WO2009120886A2

WO2009120886A2 - Design and manufacturing of bioactive implanted surgical fixation devices using injection molding of gradient cellular strucures

Info

Publication number: WO2009120886A2
Application number: PCT/US2009/038436
Authority: WO
Inventors: Gongyao Zhou; Peter I. Lelkes; Norman A. Johanson; Frederic A. Kleinbart; Donggang Yao
Original assignee: Drexel University; Philadelphia Health & Education Corporation
Priority date: 2008-03-28
Filing date: 2009-03-26
Publication date: 2009-10-01
Also published as: WO2009120886A3

Abstract

The present invention relates to compositions and methods for treating a bone defect. The present invention provides a multifunctional surgical fixation device having desirable mechanical integrity and bioactive utility. The device comprises a permeable medium containing porous materials having a gradient cellular structure.

Description

TITLE OF THE INVENTION

DESIGN AND MANUFACTURING OF BIOACTIVE IMPLANTED SURGICAL FIXATION DEVICES USING INJECTION MOLDING OF GRADIENT CELLULAR

STRUCTURES

BACKGROUND OF THE INVENTION

Around 300,000 surgical operations are performed every year to treat knee injuries in the United State; there are more than 90,000 anterior cruciate ligament (ACL) reconstructive surgeries worldwide annually. In orthopedic or spinal surgery, many fixation devices (such as plates, screws, pins, rods, anchors, staples, and others) are used in bone fractures fixation, autograft ankle stabilization, reconstruction surgery of the anterior cruciate ligament (ACL) and the posterior cruciate ligament (PCL), replacement of the intervertebral discs, posterior spinal fixation, etc. However, currently used surgical fixation devices (made by either metals or polymers) have shortages in bioactive features. They may lead to a secondary operation and bring more pain to the patient, and may cause long term adverse effects. Furthermore, current surgical fixation devices do not promote bone healing and re-growth, subsequently leaving voids in the tissue once the implanted device is pulled out (metal device) or fully degraded (biopolymer device). In current procedures, metal surgical fixation devices are used to provide high initial fixation strength and early rehabilitation in postoperative recovery. Typically, interference screws are used to secure the replacement ligament connecting femur and tibia. Repair of a fractured tibial plateau also requires the use of a contour plate and metal screws in a knee fixation operation. The metallic implant devices, however, are not degradable and thus leave cavitations in the bone once pulled out.

There are problems associated with current metal implanted surgical devices. Most surgical fixation devices are made primarily of titanium and stainless steel. The broken bones are first surgically reset into their proper position. Then a surgical device is fixed onto the broken bones to hold them in place, while the bone heals back together. While metal has the desired strength and rigidity to allow the healing process to begin, there are a number of issues associated with using permanent metallic fixation devices, such as bone growth restriction, accumulation of metals in tissues, adverse effects of metals, and potential for cross contamination. Thus, a secondary operation is often needed, bringing more pain to the patient.

In recent years, various biodegradable polymers have been used to make surgical fixation devices to overcome the drawbacks of metallic fixation devices. Biopolymeric surgical implant devices, such as biodegradable interference screws, provide a secure initial fixation of bone blocks, comparable to that of metal interference screws, while allowing controlled degradation followed by replacement with ingrowth of host tissue. The first biodegradable implants, developed in the mid-1980s, were rods and screws for the fixation of osteochondral fragments or apical fractures to cross connect two pieces of bone. Unlike their metal counterparts, biodegradable screws do not distort magnetic resonance imaging scans, do not compromise revision surgery, and do not present as great a risk of graft laceration during screw insertion. Given these advantages, the use of biodegradable surgical devices in tissue reconstruction has recently raised significant interest.

Increased interest in biodegradable surgical tools for bone, ligament, tendon and graft fixations has led to the development of some biopolymeric devices. For example, biodegradable surgical fixation devices have been made of polylactic acid

(PLA), poly-D/L-lactic acid with polyglycolic acid (PDLLA-co-PGA), ply-L-lactic acid with β-tricalcium phosphate (PLLA-TCP), poly-L-lactic acid with hydroxyapatite (PLLA-HA), and polycaprolactone (PCL) with alginate.

Compared with metal insertion devices, biodegradable non-metallic surgical fixation devices have many advantages, e.g., no long-term implant palpability or temperature sensitivity, predictable degradation to provide progressive bone loading and no stress shielding, leading to better bone healing, reduced patient trauma and cost, no second surgery required for implant removal, no imaging interference, provided sterile. Nevertheless, current biopolymeric surgical fixation devices, similar to metallic devices, do not promote bone re-growth, subsequently leaving voids in the tissue once the screw has fully degraded. Furthermore, current surgical fixation devices, no matter whether they are metallic or polymeric, lack bioactive features. That is, these devices do not actively promote bone healing and re-growth, and therefore subsequently leaving voids in the tissue once the implanted surgical fixation device is pulled out (metal device), or fully degraded (biopolymer device).

BRIEF SUMMARY OF THE INVENTION The present invention provides an implantable device comprising a gradient porous structure, wherein the structure has a gradient of pore size such that the size of pores located closer to the outer surface of the structure is smaller than the size of pores located further from the outer surface of the structure.

In one embodiment, the gradient porous structure is a permeable structure. In another embodiment, the implantable device comprises a hollow core, wherein the core can be filled with a bioactive agent.

In another embodiment, the gradient porous structure comprises a polymer selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), co- polyglycolic/lactic acid (PLGA), and any combinations thereof. In one embodiment, the gradient porous structure comprises a composite of PLGA and calcium phosphate (CaP).

In another embodiment, the gradient porous structure comprises a composite of PLA and calcium phosphate (CaP).

In one embodiment, the bioactive agent is released or controlled released within a recipient.

In one embodiment, the bioactive agent comprises at least one agent selected from the group consisting of an antibiotic, a growth factor, a drug, a cell, and any combination thereof.

In another embodiment, the gradient porous structure degrades over a period of time within a recipient.

In one embodiment, the device promotes bone growth within a recipient. The invention also provides a method of producing a structure having a gradient of pore size such that the size of pores located closer to the outer surface of the structure is smaller than the size of pores located further from the outer surface of the structure. Preferably, the method comprises injection molding a biodegradable blend, wherein the injection molding comprises spatially controlled thermal conditioning to adjust phase size of the porous structure, thereby producing the device. In one embodiment, the thermal conditioning comprises a lower temperature at areas located closer to the outer surface of the structure and a higher temperature at areas located further from the outer surface of the structure.

In one embodiment, the biodegradable blend comprises a biomaterial and a sacrificial polymer.

In one embodiment, the biomaterial is selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), co-polyglycolic/lactic acid (PLGA), and any combination thereof.

In one embodiment, the biomaterial is a composite of PLGA and CaP. In another embodiment, the sacrificial polymer is polystyrene.

The invention also provides a method of treating a bone defect in a mammal. The method comprises administering an implantable device into a mammal, wherein the device comprises a gradient porous structure, wherein the structure has a gradient of pore size such that the size of pores located closer to the outer surface of the structure is smaller than the size of pores located further from the outer surface of the structure.

In one embodiment, the gradient porous structure is a permeable structure.

In one embodiment, the device further comprises a hollow core, wherein the core can be filled with a bioactive agent. In another embodiment, the bioactive agent is released or controlled released within the mammal.

In another embodiment, the bioactive agent comprises at least one agent selected from the group consisting of an antibiotic, a growth factor, a drug, a cell, and any combination thereof. In one embodiment, the gradient porous structure degrades over a period of time within the mammal.

In another embodiment, the device promotes bone growth within the mammal.

BRIEF DESCRIPTION OF THE DRAWEVGS

For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure l is a schematic depiction of the innovative bioactive interference screw of the invention. Figure 2 is a schematic of the injection molding procedure to produce a gradient cellular structure (GCS). Figure 2 shows a GCS mold with a heated core. The core temperature and the mold temperature are controlled separately to create the desired thermal gradient.

Figure 3, comprising Figures 3A and 3B, is a series of images showing coarsening of the phase structure after a period of annealing. Figure 3 A depicts phase evolution due to interfacial tension at time zero. Figure 3B depicts the development of phase structural coarsening after a period of time.

Figure 4, comprising Figures 4A and 4B, is a series of images showing different porous structures from different processing conditions. Figure 4A depicts porous structures without dwell time inside the intruder. Figure 4B depicts porous structures with dwell time inside the intruder.

Figure 5 is an image depicting a skin-core structure in injection-molded blends.

Figure 6, comprising Figures 6A and 6B, is a series of images of scanning electron microscopy of representative fabricated biopolymer scaffolds. Figure 6A is an image of 200 μm pore size pure polycaprolactone (PCL) scaffold. Figure 6B is an image of 600 μm pore size 80:20 PCL/calcium phosphate (CaP) composite scaffold.

Figure 7 is a chart demonstrating the compressive mechanical properties (compression modulus and ultimate compression strength) of PCL-CaP composites with different concentrations of CaP (from 0% to 20%). Bars represent mean ± standard deviation. Statistical analysis indicates that the material properties are significantly different (p < 0.002, t-Test, one-tail: assuming Unequal Variances) for different concentrations of CaP.

Figure 8, comprising Figures 8A and 8B, is a series of images demonstrating bisbenzimide nuclear staining of adherent HEPM cells following 24 hours of orbital shaker seeding on PCL (Figure 8A, 20Ox) and 80:20 PCL-CaP composite (Figure 8B, 10Ox) scaffolds, 600 μm pore sizes. Images are captured by imaging the surface of a strut on the outside of the scaffold. Figure 9 is a chart summarizing degradation experiments of solid cylinders made of PCL in both a physiological buffer (DMEM) and upon admixing blood proteins (DMEM with 10%FBS). Figure 9 demonstrates that the composite degrade over time as measured by a decrease in molecular weight over time. Figure 10 is an image summarizing the experimental design for the push- out test to assess the mechanical performance of model bone screws.

Figure 11 is a graph depicting the load curve vs. push-down depth position for a polystyrene screw using the experimental design set forth in Figure 10.

DETAILED DESCRIPTION OF THE WVENTION

The present invention provides methods and compositions for designing and manufacturing bioactive surgical fixation devices. The bioactive surgical fixation devices are useful for promoting healing and regeneration of numerous osseous tissues without leaving cavitations in surgical mammals. The invention relates to a surgical bioactive fixation device. The bioactive fixation device of the invention is designed to promote bone healing and re-growth. In one embodiment, the device is strong enough to withstand the stress during insertion and post-operative activity. In another embodiment, the device possesses controllable biodegradation to promote tissue growth at a rate that is comparable to the degradation of the device. In yet another embodiment, the device can be used as a bone substitute. In yet another embodiment, the bioactive fixation device of the invention has osteoconductive and/or osteoinductive properties comparable to those of the healthy bone. In yet still another embodiment, the fixation device of the invention is biocompatible and does not cause an inflammatory or immune response in the recipient. The invention is partly based on the discovery of a novel technique comprising a unique injection molding method which can create gradient porous structures. The gradient porous structures allow for the incorporation of bioactive reagents into the surgical fixation device. Therefore the device has a mechanical property that is strong enough to endure surgical procedures as well as the ability to deliver biomaterials to a desirable locale.

