and the outer surface of the device body.
6. 6. The device of any of the preceding claims, wherein BM is selected from the group comprising polyethylen g lycole derivatives, polyvinyl alcohol derivatives, dextran derivatives, chitosan derivatives, hyalu ronic acid derivatives, collagen derivatives, alginate derivatives, polyurethane derivatives, polylactic acid (PLA) derivatives, polyglycolid acid (PGA) derivatives, derivatives of copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone derivatives, polyphosphoester derivatives, polyorthoester derivatives, poly(hydroxy butyrate) derivatives, poly(diaxanone) derivatives, poly(hydroxy valerate) derivatives, poly(hydroxy butyrate-co-valerate) derivatives, poly(glycolide-co-trimethylene carbonate) derivatives, polyanhydrides derivatives, polyphosphoester derivatives, poly(ester-amide) derivatives, polyphosphoeser derivatives, polyphosphazene derivatives, poly(phosphoester-urethane) derivatives, poly(amino acids) derivatives, polycyanoacrylates derivatives, derivatives of biopolymeric molecules such as fibrin, fibrinogen, cellulose, starch, or a mixture or a copolymer thereof.
7. 7. The device of claim 1, wherein BM includes a photoinitiator and the photoinitiator is cytocompatible.
8. 8. The device of claim 7, wherein the photoinitiator is activated by visible light or UV light.
9. 9. The device of claim 1, wherein BM further comprises cleavable sequences by natural proteases.
10. 1O.The device of claim 9, wherein the cleavable sequence comprises at least one bis-cysteine matrix metalloproteinase (MMP)-sensitive peptide.
11. 11. The device of claim 1, wherein ID is combined with BM prior to its polymerization.
12. 12.The device of claim 11, wherein the gel matrix is polymerized chair-side prior to insertion.
13. 1 3.The device of claim 1, wherein BC are drugs.
14. 1 4.The device of claim 1, wherein BC are peptides 2. The device of claim 14, wherein the peptides are selected from the group consisting of hormones, cytokines, lymphokines, chemokines, enzymes, transcription factors, second messengers, growth factors, mobilization factors, trophic factors, Zinc Finger Nucleases, or a mixture thereof.3. The device of claim 1, wherein BC are nucleic acids, selected from the group consisting of plasmids, 5iRNA, shRNA or a mixture thereof.4.The device of claims 13 to 16, wherein BC are adsorbed, encapsulated in or conjugated to BM.5.The device of claim 17, wherein conjugation occurs through covalent bond.6.The device of claim 18, wherein conjugation occurs through a crosslinker.7.The device of claim 19, wherein the crosslinker comprises cleavable sequences by natural proteases.8.The device of claim 20, wherein the cleavable sequence comprises at least one matrix metalloproteinase (MMP)-sensitive peptide.9.The device of claim 1, wherein BC are viral particles.10.The device of claim 22, wherein the viral particles are selected from the group: retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirus vectors, as well as any combination thereof.11.The device of claim 23, wherein the viral particles contain the genome of a viral vector carrying genes encoding marker and/or therapeutic genes.12. The device of claim 1, wherein BC are nanoparticles, selected from the group consisting of polymeric micelles, dendrimers, polymeric nanoparticles, metal nanoparticles, ceramic nanoparticles, protein cage architectures, viral-derived capsid nanoparticles, DNA nanoparticles, polyplexes, solid lipid particles and liposomes.13.The device of claim 25, wherein the nanoparticles are functionalized with nucleic acids, proteins or drugs.14.The device of any claims from 22 to 26, wherein BM further comprises drugs or bioactive peptides.
15. 15. The device of any of the preceding claims, wherein BM further comprises extracellular matrix components or fragments thereof.
16. 16. The device of any of the preceding claims, wherein BM further comprises adhesion peptides.
17. 17.A pharmaceutical composition wherein the composition of any of the preceding claims further comprises pharmaceutically acceptable excipients.
18. 18. The pharmaceutical composition of claim 30, wherein the excipients are selected from the group comprising diluents, carriers, suspending agents, buffering agents, isotonicity agents, co-solvents, detergents, solubilizers, thickening agents, stabilizers, preservatives, antioxidants or a mixture thereof.
19. 19.A method for treating a subject having a tissue or organ defect, comprising: implanting the microstructured ID of any of the preceding claims into a subject in need thereof; optionally, determining an appropriate BC according to a diagnosis of the subject; optionally, providing the appropriate BC in a BM and combining ID, BC and BM for clinical use.