The invention provides a gradient cellular structure (GCS) manufacturing process which is useful for engineering applications wherever structural integrity and permeability are both required in the design. In addition, the process allows for the ability to optimize and control fabrication of pore gradients and allow for fine tuning of the modulus at the interface between dissimilar materials.

The invention also provides a method of alleviating or treating a bone defect in a mammal, preferably a human. The method comprises administering to the mammal in need thereof a therapeutically effective amount of a composition comprising a surgical bioactive fixation device of the invention, thereby alleviating or treating the bone defect in the mammal.

Definitions Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Ausubel et al., 2002, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), which are provided throughout this document.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. The term "about" will be understood by persons of ordinary skill in the art and will vary to some extent based on the context in which it is used.

As used herein, the term "biodurable" describes products that are stable for extended periods of time in a biological environment. Such products should not exhibit significant symptoms of breakdown or degradation, erosion or significant deterioration of mechanical properties relevant to their employment when exposed to biological environments for periods of time commensurate with the use of the implantable device. The period of implantation may be weeks, months or years; the lifetime of a host product in which the products of the invention are incorporated, such as a graft or prosthetic; or the lifetime of a patient host to the product. The terms "precursor cell," "progenitor cell," and "stem cell" are used interchangeably in the art and as used herein refer either to a pluripotent or lineage- uncommitted progenitor cell, which is potentially capable of an unlimited number of mitotic divisions to either renew itself or to produce progeny cells which will differentiate into the desired cell type. In contrast to pluripotent stem cells, lineage-committed progenitor cells are generally considered to be incapable of giving rise to numerous cell types that phenotypically differ from each other. Instead, progenitor cells give rise to one or possibly two lineage-committed cell types.

The term "dedifferentiation", as used herein, refers to the return of a cell to a less specialized state. After dedifferentiation, such a cell will have the capacity to differentiate into more or different cell types than was possible prior to re -programming. The process of reverse differentiation (i.e., de-differentiation) is likely more complicated than differentiation and requires "re-programming" the cell to become more primitive. As used herein, "scaffold" refers to a structure, comprising a biocompatible material, that provides a surface suitable for adherence and proliferation of cells. A scaffold may further provide mechanical stability and support. A scaffold may be in a particular shape or form so as to influence or delimit a three-dimensional shape or form assumed by a population of proliferating cells. Such shapes or forms include, but are not limited to, films (e.g. a form with two-dimensions substantially greater than the third dimension), ribbons, cords, sheets, flat discs, cylinders, spheres, 3 -dimensional amorphous shapes, etc.

As used here, "biocompatible" refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal. A biocompatible material, when introduced into an individual, is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the mammal.

As used herein, the term "biocompatible lattice," is meant to refer to a substrate that can facilitate formation of three-dimensional structures conducive for tissue development. Thus, for example, cells can be cultured or seeded onto such a biocompatible lattice, such as one that includes extracellular matrix material, synthetic polymers, cytokines, growth factors, etc. The lattice can be molded into desired shapes for facilitating the development of tissue types. Also, at least at an early stage during culturing of the cells, the medium and/or substrate is supplemented with factors (e.g., growth factors, cytokines, extracellular matrix material, etc.) that facilitate the development of appropriate tissue types and structures. "Bioactive agents," as used herein, can include one or more of the following: chemotactic agents; therapeutic agents (e.g., antibiotics, steroidal and nonsteroidal analgesics and antiinflammatories (including certain amino acids such as glycine), anti-rejection agents such as immunosuppressants and anti-cancer drugs); various proteins (e.g. , short term peptides, bone morphogenic proteins, collagen, hyaluronic acid, glycoproteins, and lipoprotein); cell attachment mediators; biologically active ligands; integrin binding sequence; ligands; various growth and/or differentiation agents and fragments thereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factors (VEGF), fibroblast growth factors (e.g., bFGF), platelet derived growth factors (PDGF), insulin derived growth factor (e.g., IGF- 1, IGF-II) and transforming growth factors (e.g., TGFβ I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4; BMP-6; BMP-7; BMP-12; BMP-13; BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52, and MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-I ; CDMP-2, CDMP-3)); small molecules that affect the upregulation of specific growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate; fibronectin; decorin; thromboelastin; thrombin-derived peptides; heparin-binding domains; heparin; heparan sulfate. Suitable effectors likewise include the agonists and antagonists of the agents described above. The growth factor can also include combinations of the growth factors described above. In addition, the growth factor can be autologous growth factor that is supplied by platelets in the blood. In this case, the growth factor from platelets will be an undefined cocktail of various growth factors. If other such substances have therapeutic value in the orthopedic field, it is anticipated that at least some of these substances will have use in the present invention, and such substances should be included in the meaning of "bioactive agent" and "bioactive agents" unless expressly limited otherwise. Preferred examples of bioactive agents include culture media, bone morphogenic proteins, growth factors, growth differentiation factors, recombinant human growth factors, cartilage-derived morphogenic proteins, hydrogels, polymers, antibiotics, anti-inflammatory medications, immunosuppressive mediations, autologous, allogenic or xenologous cells such as stem cells, chondrocytes, fibroblast and proteins such as collagen and hyaluronic acid. Bioactive agents can be autologus, allogenic, xenogenic or recombinant. The term "biologically compatible carrier" or "biologically compatible medium" refers to reagents, cells, compounds, materials, compositions, and/or dosage formulations which are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio.

As used herein, the term "bone condition (or injury or disease)" refers to disorders or diseases of the bone including, but not limited to, acute, chronic, metabolic and non-metabolic conditions of the bone. The term encompasses conditions caused by disease, trauma or failure of the tissue to develop normally. Examples of bone conditions include, but are not limited, a bone fracture, a bone/spinal deformation, osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis, osteomalacia, rickets, fibrous osteitis, renal bone dystrophy, and Paget's disease of bone.

As used herein, "autologous" refers to a biological material derived from the same individual into whom the material will later be re-introduced. As used herein, "allogeneic" refers to a biological material derived from a genetically different individual of the same species as the individual into whom the material will be introduced.

As used herein, a "graft" refers to a cell, tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect. A graft may further comprise a scaffold. The tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: "autograft", "autologous transplant", "autologous implant" and "autologous graft". A graft comprising cells from a genetically different individual of the same species is referred to herein by the following interchangeable terms: "allograft", "allogeneic transplant", "allogeneic implant" and "allogeneic graft". A graft from an individual to his identical twin is referred to herein as an "isograft", a "syngeneic transplant", a "syngeneic implant" or a "syngeneic graft". A "xenograft", "xenogeneic transplant" or "xenogeneic implant" refers to a graft from one individual to another of a different species.

As used herein, the terms "tissue grafting" and "tissue reconstructing" both refer to implanting a graft into an individual to treat or alleviate a tissue defect, such as a bone defect or a soft tissue defect.

As used herein, to "alleviate" a disease, defect, disorder or condition means reducing the severity of one or more symptoms of the disease, defect, disorder or condition. As used herein, to "treat" means reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.

As used herein, a "therapeutically effective amount" is the amount of a composition of the invention sufficient to provide a beneficial effect to the individual to whom the composition is administered.

As used herein, the term "growth medium" is meant to refer to a culture medium that promotes growth of cells. A growth medium will generally contain animal serum. In some instances, the growth medium may not contain animal serum. "Differentiation medium" is used herein to refer to a cell growth medium comprising an additive or a lack of an additive such that a stem cell, fetal pulmonary cell or other such progenitor cell, that is not fully differentiated, develops into a cell with some or all of the characteristics of a differentiated cell when incubated in the medium.

As used herein, the term "growth factor product" refers to a protein, peptide, mitogen, or other molecule having a growth, proliferative, differentiative, or trophic effect on a cell. Growth factors include, but are not limited to, fibroblast growth factor (FGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-T), insulin-like growth factor-II (IGF-II), platelet-derived growth factor (PDGF), vascular endothelial cell growth factor (VEGF), activin-A, bone morphogenic proteins (BMPs), insulin, growth hormone, erythropoietin, thrombopoietin, interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 7 (IL-7), macrophage colony stimulating factor, c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, nerve growth factor, ciliary neurotrophic factor, cytokines, chemokines, morphogens, neutralizing antibodies, other proteins, and small molecules.

As used herein, "osteogenic medium" refers to a differentiation medium that induces development of some or all of the characteristics of an osteoblast or osteocyte.

As used herein, an "osteogenic stimulant" refers to an additive that is capable of inducing some or all of the characteristics of an osteoblast or osteocyte in a stem cell, adipose-derived adult stem cell or other such progenitor cell that is not fully differentiated. An "isolated cell" refers to a cell which has been separated from other components and/or cells which naturally accompany the isolated cell in a tissue or mammal.

As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. Thus, a substantially purified cell refers to a cell which has been purified from other cell types with which it is normally associated in its naturally- occurring state.

"Expandability" is used herein to refer to the capacity of a cell to proliferate, for example, to expand in number or, in the case of a population of cells, to undergo population doublings.

"Proliferation" is used herein to refer to the reproduction or multiplication of similar forms, especially of cells. That is, proliferation encompasses production of a greater number of cells, and can be measured by, among other things, simply counting the numbers of cells, measuring incorporation of ^H-thymidine into the cell, and the like. As used herein, "tissue engineering" refers to the process of generating tissues ex vivo for use in tissue replacement or reconstruction. Tissue engineering is an example of "regenerative medicine," which encompasses approaches to the repair or replacement of tissues and organs by incorporation of cells, gene or other biological building blocks, along with bioengineered materials and technologies. As used herein "endogenous" refers to any material from or produced inside an organism, cell or system.

"Exogenous" refers to any material introduced into or produced outside an organism, cell, or system.

"Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally-occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The phrase "under transcriptional control" or "operatively linked" as used herein means that the promoter is in the correct location and orientation in relation to the polynucleotides to control RNA polymerase initiation and expression of the polynucleotides.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (i.e., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.

The term "patient" as used herein includes human and veterinary subjects.

Description of the Invention

The invention includes a novel surgical fixation device (e.g., screws, anchors, plates, pins, staples, etc.) capable of securing a graft in place. The device can also incorporate bioactive materials including but is not limited to such as growth factors, drugs, and cells, intended to promote/accelerate bone tissue growth. For example, the device is multifunctional with respect to mechanical integrity in that it can withstand a sufficient amount of physical stress and with respect to bioactive efficacy in that it can provide a therapeutic effect to a recipient. The surgical fixation device of the invention is generated by application of gradient cellular structure (GCS) injection molding procedure. The GCS injection molding procedure is useful for generating a porous conductive material. The porous material is capable of incorporated bioactive material and release the material locally in a controlled release manner. The porous material is also able to mimic tissue, extracellular matrix (ECM) architecture, support cell proliferation, and the like.

In a preferred embodiment, the surgical fixation device of the invention is a bioactive surgical screw. The screw can be used in orthopedic, spinal and other surgeries to provide both mechanical fixations and bioactive bone structure healing, growing and regeneration. Accordingly, the surgical fixation device can be extended to other fixation devices (e.g., pins, rods, anchors and staples), and even to many other bone structure grafts (e.g., femur, hip, etc.).

The implantable devices of the invention are also suitable for use as tissue engineering scaffolds, or other comparable substrates, to support in vivo cell propagation applications, for example in a large number of orthopedic applications especially in soft tissue attachment, regeneration, augmentation, support and ingrowth of a prosthetic organ. Without being bound by any particular theory, having a high porous content is believed to allow the implantable device to become at least partially ingrown and/or proliferated, in some cases substantially ingrown and proliferated, in some cases completely ingrown and proliferated, with cells including tissues such as fibroblasts, fibrous tissues, synovial cells, bone marrow stromal cells, stem cells and/or fibrocartilage cells. The ingrown and/or proliferated tissues thereby provide functionality, such as load bearing capability, for defect repair of the original tissue that is being repaired or replaced.

Composition

It is preferred that the bioactive surgical device of the invention is multifunctional with respect to mechanical integrity and bioactive efficacy. With respect to mechanical integrity, it is preferred that the device possess a high bending strength that is equivalent to, or greater than, that of natural bone, preferably normal human bone. With respect to bioactive efficacy, it is preferred that the device of the invention is capable of being embedded with bioactive reagents such as growth factors, antibiotics/anti-inflammatory drugs, and/or cells to provide fast bone/tissue healing and recovery. In some aspects, the device is able to provide osteoinductivity and osteoconductivity, which promotes bone growth and bonding to the surrounding normal bone without intervening fibrous tissue to promote bone/tissue healing and recovery.

In some instances, it is preferred that the implantable devices of the invention be able to occupy a site of the recipient for extended periods of time without being harmful to the host. In one embodiment, such implantable devices can also eventually become integrated, such as biointegrated, e.g., ingrown with tissue or bio- integrated.

The surgical fixation device of the invention comprises a core reservoir to store bioactive materials such as, for example, bone healing drugs, growth factors, and cells surrounded by a wall having interconnected pores. The wall has a controlled porosity and allows the bioactive materials contained in the core to pass through. The pores have a gradient of pore's diameter such that pore diameters diminish in the direction from the core to the outer surface.

The wall porosity may vary from 0-90 vol % and any and all whole or partial integers therebetween (or from about 3% to about 90%, about 5% to about 85%, about 10% to about 90%, about 15% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 75% to about 90%, about 5% to about 75%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30% about 5% to about 25%). In certain embodiments, the device comprises a gradient of pore size, porosity, and material composition extending from the surface region into or throughout the interior of the device, wherein the gradient transition is continuous or seamless and the growth of cells extending from the surface region inward is promoted.

In some embodiments, the surface region comprises nanoscale or microscale pores ranging from about 1 nm to about 500 nm in diameter, or from about 1 nm to about 1 μm. In certain implementations, the member/material structure comprises a microporous or macroporous pattern having pore sizes in the range of about 1 μm to about 5 mm. In certain embodiments, the method comprises depositing a chemical or biological agent deposited in or on the composite member/material or in one or more pores thereof to operatively provide for release or controlled release of the agent within a recipient. In certain embodiments, the chemical or biological agent is deposited in or on one or more surface structures or pores thereof. In certain aspects, the agent comprises at least one agent suitable to provide a beneficial biological or physiological effect. In particular embodiments, the at least one agent suitable to provide a beneficial biological or physiological effect comprises an antimicrobial agent. In certain embodiments, the agent comprises at least one agent selected from the group consisting of antibiotics, growth factors, and drugs. In particular embodiments, at least one of the pore size, porosity and material composition is selected to provide a device having an optimal density, elastic modulus or compression strength for a specific recipient. In certain aspects, the macroporous structure is selected to provide a device having an optimal density, elastic modulus or compression strength for a specific recipient.

In one embodiment relating to orthopedic applications and the like, to encourage cellular ingrowth and proliferation and to provide adequate permeability, the average diameter or other largest transverse dimension of pores is at least about 10 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 20 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 50 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 100 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 150 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 200 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 250 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 300 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 350 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 400 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least 450 μm. In another embodiment, the average diameter or other largest transverse dimension of pores is at least about 500 μm. In some embodiments, the devices of the invention comprise continuous or step-wise pore size distribution. The continuous or step-wise pore size distribution variations, with or without pore orientation, can be important characteristics for in a number of orthopedic applications, especially in soft tissue attachment, repair, regeneration, augmentation and/or support encompassing the spine, shoulder, knee, hand or joints, and in the growth of a prosthetic organ.

Bioactive implanted surgical fixation devices of the invention have multiple functions. For example, the implant preferably comprises a gradient porous structure wherein the gradient porous structure is used as a permeable medium in order to deliver bioactive agents to the environment of the implant. In addition, the implant is mechanically stiff and strong to endure the stresses imposed on the implant during surgical procedures. Accordingly, the compositions of the invention are based on the principles of natural porous materials available in nature that possess multifunctional structures, e.g., wood, egg shell, wheat and rice, bone, skin, and others. These natural materials have a gradient cellular structure (GCS), such that their porosity is not uniform.

The compositions of the invention are based on application of GCS principles to an orthopedic implant to create a bioactive fixation device and development of a permeable surgical fixation device with structural integrity. The novel surgical fixation devices (e.g., screw, anchor, plate, pin, staple) of the invention provides a dual need by securing a graft in place and promoting bone tissue growth by delivering incorporated bioactive materials such as growth factors, drugs and cells. Suitable materials for making the surgical fixation devices of the invention comprise (1) structure materials for making an outer surface having sufficient strength (e.g., PLGA and CaP composite) and (2) bioactive materials for efficient bone/tissue healing and growth. A gradient cellular structure (GCS) technique is used to create interconnective porous structure to assure the screw mechanical strength and assist bioactive materials delivery. A non-limiting example of a surgical fixation device of the invention is a bioactive interference screw. The interference screw comprises a hollow core which serves as a reservoir and gradient porous walls to allow for delivery of bioactive materials. Accordingly, the interference screw is a non-limiting example of a surgical fixation device having mechanical integrity and bioactive utility.

Suitable structure materials used in the manufacture of the devices described herein are materials which are biocompatible, bioresorbable over periods of weeks or longer, and generally encourage cell attachment. The term "bioresorbable" is used herein to mean that the material degrades into components which may be resorbed by the body and which may be further biodegradable. Biodegradable materials are capable of being degraded by active biological processes such as enzymatic cleavage. Other properties desirable for materials to be used in the manufacture of the devices described herein include (1) solubility in a biologically acceptable solvent that can be removed to generally accepted safe levels, (2) capability of being milled to particles of less than 150 microns, and (3) elasticity and compressive and tensile strength.

Biocompatible Polymers:

Components of the surgical device of the invention are made from biocompatible materials. The ideal properties of the biocompatible materials for use in the instant invention include at least one of: mechanical integrity, thermal stability, non- immunogenic, bioresorbable, slow degradation rate, capacity to be functionalized with, for instance, cell growth factors, and plasticity in terms of processing into different structural formats.

The physical characteristics of the biomaterial are carefully considered when designing a substrate to be used in tissue engineering or repair. In order to promote tissue growth, the scaffold must have a large surface area to allow cell attachment. This is usually done by creating highly porous scaffolds wherein the pores are large enough such that cells can penetrate the pores. Furthermore, the pores can be interconnected to facilitate nutrient and waste exchange by the cells. These characteristics, i.e., interconnectivity and pore size, are often dependent on the method of fabrication.

An initial characteristic to consider when manufacturing composites and scaffolds is the choice of materials. It is understood that if the composites or scaffolds are manufactured for therapeutic use, all components used must be biocompatible. Accordingly, in considering substrate materials, it is imperative to choose one that exhibits clinically acceptable biocompatibility. In addition, the mechanical properties of the scaffold must be sufficient so that it does not collapse during the patient's normal activities. Both natural (e.g., collagen, elastin, poly(amino acids), and polysaccharides such as hyaluronic acid, glycosamino glycan, carboxymethylcellulose); and synthetic polymer materials may be used to manufacture the composites and scaffolds of the present invention. The polymer material may be in the form of one or more of sheet(s), blocks(s), pellets, granules, or any other desirably shaped polymer material. A variety of biocompatible polymers can be used to make the device. The biocompatible polymers can be synthetic polymers, natural polymers or combinations thereof. As used herein the term "synthetic polymer" refers to polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. The term "natural polymer" refers to polymers that are naturally occurring. Synthetic polymers which have been found to be particularly suited to the present use include: poly(alpha)esters, such as: poly(lactic acid) (PLA) and poly(DL- lactic-co-glycolic acid) (PLGA). Other suitable materials include: poly(ε-caprolactone) (PCL), polyanhydrides, polyarylates, and polyphosphazene. Natural polymers which are suitable in combination with synthetic polymers include: polysaccharides such as cellulose, dextrans, chitin, chitosan, glycosaminoglycans; hyaluronic acid or esters, chondroitin sulfate, and heparin; and natural or synthetic proteins or proteinoids such as elastin, collagen, agarose, calcium alginate, fibronectin, fibrin, laminin, gelatin, albumin, casein, silk protein, proteoglycans, Prolastin, Pronectin, or BetaSilk. Mixtures of any combination of polymers may also be used. Preferred synthetic polymers include: poly(hydroxy alkanoates), polydioxanone, polyamino acids, poly(gamma-glutamic acid), poly(vinyl acetates), poly(vinyl alcohols), poly(ethylene-imines), poly(orthoesters), polypohosphoesters, poly(tyrosine-carbonates), poly(ethylene glycols), poly(trimethlene carbonate), polyiminocarbonates, poly(oxyethylene-polyoxypropylene), poly(alpha- hydroxy-carboxylic acid/polyoxyalkylene), polyacetals, poly(propylene fumarates), and carboxymethylcellulose.

In one embodiment, the device includes at least one natural polymer. Suitable examples of natural polymers include, but are not limited to, fibrin-based materials, collagen-based materials, hyaluronic acid-based materials, glycoprotein-based materials, cellulose-based materials, silks and combinations thereof.

One of ordinary skill in the art will appreciate that the selection of a suitable material for forming the biocompatible tissue repair device of the present invention depends on several factors. These factors include in vivo mechanical performance; cell response to the material in terms of cell attachment, proliferation, migration and differentiation; biocompatibility; and optionally, bioabsorption (or bio- degradation) kinetics. Other relevant factors include the chemical composition, spatial distribution of the constituents, the molecular weight of the polymer, and the degree of crystallinity.

The materials used to make the reinforcing component can include monofilaments, yarns, threads, braids, or bundles of fibers. These fibers can be made of any biocompatible material including bioabsorbable materials such as polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC), copolymers or blends thereof. These fibers can also be made from any biocompatible materials based on natural polymers including silk and collagen-based materials. These fibers can also be made of any biocompatible fiber that is nonresorbable, such as, for example, polyethylene, polyethylene terephthalate, poly(tetrafluoroethylene), polycarbonate, polypropylene and poly(vinyl alcohol). In one embodiment, the fibers are formed from 95:5 copolymer of lactide and glycolide. In a preferred embodiment of the invention, the surgical fixation device is produced from the combination of PLGA and CaP. In the bioactive surgical devices of the invention, one considerations for the material selection is whether it is strong enough to withstand the stress during implantation and post-operative activity. PLGA is desirable due to its adjustable biodegradability and high mechanical strength although it is not bioactive. In order to improve the osteoconductivity and bioactivity of the PLGA screws, CaP powder is compounded with PLGA to make the PLGA-CaP composite screws. With alteration in the CaP/PLGA and PLA/PGA ratio, the morphology, mechanical properties, and biodegradation behavior can be changed. Preferably, the CaP used herein has not been subjected to calcination or sintering, and has a chemical composition similar to that which is synthesized in the body. CaP causes minimal physical irritation of the surrounding tissues, gradually biodegrades and is resorbed. Also, it exhibits superior bioactivity, such as osteoconductivity and bone-binding ability, and provides excellent biocompatibility and safety. The combination of PLGA and CaP produces a surgical device having ideal characteristics which improves biocompatibility associated with controlled degradation rate and promotes new bone formation together with longer strength retention.

The excellent biocompatibility of calcium phosphate (CaP) due to its close mimicking of the inorganic phase of the natural bone mineral has led to the widespread use as a bone substitute in the field of dentistry, orthopedic and reconstructive surgery. The incorporation of a biocompatible CaP having the same ions as those found in the human skeleton carries major advantages when applied to a resorbable orthopedic device. CaP has a relatively high mechanical strength comparing with PLGA and shows a great osteoconductive characteristic, but in general it degrades relatively slow.

Fabrication of surgical device

The present invention provides methods for the production of 3- dimensional porous silk scaffolds that can be used in tissue engineering. The scaffolds described herein are particularly suited for tissue engineering as the porosity of the scaffold can be adjusted throughout mimicking the gradient of densities found in natural tissue. Methods for producing 3-dimensional tissue using the gradient cellular structure based scaffolds are also provided.

A composition according to the present invention may be obtained by mixing or blending respective constituents in the desired amounts. This may be performed by applying a two-step approach to the injection molding procedure. In the first step, the flow and heat transfer process of a standard injection molding procedure is applied to a 3-D model. In the second step, the phase evolution process procedure is carried out by solving a surface tension driven flow problem/equation with the thermomechanical history obtained in the first step as the boundary condition as described in more detail in the Examples.

The implantable devices of the invention (e.g., a screw) are fabricated applying molding techniques, such as injection molding to generate a gradient cellular structure (GCS). The GCS concept can be used to overcome some design conflicts in bioactive surgical devices. The invention provides a method of controling pore sizes and gradient porous structures during fabrication. The method includes controlling the thermomechanical history of the blend during processing. The result is a gradient and interconnective structure.

Gradient materials are prepared by applying injection molding techniques to an immiscible polymer blend, with spatially controlled thermal conditioning to adjust the phase size from core to surface. For example, polylactides/glycolides (PLA, PGA, or PLGA) and a sacrificial, immiscible polymer (e.g., polystyrene) are initially melt-blended in a batch mixer to ensure the development of a fine blend structure with phase size in the micrometer regime. The blend is then injection molded into the mold cavity of a desired structure, for example a screw. While inside the mold, the blend is thermally conditioned with higher temperature at the core and lower temperature at the surface, resulting in the development of coarse phases at the core but fine phases at the surface. The interfacial surface tension between the two polymers causes coarsening of the phase structure at high-temperature regions. The gradient in phase size can be regulated by adjusting the thermal history. Fabricating the composite surgical fixation device component preferably is carried out by utilizing an injection molding technique to fabricate the polymeric portion. For example, an injection molding technique can be used to fabricate a composite fixation component comprised of an outer shell with an inner cavity. After molding, the inner cavity may be filled with polymer. Other techniques suitable for fabricating the composite fixation components described herein also can be used, as will be appreciated by those skilled in the art upon review of the guidelines provided herein. A composite comprising any of the materials disclosed herein may be used to fabricate various components of fixation devise, such as rods, screws, or plates.

Bioactive agents

Surface chemistry modifiers or biological factors or growth factors can be positioned on or in the device, which can be releasable in a physiological environment for the purpose of stimulating cell attachment, growth, maturation, and differentiation in the area of the device. Those bioactive agents which can be directly dissolved in a biocompatible solvent are highly preferred. Examples generally include proteins and peptides, polysaccharides, nucleic acids, lipids, and non-protein organic and inorganic compounds, referred to herein as "bioactive agents" unless specifically stated otherwise. These materials have biological effects such as growth factors, differentiation factors, steroid hormones, cytokines, lymphokines, antibiotics, and angiogenesis promoting or inhibiting factors.

Bioactive agents also include compounds having principally a structural role, for example, hydroxyapatite crystals in a matrix for bone regeneration. The particles may have a size of greater than or less than the particle size of the polymer particles used to make the matrix.

It is also possible to incorporate materials not exerting a biological effect such as air, radiopaque materials such as barium, or other imaging agents for the purpose of monitoring the device in vivo.

In order to promote cell attachment, cell adhesion factors such as laminin, pronectin, or fibronectin or fragments thereof, e.g. arginine-glycine-aspartate, may be coated on or attached to the device. The device may also be coated or have incorporated cytokines or other releasable cell stimulating factors such as; basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), nerve growth factor (NGF), insulin-like growth factor- 1 (IGF-I), growth hormone (GH), multiplication stimulating activity (MSA), cartilage derived factor (CDF), bone morphogenic proteins (BMPs) or other osteogenic factors, anti-angiogenesis factors (angiostatin), and the like.

Some bioactive agents or materials are osteogenic and stimulate the growth of bone forming cells; some materials are osteoconductive, encouraging bone- forming cell migration and incorporation; and some are osteoinductive, inducing the differentiation of mesenchymal stem cells into osteoblasts. Materials which have been found to be osteogenic usually contain a natural or synthetic source of calcium phosphate. Osteoinductive materials include molecules derived from members of the transforming growth factor-beta (TGFβ) gene superfamily including: bone morphogenetic proteins (BMPs) and insulin-like growth factors (IGFs).

In one embodiment for repair or replacement of bone, a gradient is formed of osteogenic and osteoconductive materials, such as calcium phosphates, to materials which are synthetic biocompatible polymers, such as poly(alpha)esters, which are particularly well suited for attachment of cells and controlled biodegradation. In another embodiment, the devices have a gradient in macroarchitecture. The macroarchitecture, or overall shape, can be of a design which allows fluid flow through and/or around one region and a different shape in another region with a gradient from one shape to the other. In another embodiment, the microarchitecture may be from an osteoinductive system of interconnected pores to a system of staggered channels inductive to chondrocyte colonization. In another aspect, the gradient may relate to mechanical properties such as tensile or compressive strength. The gradient of properties may be from that which is suitable for weight bearing loads to one which is suitable for soft tissue regeneration.

In another embodiment, materials such as growth factors, which selectively encourage or enhance the growth or differentiation of cells that form tissues, can be incorporated on or in the device. A particularly favored method of fabricating the devices includes incorporating the factors in the structure of the device.

In addition, either exogenously added cells or exogenously added factors including genes may be added to the implant before or after its placement in the body. Such cells include autografted cells which are derived from the patients tissue and have (optionally) been expanded in number by culturing ex vivo for a period of time before being reintroduced. Cartilage tissue may be harvested and the cells disaggregated therefrom, and cultured to provide a source of new cartilage cells for seeding the devices. The devices may also be seeded with cells ex vivo and placed in the body with live cells attached thereto.

Genetic modification

Cells applicable to the device of the present invention can also be used to express a foreign protein or molecule for a therapeutic purpose. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into the cells with concomitant expression of the exogenous DNA in the cells. Methods for introducing and expressing DNA in a cell are well known to the skilled artisan and include those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

The term "genetic modification" as used herein refers to the stable or transient alteration of the genotype of a cell by intentional introduction of exogenous DNA. The DNA may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful DNA sequences. The term "genetic modification" as used herein is not meant to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like.

Exogenous DNA may be introduced to a cell using viral vectors (retrovirus, modified herpes viral, herpes-viral, adenovirus, adeno-associated virus, lentiviral, and the like) or by direct DNA transfection (lipofection, calcium phosphate transfection, DEAE-dextran, electroporation, and the like).

One purpose of genetic modification of the cell is for the production aggrecan and/or components thereof. However, the cells can also be genetically modified for the purpose of producing of a biological agent. Examples of biological agents include, but are not limited to, chemotactic agents; therapeutic agents (e.g., antibiotics, steroidal and non-steroidal analgesics and antiinflammatories (including certain amino acids such as glycine), anti -rejection agents such as immunosuppressants and anti-cancer drugs); various proteins (e.g., short term peptides, bone morphogenic proteins, collagen, hyaluronic acid, glycoproteins, and lipoprotein); cell attachment mediators; biologically active ligands; integrin binding sequence; ligands; various growth and/or differentiation agents and fragments thereof (e.g., epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factors (VEGF), fibroblast growth factors (e.g., bFGF), platelet derived growth factors (PDGF), insulin derived growth factor (e.g., IGF- 1, IGF-II) and transforming growth factors (e.g., TGFβ I-III), parathyroid hormone, parathyroid hormone related peptide, bone morphogenic proteins (e.g., BMP-2, BMP-4; BMP-6; BMP-7; BMP-12; BMP-13; BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52, and MP-52 variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-I; CDMP-2, CDMP-3)); small molecules that affect the upregulation of specific growth factors; tenascin-C; hyaluronic acid; chondroitin sulfate; fibronectin; decorin; thromboelastin; thrombin-derived peptides; heparin-binding domains; heparin; heparan sulfate.

A preferred bioactive agent is a substance that is useful for the treatment of a given bone disorder. For example, it may be desired to genetically modify cells so that they secrete a certain growth factor product associated with bone formation.

The cells can be genetically modified by introducing exogenous genetic material into the cells to produce a molecule such as a trophic factor, a growth factor, a cytokine, and the like. In addition, the cell can provide an additional therapeutic effect to the mammal when transplanted into a mammal in need thereof. For example, the genetically modified cell maybe modified to secrete a molecule that is beneficial to neighboring cells in the mammal and ultimately cause a beneficial effect in the mammal.

As used herein, the term "growth factor product" refers to a protein, peptide, mitogen, or other molecule having a growth, proliferative, differentiative, or trophic effect on a cell. Specific growth factors useful in the treatment of bone disorders include, but are not limited to, FGF, TGF- β, insulin-like growth factor, and bone morphogenetic protein.

According to some aspects of the invention, cells obtained from the mammal to be treated or from another donor mammal, may be genetically altered to replace a defective gene and/or to introduce a gene whose expression has therapeutic effect in the mammal being treated.

In all cases in which a gene construct is transfected into a cell, the heterologous gene is operably linked to regulatory sequences required to achieve expression of the gene in the cell. Such regulatory sequences typically include a promoter and a polyadenylation signal.

The gene construct is preferably provided as an expression vector that includes the coding sequence for a heterologous protein operably linked to essential regulatory sequences such that when the vector is transfected into the cell, the coding sequence will be expressed by the cell. The coding sequence is operably linked to the regulatory elements necessary for expression of that sequence in the cells. The nucleotide sequence that encodes the protein may be cDNA, genomic DNA, synthesized DNA or a hybrid thereof, or an RNA molecule such as mRNA. The gene construct includes the nucleotide sequence encoding the beneficial protein operably linked to the regulatory elements and may remain present in the cell as a functioning cytoplasmic molecule, a functioning episomal molecule or it may integrate into the cell's chromosomal DNA. Exogenous genetic material may be introduced into cells where it remains as separate genetic material in the form of a plasmid. Alternatively, linear DNA which can integrate into the chromosome may be introduced into the cell. When introducing DNA into the cell, reagents which promote DNA integration into chromosomes may be added. DNA sequences which are useful to promote integration may also be included in the DNA molecule. Alternatively, RNA may be introduced into the cell.

The regulatory elements for gene expression include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. It is preferred that these elements be operable in the cells of the present invention. Moreover, it is preferred that these elements be operably linked to the nucleotide sequence that encodes the protein such that the nucleotide sequence can be expressed in the cells and thus the protein can be produced. Initiation codons and stop codons are generally considered to be part of a nucleotide sequence that encodes the protein. However, it is preferred that these elements are functional in the cells. Similarly, promoters and polyadenylation signals used must be functional within the cells of the present invention. Examples of promoters useful to practice the present invention include but are not limited to promoters that are active in many cells such as the cytomegalovirus promoter, SV40 promoters and retroviral promoters. Other examples of promoters useful to practice the present invention include but are not limited to tissue-specific promoters, i.e. promoters that function in some tissues but not in others; also, promoters of genes normally expressed in the cells with or without specific or general enhancer sequences. In some embodiments, promoters are used which constitutively express genes in the cells with or without enhancer sequences. Enhancer sequences are provided in such embodiments when appropriate or desirable.

The cells can be transfected using well known techniques readily available to those having ordinary skill in the art. Exogenous genes may be introduced into the cells using standard methods where the cell expresses the protein encoded by the gene. In some embodiments, cells are transfected by calcium phosphate precipitation transfection, DEAE dextran transfection, electroporation, microinjection, liposome-mediated transfer, chemical-mediated transfer, ligand mediated transfer or recombinant viral vector transfer. In some embodiments, recombinant adenovirus vectors are used to introduce DNA with desired sequences into the cell. In some embodiments, recombinant retrovirus vectors are used to introduce DNA with desired sequences into the cells. In other embodiments, standard CaPO₄, DEAE dextran or lipid carrier mediated transfection techniques are employed to incorporate desired DNA into dividing cells. In some embodiments, DNA is introduced directly into cells by microinjection. Similarly, well- known electroporation or particle bombardment techniques can be used to introduce foreign DNA into the cells. A second gene is usually co-transfected or linked to the therapeutic gene. The second gene is frequently a selectable antibiotic-resistance gene. Standard antibiotic resistance selection techniques can be used to identify and select transfected cells. Transfected cells are selected by growing the cells in an antibiotic that will kill cells that do not take up the selectable gene. In most cases where the two genes co-transfected and unlinked, the cells that survive the antibiotic treatment contain and express both genes.

Administration

There are numerous bone defects for which the inventive method is applicable. Such defects include, but are not limited to, segmental bone defects, nonunions, malunions or delayed unions, cysts, tumors, necroses or developmental abnormalities. Other conditions requiring bone augmentation, such as joint reconstruction, cosmetic reconstruction or bone fusion, such as spinal fusion or joint fusion, are treated in an individual by administering, for example, into the site of the bone defect, a composition of the invention to an extent sufficient to augment bone formation therefrom, thereby alleviating or treating the defect. The composition can also contain one or more other components which degrade, resorb or remodel at rates approximating the formation of new tissue. In a typical application, the composition is inserted in the defect and results in osteogenic healing of the defect.

Numerous soft tissue defects may also be alleviated or treated using the compositions and methods of the invention. Non-limiting examples of soft tissue reconstruction include breast reconstruction after mastectomy, breast augmentation, and soft tissue reconstruction after tumor resection, such as facial tissue. A composition of the invention is administered to an extent sufficient to achieve alleviation or treatment of the soft tissue defect. Advantageously, the composition and method of the invention improve on prior art methods of soft tissue defect in reducing the extent of undesirable outcomes, such as dimpling.

In one embodiment, the present invention provide methods for regenerating articular (e.g., hyaline) cartilage. Articular cartilage lines the bony surface of joints in mammals. Articular cartilage functions to distribute force in contact areas between the bones, such as for example knee joints. Articular cartilage is also found in other joint structures including by not limited to hips, shoulders, elbows, wrists, the interphalangeal joint of the hand, cartilaginous areas of costal joints such as ribs. In another embodiment, the present invention provides methods for regenerating fibrocartilage which functions as structural support structures including but not limited to the knee meniscus, vertebral disc, nose, annulus fibrosis of intervertebral disc, pubis symphisis, and certain areas of bone ligament junctions.

The tissue defect can relate to an orthopedic application, general surgical application, cosmetic surgical application, tissue engineering application, or any mixture thereof. The orthopedic application can relate to a repair, reconstruction, regeneration, augmentation, gap interposition, or any mixture thereof of a tendon, ligament, cartilige, meniscus, spinal disc, or any mixture thereof. The general surgical application can relate to an inguinal hernea, a ventral abdominal hernea, a femoral hernea, an umbilical hernea, or any mixture thereof. The surgical fixation device is also applicable to treating a bone defect.

ACL is most commonly reconstructed with either a middle-third bone-patellar tendon- bone (BPTB) autograft or a combination of the semitendinosus and gracilis hamsting autografts. These grafts can be fixed in the bone tunnels using a wide range of surgical devices which include plates, staples, buttons, posts/rods, anchors, interference screws and transverse pins. Good initial fixation of the properly positioned and tensioned graft is critical, but the importance of this initial fixation decreases as the graft is incorporated into the adjacent bone tunnels. As a non-limiting example, the surgical fixation devices of the present invention are used in ACL reconstruction. However, the devices can be used in numerous procedures. In the ACL reconstruction example, the device of the invention having the multifunctional characteristic of bioactive efficacy and mechanical integrity is able to be used as a bioactive interference screw in bone/tissue structure fixation.

An example of a procedure to treat a bone defect is ACL reconstructive surgery. This procedure involves replacing the torn ligament with new tissue (a graft), repairing and/or replacing the damaged ACL and dealing with knee instability, pain and recurrent swelling. A surgeon often uses an arthroscope and surgical tools to treat any other injuries. Then small holes are drilled in the bone. The graft is passed through the drilled holes to position the graft/repaired ligament. Interference screws or anchors of the invention are used to secure the graft in place.

Accordingly, the invention provides for methods of treating a patient by implanting a surgical fixation device having at least multifunctional properties (e.g., mechanical integrity and bioactive efficacy) to promote bone healing and re-growth. In some instance, after implantation, the device comprises cells, wherein the grafted cells can respond to environmental cues that will cause it to develop characteristics of the endogenous tissue. For example, if the cells are implanted into the ACL, it will be induced to synthesize components of the ACL. The grafted cells can also secret growth factors to the environment wherein the growth factors contribute to bone healing and re- growth. In other instance, the grafted cells form bone structures, comprised of differentiated bone cells. Thus, the implanted cells can develop characteristics that liken it to the surrounding tissue. By these methods, the implant can augment the tissue; the biological implant of the invention can be used for tissue engineering and in any conventional tissue engineering setting.

In other embodiments, the device of the invention can also comprise a bioactive agent. A preferred bioactive agent provides osteoinductivity to the bone and/or soft tissue. In another embodiment, the bioactive agent provides osteoconductivity to the bone and/or soft tissue. In yet another embodiment, the device comprises a combination of bioactive agents that provide osteoinductivity and osteocondictivity to the bone and/or soft tissue to promote healing and re-growth. Accordingly, the invention encompasses tissue regeneration applications.

The objective of the tissue regeneration therapy approach is to deliver high densities of repair-competent cells (or cells that can become competent when influenced by the local environment) to the defect site in a format that optimizes both initial wound mechanics and eventually new tissue production. The composition of the instant invention is particularly useful in methods to alleviate or bone defects and/or soft tissue defects in individuals. Advantageously, the composition of the invention provides for improved bone and/or soft tissue regeneration. Specifically, the tissue regeneration is achieved more rapidly as a result of the inventive composition. In one embodiment, the surgical fixation device of the invention is a bioactive interference screw. The screw is applicable for ACL and bone-tendon-bone (BTB) fixations. The screw is tapered without head so it does not protrude out of the drilled hole. The tapered feature is specifically designed to provide maximum pull-out strength in ACL reconstruction and it is slightly more tapered at the tip to facilitate insertion when graft is tightly fitting. Also the screw possesses a rounded blunt posterior aspect to prevent graft damage. The screw is molded with a high gloss finish using injection mold technique to aid insertion. In addition, these screws include full-length tapers so that the highest insertion torque is only realized once the screw is fully inserted. A full-length taper has been shown to increase fixation and pullout strength. A reduced thread pitch eases insertion by effectively reducing the screw "lead" (the axial distance the screw travels into the bone tunnel per screw turn). This difference in "lead" reduces the stress within the screw as it is turned, ensuring that the screw is not subjected to forces beyond the material limits. The design incorporates a tapered screw design and tapered driver, thus decreasing insertion stress while maintaining fixation strength. The innovative new design minimizes driver stripping and screw breaking by optimizing stress distribution and force transfer. In addition, the designed tapered design and enhanced surface finish can reduce insertion torque, making insertion easier.

The hollow core of these innovative screws can be embedded with additional materials like growth factor and drugs by the surgeon to stimulate and allow for bone growth in the tunnel. The surrounding environment is connected with the driver shaft using gradient cellular holes. This entire system is continuous, and therefore maximizes bone re-growth after insertion.

The composition of the invention may be administered to an individual in need thereof in a wide variety of ways. Preferred modes of administration include intravenous, intravascular, intramuscular, subcutaneous, intracerebral, intraperitoneal, soft tissue injection, surgical placement, arthroscopic placement, and percutaneous insertion, e.g. direct injection, cannulation or catheterization. Most preferred methods result in localized administration of the inventive composition to the site or sites of tissue defect. Any administration may be a single application of a composition of invention or multiple applications. Administrations may be to single site or to more than one site in the individual to be treated. Multiple administrations may occur essentially at the same time or separated in time. EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

The experiments presented herein were designed to generate a bioactive fixation device that promotes a therapeutic effect (e.g., bone healing and re-growth). For example, the surgical fixation devices encompass a core reservoir to bioactive materials (e.g., bone healing drugs, growth factors, cells, and the like) and an inter-connective porous wall, which allows for locally controlled delivery of the desired biomaterials.

Some of the experimental were designed based on the following criteria: the device should be strong enough to withstand the mechanical stress during insertion and post-operative activity; the device should possess controllable biodegradation to promote tissue growth at a rate that is comparable to the degradation of the device, and the device is capable of being substituted by bone which can fill any defects of the device. The use of the fixation devices also avoids the need for future implant removal operation owing to adverse reactions or implant loosening. The fixation devices are also biocompatible, i.e., they do not cause an inflammatory or immune response in the body. To fulfill the aforementioned design criteria, the surgical fixation devices were designed to comprise at least the following components: (1) a core that is first used as the screw driver interface and then after the screw insertion is used as a reservoir to store syringe-injected hydrogel embedded with bioactive materials, such as bone healing drugs, growth factors, such as bone morphogenetic protein 2 (BMP2), or bone cells/stem cells; and (2) an interconnective porous wall, which allows for local, controlled delivery of the desired biomaterials. The porous screw also serves as a scaffold for osteoblasts to migrate into the screws, where they can proliferate/differentiate and form new bone tissue as the screw degrades.

Example 1 : Biomaterials

In order to develop novel surgical fixation devices (screws, anchors, plates, pins, staples, etc.) that not only secure a graft in place, but incorporate bioactive materials such as growth factors, drugs and cells, intended to promote/accelerate bone tissue growth, it is desirable to conduct biomaterials research to identify and optimize suitable structural materials for use in generating a strong screw body and bioactive materials for efficient bone/tissue healing and growth. By way of example, co- polyglycolic/lactic acid (PLGA) and calcium phosphate (CaP) were initially tested as candidates of composites for generating a screw body material.

The following experiments were designed to select suitable biomaterials for generating bioactive surgical fixation devices. The desirable characteristics of these materials are biocompatibility (i.e., not to produce any unwanted tissue response to the implant, and at the same time to possess the right surface chemistry to promote cell attachment and function) and biodegradability (i.e., degradable into nontoxic products), leaving the desired living tissue with desired mechanical properties (Weiler et al., 1998, Am J Sports Med. 26(1): 1 19-26; Barber et al., 2000, Biomaterials 21(24):2623-2629; Lee et al., 2005, Biomaterials, 26(16):3249-3257). Polylactic acid (PLA), polyglycolic acid (PGA), and co-polyglycolic/lactic acid (PLGA) are amongst the most commonly used synthetic, biodegradable polymers, with an extensive U.S. Food and Drug Administration (FDA) approval history (Ella et al., 2005, J Mater Sci Mater Med. 16(7):655-662, Huh et al., 2003, Drug Delivery Technology 3(5):52-58, Kolybaba, M., et al. Biodegradable Polymers: Past, Present, and Future, in CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE. October 3-4, 2003. Quality Inn & Suites 301 3rd Avenue North Fargo, North Dakota, USA). PGA is a highly crystalline hydrophilic polymer, which tends to lose its mechanical strength rapidly (50%) over a period of 2 weeks. Upon implantation, PGA degrades in about 4 weeks and can be completely absorbed in 4-6 months (Kolybaba, M., et al. Biodegradable Polymers: Past, Present, and Future, in CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE. October 3-4, 2003. Quality Inn & Suites 301 3rd Avenue North Fargo, North Dakota, USA; Grayson et al., 2005, Biomaterials, 26(14):2137-2145; Ouyang et al., 2002, Mat Sci Eng C-Bio S. 20(l-2):63-69; Zhang et al., 2006, Polym Degrad Stabil, 91(9): 1929-1936; Panyam et al., 2003, J Control Release 92(1-2): 173- 187; Oh et al., 2006, J Mater Sci Mater Med. 17(2): 131-137; Valimaa et al., 2004, Biomaterials 25(7-8): 1225-1232; Habraken et al., 2006, J Biomater Sci Polym Ed

17(9): 1057- 1074). Although structurally similar to PGA, PLA is quite different in terms of its chemical, physical, and mechanical properties because of the presence of a pendant methyl group on the alpha carbon (Kolybaba, M., et al. Biodegradable Polymers: Past, Present, and Future, in CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE. October 3-4, 2003. Quality Inn & Suites 301 3rd Avenue North Fargo, North Dakota, USA). PLA has a high modulus that makes it more suitable for load-bearing applications. For PLGA copolymers, the mechanical strength and, especially, the degradation rate, depend on the ratio of PLA/PGA. The higher the contents of PLA in the PLGA copolymer, the higher will be the mechanical properties. PGA is more hydrophilic than PLA; therefore, a higher percentage of PGA leads to a faster degradation rate. PLGA degradation is also influenced by other factors including the polymer chain length and characteristics of the surrounding medium (Kolybaba, M., et al. Biodegradable Polymers: Past, Present, and Future, in CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE. October 3-4, 2003. Quality Inn & Suites 301 3rd Avenue North Fargo, North Dakota, USA; Grayson et al.,

2005, Biomaterials, 26(14):2137-2145; Ouyang et al., 2002, Mat Sci Eng C-Bio S. 20(1- 2):63-69; Zhang et al., 2006, Polym Degrad Stabil, 91 (9): 1929-1936; Panyam et a]., 2003, J Control Release 92(1-2):173-187; Oh et al., 2006, J Mater Sci Mater Med. 17(2): 131-137; Valimaa et al., 2004, Biomaterials 25(7-8): 1225-1232; Habraken et al.,

2006, J Biomater Sci Polym Ed 17(9): 1057-1074]. PLGA degrades by hydrolysis into lactic and glycolic acids, both of which are harmless physiological metabolites.

PLGA has been extensively investigated for use in a wide range of applications based on its good mechanical properties, versatile degradation kinetics, non- toxicity, and biocompatibility (Kolybaba, M., et al. Biodegradable Polymers: Past,

Present, and Future, in CSAE/ASAE Annual Intersectional Meeting Sponsored by the Red River Section of ASAE. October 3-4, 2003. Quality Inn & Suites 301 3rd Avenue North Fargo, North Dakota, USA; Ouyang et al., 2002, Mat Sci Eng C-Bio S. 20(l-2):63- 69; Zhang et al., 2006, Polym Degrad Stabil, 91(9):1929-1936; Panyam et al., 2003, J Control Release 92(1-2): 173-187; Oh et al., 2006, J Mater Sci Mater Med. 17(2): 131- 137; Valimaa et al., 2004, Biomaterials 25(7-8): 1225-1232; Habraken et al., 2006, J Biomater Sci Polym Ed 17(9): 1057- 1074). PLGAs have been in use for over 20 years in surgical sutures, and have a long and favorable clinical history. They have been extensively used as implantable materials, e.g., as controlled drug delivery microcapsules, and tissue engineering scaffolds because of their wide range of physical properties, degradation rate, and their controllable degradation kinetics, ranging from weeks to months or even longer.

Inorganic calcium phosphate (CaP) is another biocompatible biomaterial frequently used in bone substitutes. Due to its close resemblance to the inorganic phase of the natural bone mineral, CaP has been widely used as a bone substitute in dental, orthopedic, and reconstructive surgery [Koerten et al., 1999, J Biomed Mater Res. 44(l):78-86; Korventausta et al., 2003, Biomaterials 24(28): 5173-5182;]. The incorporation of a biocompatible CaP, having the same ions as those found in the human skeleton, offers major advantages when applied to a resorbable orthopedic device

[Habraken et al., 2006, J Biomater Sci Polym Ed 17(9):1057-1074; Koerten et al., 1999, J Biomed Mater Res. 44(l):78-86; Korventausta et al., 2003, Biomaterials 24(28): 5173- 5182]. CaP has a relatively high mechanical strength, as compared to PLGA, and shows advantageous osteoinductive and osteoconductive characteristics. In general, CaP degrades relatively slowly (in 2-3 years) (Schnettler R et al., 2004 Eur J Trauma 30: 219- 229). When implanted in the body, CaP slowly dissolves to total resorption with the Ca and P ions re-precipitating as apatite crystals, producing a thin layer of hydroxyapatite (the mineral phase of bone) which then forms around the implant [Korventausta et al., 2003, Biomaterials 24(28): 5173-5182; Verheyen et al., , C.C.P.M., et al., Mechanical behaviour of hydroxylapatite/poly(L-lactide) composites, in Ceramics in substitutive and reconstructive surgery, P. Vincenzini, Editor. 1991, Elsevier Science Publishers: Amsterdam, p. 275-285]. To adjust the degradation rate to a physiologically required value, faster degrading polymers, such as PLGA, can be mixed with CaP [Korventausta et al., 2003, Biomaterials 24(28): 5173-5182; Verheyen, C.C.P.M., et al., Mechanical behaviour of hydroxylapatite/poly(L-lactide) composites, in Ceramics in substitu-tive and reconstructive surgery, P. Vincenzini, Editor. 1991, Elsevier Science Publishers: Amsterdam, p. 275-285; Ruhe et al., 2006, Tissue Eng 12(4):789-800]. Studies investigating the potential of CaP when used as a composite material (Schnettler R et al., 2004 Eur J Trauma 30: 219-229); Verheyen, C.C.P.M., et al., Mechanical behaviour of hydroxylapatite/poly(L-lactide) composites, in Ceramics in substitu-tive and reconstructive surgery, P. Vincenzini, Editor. 1991, Elsevier Science Publishers: Amsterdam, p. 275-285; Ruhe et al., 2006, Tissue Eng 12(4):789-800], have shown significant improvements in the surgical outcome.

In using the proposed bioactive surgical device(s) as a load bearing body, the first consideration for the material selection is whether it is strong enough to withstand the stress during implantation and post-operative activities. PLGA is attractive due to its adjustable biodegradability and high mechanical strength, although it is not bioactive. In order to improve the osteoconductivity and bioactivity of the PLGA screws, CaP powder is compounded with PLGA to make PLGA-CaP composite screws. With alteration in the CaP/PLGA and PLA/PGA ratio, mechanical properties and biodegradation behavior can be changed in a controlled fashion. In some instances, the CaP used in these experiments were not subjected to calcination or sintering, and has a chemical composition similar to that which is synthesized in the body. CaP causes minimal physical irritation of the surrounding tissues, gradually biodegrades and is resorbed [Verheyen, C. C. P. M., et al., Mechanical behaviour of hydroxylapatite/poly(L-lactide) composites, in Ceramics in substitu-tive and reconstructive surgery, P. Vincenzini, Editor. 1991 , Elsevier Science Publishers: Amsterdam, p. 275-285; Ruhe et al., 2006, Tissue Eng 12(4):789-800], and it also exhibits superior bioactivity, such as osteoinductivity, osteoconductivity and bone- binding ability, together with excellent biocompatibility and safety. Therefore, the surgical device can provide the surgeon with the capability of returning the tissue at the fixation site to its pre-injury condition [Ella et al., 2005, J Mater Sci Mater Med. 16(7):655-662; Habraken et al., 2006, J Biomater Sci Polym Ed 17(9):1057-1074]. The combination of PLGA and CaP is an example of a composition that is ideal for generating an implantable fixation device which offers improved biocompatibility associated with controlled degradation rate. An advantage of using the combination of PLGA and CaP for generating the implantable device is the enhanced gradual formation of new tissues concomitant with longer strength retention [Verheyen, C. C. P. M., et al., Mechanical behaviour of hydroxylapatite/poIy(L-lactide) composites, in Ceramics in substitu-tive and reconstructive surgery, P. Vincenzini, Editor. 1991 , Elsevier Science Publishers: Amsterdam, p. 275-285; Ruhe et al., 2006, Tissue Eng 12(4):789-800; Ooms et al., 2003, Biomaterials 24(6):989-1000; Baxter et al., 2002, Eur Cell Mater 4: 1-17].

Example 2: Bioactive Screw Design:

The following experiments were designed to generate a novel bioactive interference screw for ACL fixations. It is desirable that the bioactive interference screw is multifunctional with respect to mechanical integrity and bioactive efficacy. Preferably, the screw is designed with a hollow core as reservoir and gradient porous wall as bioactive materials delivery channels.

Without wishing to be bound by any particular theory, it is believed that the screw should be tapered without a head so that it will not protrude out of the drilled hole. The tapered feature is designed to provide maximum pull-out strength in ACL reconstruction, the screw is slightly more tapered at the tip to facilitate insertion taking into account the tight fit at this locale. The screw also possesses a rounded blunt posterior portion to prevent graft damage. In addition, these screws include full-length tapers so that the highest insertion torque is only realized once the screw is fully inserted. A reduced thread pitch (the axial distance per screw turn) eases insertion by effectively reducing the screw "lead", which the screw uses to travel into the bone tunnel. This difference in "lead" reduces the stress within the screw as it is turned, ensuring that the screw is not subjected to forces beyond the material limits.

The screw design incorporates a tapered screw and a tapered driver, thus decreasing the insertion stress while maintaining fixation strength. This design minimizes driver stripping and screw breaking by optimizing stress distribution and force transfer. As a result, the tapered design reduces insertion torque, making insertion easier.

The hollow core of these innovative screws (after the screws are inserted) can additionally be filled with bioactive materials like growth factors, drugs and/or cells at the time of surgery in the operating room setting to stimulate bone growth. For maximum effectiveness, the bone adjacent to the screw and the bio-reagents in the core will be connected with gradient cellular pores, creating a continuous system that accelerates bone re-growth upon insertion and filling the void upon degradation of the screw. Figure l is a schematic depiction of the innovative bioactive interference screw of the invention.

Example 3: Application of Gradient Cellular Structure (GCS)

The following experiments were designed to generate bioactive surgical fixation implant devices that are multifunctional. Without wishing to be bound by any particular theory, it is believed that the device should have the necessary stiffness and strength. It is also desirable to have the device be permeable so that the device can deliver bioactive agents to the environment.

Nature is an excellent producer of similar multifunctional structures, e.g., wood, eggshell, wheat and rice, bone, skin, and others. These naturally porous materials all have a gradient cellular structure (GCS), meaning that the porosity is not uniform. Rather, it is distributed in space so as to maximize the overall performance of the structure. Often, the pore size continuously varies from the surface to the core, as in bone and in eggshell [Zhang et al., 2005, Journal of Zhejiang University Science 6a(10):1095- 1099]. In bone, regions of dense "cortical" bone neighbor regions of low-density "trabecular" bone. With pore sizes decreasing from the core to the exterior, bone is able to maintain a highly permeable core and yet provide outer wall structural integrity. The increase of stiffness and strength with the reduction in pore size is understandable from both experimental and theoretical perspectives. Similarly, the avian eggshell utilizes a GCS to achieve desired mechanical performance and in the meantime a necessary permeability. From inside to outside, the pore size varies from several 100 nm to a couple of microns [Zhang et al., 2005, Journal of Zhejiang University Science 6a(10):1095-1099; Kitimasak, et al., 2003, Science Asia. 29:95-98]. These graded pores serve for the exchange of matter between the outside and inside of the eggshell while having enough strength to prevent the shell from cracking caused by collision or impact. This GCS further improves the toughness of the eggshell. In view of the unique capabilities of natural GCS, particularly the ability to integrate different functions (often contradictory) and achieve an optimized design, the experiments were designed to utilize GCS for the realization of innovative biomimetic devices. By applying such a bionic design, a "permeable" bioactive surgical fixation device with high structural integrity can be developed. With GCS, variation of permeability throughout the device can also be regulated. Further, variation of mechanical properties of the structure to match other materials at the interface is made possible. Therefore, implementation of the GCS design allows for the development of innovative bioactive surgical fixation implant devices that are multifunctional.

The next set of experiments were designed to apply the GCS principle for creating an interconnective porous structure. It is believed that the porous structure assures the screw's mechanical strength and assists in the delivery of bioactive materials deposited within the screw and facilitates bone ingrowth. The fundamental principles of process modeling and simulation, and realistic/applicable injection molding procedures are applicable to the generation of the bioactive surgical fixation implant device.

The GCS concept can be used to overcome some design conflicts in bioactive surgical devices. GCSs have been provided by nature for millions of years; however, they are difficult to reproduce synthetically. Porous structures are traditionally produced by salt leaching [Wang et al., 2007 Cell Polym 26(1): 11-35; Yang et al., 2006, J Macromol Sci B 45(6):1171-1181], gas foaming [Heijkants et al., 2006, J Mater Sci 41 (8):2423-2428; Lee et al., 2005, Polymer Korea, 29(2): 198-203] and phase inversion processes [Nishikawa et al., 2002 Int. J. Nanoscience 1 : 415-418; Sato et al., 2002,. Int. J. Nanoscience 1 :689-694], which are physically, thermally or chemically induced, respectively. With careful process tuning, the pore size can be adjusted in these processes, but a gradient and interconnective structure can not be obtained. Recent developments in microcellular processing [Kumar, 1993, Cellular Polymers, 12(3):207- 223; Suh, 2003, Macromolecular Symposia 201 : 187-201 ; Gong et al., 2005, International Polymer Processing, 20(2):202-214] allow effective production of materials with micron to submicron pores, but again, a gradient pore size and interconnectivity cannot be created. On the other hand, rapid prototyping methods, e.g., fusion deposition modeling and laser sintering, can be used to produce gradient structures but they have a low resolution, prohibiting the production of fine GCSs. Recently, GCSs with pore diameters of- 200 μm have been created. Although such a coarse GCS can be used to optimize mechanical properties, the structure had an undesirably high permeability; thus, exchange of matter from inside and outside cannot be regulated.

Preliminary results show that through control of the thermomechanical history of the blend during processing, controllable pore sizes and gradient porous structures can be obtained.

Example 4: GCS fabrication

Gradient materials are be prepared by injection molding an immiscible polymer blend, with spatially controlled thermal conditioning to adjust the phase size from core to surface. For example, polylactides/glycolides (PLA, PGA, or PLGA) and a sacrificial, immiscible polymer (e.g., polystyrene) are initially melt-blended in a batch mixer to ensure the development of a fine blend structure with phase size in the micrometer regime. The blend is then injection molded into the mold cavity for the bioactive screw. While inside the mold, the blend are thermally conditioned with higher temperature at the core and lower temperature at the surface, resulting in the development of coarse phases at the core but fine phases at the surface (Figure 1). The interfacial surface tension between the two polymers causes coarsening of the phase structure at high-temperature regions. The gradient in phase size can be regulated by adjusting the thermal history. When the desired growth is reached, the mold is cooled and the screw can be ejected. The sacrificial polymer in the screw is selectively dissolved or degraded to obtain an interconnected GCS for the screw.

Example 4: Injection molding of GCS surgical screws The steps involved in preparing a screw with a gradient cellular structure include: 1) blending the biomaterial with a sacrificial polymer to form a well dispersed but immiscible blend; 2) GCS injection molding of the blend using a gradient thermal boundary; and 3) selective dissolution of the sacrificial polymer and drying to form a GCS. For example, a Brabender™ batch mixer can be used to develop a well dispersed immiscible blend, with a co-continuous structure and a pore size of several microns or smaller.

During selective dissolution, water and biocompatible solvents are preferably used. However, other solvents can be used. If other solvents are needed, the extraction is performed by flushing in water to remove all solvent residues. For example, a PLA/PCL system is tested for its suitability. Polycaprolactone (PCL) is another biocompatible and biodegradable polymer. The PCL phase can be extracted with acetic acid. This polymer system can be used to create well-defined co-continuous phase structure. However, the two polymers have very different melting temperature, thus increasing the processing difficulty. Other more polar polymers such as polystyrene (PS) can also be tested. In the case of PS, extraction can start with cyclohexanone, followed by an alcohol based solvent, and finally water. In addition, PLA (or PLGA) and PGA for forming a co-continuous phase morphology and subsequently degrading the PGA phase to form a porous PLA structure are tested. PGA has a much higher degradation rate than PLA. Therefore, such a system is fully biocompatible since no solvent is involved.

Another advantage of this method is that degradation of the PGA phase can be induced in vivo. This allows the use of a fully dense screw during insertion.

An important step in GCS preparation is the injection molding step. Special handling is needed to create a differential thermal boundary for the formation of a GCS (Figure 2). For this purpose, a thin heater (e.g., a thin cartridge heater) is embedded in the hexagonal core to control the core temperature. A separate heating/cooling unit is employed in the mold base. This setup allows differential control of the thermal conditions from the surface to the core of the molding polymer. The different thermal history results in different degrees of coarsening in the phase structure; particularly, it results in a coarser structure near the core and a finer one at the surface. From preliminary thermal conditioning experiments with polymer blends, it is believed that a dwell time of a couple of minutes at an elevated temperature can result in a significant amount of structural coarsening. The desired structure can be approached through adjustment of the two temperatures, i.e. core and base temperatures. Rapid mold heating techniques [e.g., Yao et al, 2002, Polym Eng Sci 42(12):2471-2481 ; Yao et al, 2002, Polym-Plast Technol 41 (5):819-832; Yao, et al., 2006, Polym Eng Sci 46(7):938-945] can be used to rapidly heat the core and the mold, thus facilitating in situ control to yield the desired thermal history. In GCS molding, a normal filling process with a filling time on the order of one second can be used, followed by a prolonged holding stage, e.g., several minutes, to thermally condition the phase structure. The actual mold and core temperatures and the hold time depend on the desired phase structure that needs to grow, and is determined using an inverse design approach.

Example 5: Simulation of GCS Injection Molding

A two-step approach is used to simulate the structural evolution processing in GCS injection molding. In the first step, the flow and heat transfer process in injection molding is simulated. Given the typical 3-D geometry of a surgical device, a 3-D model, rather than a classical 2.5-D Hele-Shaw model, is adopted for simulating GCS injection molding. Specifically, this model implements the 3-D version of the conservation equations with the inclusion of inertia and possible gravitational effects (if jetting is important), by solving the following conservation equations (in tensor and vector format) [Mavridis et al., 1986, Advances in Polymer Technology 6(4):457-466]: 0 = dp/dt + V - pv . _{Mass Conservation} ( i )

~^~ ; Momentum Conservation (2) pc_p{dτ/dt + v - vτ)= v - kvτ + ±_η7k fi

= = ; Energy Conservation (3) where ^p , ^k , ^Cp and ^η are density, thermal conductivity, specific heat, and viscosity,

respectively, p is pressure, T is temperature, — is gravitational acceleration, - is a

velocity vector, and = is a strain rate tensor. The dependency of viscosity on

*, T and p is accommodated by using a 7-constant Cross model, as well-documented in the literature [Chiang et al., 1991, Polym Eng Sci, 31 :116-124; Cross, 1979, Rheologica Acta, 18:609-614]. These equations are used to model both filling and holding stages.

In the second step, the phase evolution process at each individual material point are predicted by solving a surface tension driven flow problem with the thermomechanical history obtained in the first step as the boundary condition. Since in GCS molding the filling stage is considerably shorter than the holding stage, the phase evolution process is considered starting from the beginning of holding. The morphology at the end of filling is be used as a starting morphology. This starting morphology is approximated based on the morphology of the initial blend (determined by mercury intrusion) and the deformation of a local material point in the filling stage. In other words, the morphology at the end of filling have a similar mean size of pores, but the pore shape is changed due to flow. The evolution of the phase structure during the holding stage is predicted using a surface tension driven flow model, - Vp + V - ηfi+ λ>c^ή = O

where ⁿ is a unit normal, ^ = V ^• « j_s the local curvature, and ^ is surface tension. Due to the nature of creep flow during the holding stage, the inertia effects are negligible. Both viscosity and surface tension depend on the temperature history during the holding stage, which is solved separately in step one. Figure 3 shows coarsening of the phase structure after a period of annealing. The surface tension and viscosity at different temperatures are measured using a standard surface tension measurement unit and a capillary rheometer.

A modified diffusion law is used to simulate diffusion of drugs and growth factors in GCS, as given in the following equation:

dt \ (5) where c is concentration and^α is a diffusivity parameter. For a GCS, ^a is location dependent; that is, ^a is larger in larger pore areas and smaller in smaller pore areas. Materials with a uniform pore size are prepared using isothermal conditioning. Such porous materials are used to characterize the diffusivity parameter for different pore sizes. An inverse design approach is used to find the suitable temperature history of the core and the mold base in order to achieve a desired morphology. In order to achieve a high strength and stiffness, small pore size is needed. The pore size in the sample prepared by batch mixing is in the 1-5 micron range. Such a pore size is considered fine enough to achieve the desired mechanical strength [Kumar, 1993, Cellular Polymers, 12(3):207-223; Suh, 2003, Macromolecular Symposia 201 : 187-201 ; Gong et al., 2005, International Polymer Processing, 20(2):202-214]. Thus, the design objective is to enlarge the thickness of the skin section with this small pore size and to grow the pore size only in the core section, in order to optimize the permeation rate in the diffusion process. The inverse design starts from the determination of a desired pore size distribution based on the permeability requirement. Using the desired permeation rate as a constraint, Equation (5) is used to find a pore size distribution according to the following inverse design scheme, d[m(a) - m] _Q

3(Vs) ^" _{; (6)} where s is pore size, m is the permeability calculated based on the pore size gradient^ , and ^m is the desired permeability. By solving Equation (6), the desired pore size distribution can be obtained. This distribution is used to determine the needed core temperature history in the next step. The inverse design scheme for this step is:

where ^s'^x' is the desired pore size distribution and ^s'^x' is the predicted pore size distribution based on the core temperature history ^c' ' . The mold base temperature history can also be included in Equation (7) if the pore size near the mold surface also needs to be controlled. Similar inverse design approaches have been used in solving a number of engineering problems, including injection molding [Kang et al., 1998, J Therm Stresses, 21(2):141-155].

Example 6: Mechanical and Biological Testing Mechanical testing:

The most frequently reported complication associated with biodegradable screws is screw breakage during insertion [Evans et al., 2002, J Mater Sci Mater Med.l3(12):l 143-1145; Barber et al., 2000, Biomaterials 21(24):2623-2629; Lee et al., 2005, Biomaterials, 26(16):3249-3257]. Material studies on PLGA/CaP composite is conducted to determine mechanical properties of the screw. Mechanical properties of the PLGA/CaP surgical devices including compression strength, tensile strength, Young's modulus, and pull-out strength are determined. The compressive and tensile tests can be performed using a Tenius Olsen H25KT single column materials testing machine, which we previously used it to characterize PCL/CaP scaffolds. A torque meter is used to determine the maximum torque that can be applied to the screw. Pullout/pushout experiments are performed using wood, and model bon (polyurethane foams, Sawbones), both of which are established tests that approximate the mechanical properties of real bone.

Upon implantation of the bioactive surgical devices, serum and interstitial body fluids (electrolyte containing bioactive proteins and peptides) come into contact with the device. Once these body fluids enter the device via the pores in the device, the device degrades/hydrolyzes. The resorption of the device depends on the shape, size and site of implantation of the device. Without wishing to be bound by any particular theory, the phosphate/polymer resorbable composite degrades in vivo in a manner consistent with healthy bone regeneration in bone augmentation and repair procedures. It is believed that the fine particles of CaP is actively released together with fine particles of reduced- molecular weight PLGA. The structure therefore gradually degrades and shrinks. At the same time, CaP continuously induces/conducts new bone formation (with additional help from admixed, bone-regenerating growth factors, such as BMP2) in the degrading sections of the screw in vivo. This system leads to total repair of the bone once the device is completely disintegrated. This in vivo situation is simulated in vitro by incubating and maintaining the bioactive screws under aseptic conditions in complete cell culture medium supplemented with 20% bovine serum. The decrease in molecular weight of the PLGA polymer over time indicates the progression of degradation. The weight loss is measured over time and a comparison of cross-sectional images of the degrading bioactive device is made to determine the degradation rate of the device in in vitro testing.

Toxicity screening:

Material toxicity is tested using extraction assays. Extracts are prepared by incubating surgical device material for up to 30 days in protein-containing culture medium. Human osteoblasts (ATCC, CRL-11372) are cultured in MEM culture medium supplemented with 10% fetal calf serum and antibiotics, with cell-free medium as negative controls. Potential toxicity-induced changes in cell morphology and detachment can be monitored by fluorescent and scanning electron microscopy. Cell viability and proliferation are quantified over a 30 day period by using the alamar blue™ assay [Mondrinos et al., 2006, Biomaterials 27(25):4399-4408; Nikolaychik et al., 1996, J Biomater Sci Polym Ed 7:881-891].

Cell attachment and proliferation: Cell attachment and proliferation are assessed by seeding composite disks with human osteoblasts, as discussed elsewhere herein. Composite materials are cast under aseptic conditions into 10 mm diameter 1.5 mm thick disks, to fit in 24-well plates. Following seeding, cell viability are assayed for up to 30 days using the Alamar Blue assay [Nikolaychik et al., 1996, J Biomater Sci Polym Ed 7:881-891]. Cell distribution in the disks are quantified after fixation and sectioning (lOμm) by light microscopy, following staining with hematoxylin and eosin, as well as by fluorescence microscopy of thicker sections (60μm) after staining the nuclei and microfilaments with, Hoechst 33258 and rhodamine phalloidin, respectively [Li et al., 2006, Biomaterials, 27:2705-2715; Li et al., 2005, Biomaterials, 26:5999-6008].

Example 5: Bioactive implanted surgical fixation device GCS Results: Polycaprolactone (PCL) and polyethylene oxide (PEO) were used as a model system for the development of continuous, gradient porous structures. The blend was first mixed in a Brabender™ batch mixer, resulting in a co-continuous structure, with a phase size in the micrometer range. To produce a structure with continuous pores, the blend was extruded with different dwell times in the extruder for thermal conditioning, and finally selectively dissolved in water and dried. It was observed that thermal conditioning can be effectively used to control the phase structure and thus the porous structure of the PCL after dissolution of the PEO phase (Figure 4). The blend was also injection molded into a cold mold. It was found that for a 6 mm circular channel, a gradient structure was developed, with finer structure at the surface and coarser structure in the center (Figure 5).

Scaffold Fabrication:

The scaffolds of the invention can be fabrication using different biomaterials, including pure polycaprolactone (PCL), calcium phosphate cement (CPC) and homogeneous composites of PCL and calcium phosphate (CaP, 10% or 20% w/w). The scaffolds of the present invention is partly based on a novel porogen method for tissue engineering scaffold fabrication. The porogen method is based on injectable porogens fabricated by drop on demand printing (DDP). Thermoplastic porogens were designed using Pro/Engineer and fabricated with a commercially available DDP machine. Then, molten polymer-ceramic composites were injection-molded into the porogen structure. The precisely molded scaffold was separated from the porogen in an agitated ethanol bath. Attainable scaffold pore sizes using the porogen-based method were found to be 200 μm for pure PCL. Figure 6 shows SEM photographs of the fabricated scaffolds. The mechanical and biological properties of the scaffold (e.g., the compressive and tensile strengths of pure PCL, and PCL-CaP composite materials (90: 10 and 80:20)) were measured according to ASTM standards. In addition to testing the mechanical properties of solid cylinders and dog-bone-shape specimens of different scaffold materials, compressive and tensile strengths of scaffolds with 600μm pore size, made of pure PCL, and 90:10 and 80:20 PCL-CaP were tested. The increase in CaP content resulted in a statistically significant increase in compressive modulus and ultimate compressive strength of the samples (Figure 7). However, the relatively low modulus and strength of the PCL and PCL-CaP composites indicated that stronger polymers such as PLGA desirable. The scaffolds of the invention are capable of supporting the growth of cells. For example, Figure 8 demonstrates that our scaffolds supported cell attachment, as visualized by nuclear staining with bisbenbenzimide (Hoechst 33258). Specifically, Figure 8 depicts the assessment of cytocompatibility of bone-scaffolds using human embryonic palatal mesenchymal (HEPM) cells. The next set of experiments were designed to assess the degradation levels of the scaffolds. Degradation experiments were conducted by incubating solid cylinders made of PCL, 90/10 and 80/20 PCL-CaP composites in both a physiological buffer (DMEM) and upon admixing blood proteins (DMEM with 10%FBS). The cylinders were harvested at different times, and their molecular weight distribution was analyzed using High Performance Liquid Chromatography (HPLC, Waters, USA) equipped with a refractive index detector. HPLC measurements were carried out at 25 °C and at a flow rate of 0.8 ml/min using THF (tetrahydrofuran) as an HPLC solvent. Polystyrene standards (Polyscience Co.) were used for calibration. The results indicated that the molecular weight of PCL decreases over time and that the effect was more pronounced in the present of blood proteins (Figure 9). During the time period tested (up to 6 weeks) the observed change was small (< 1%) but measurable; the small change was consistent with the fact that PCL has a long degradation time (~ 2 years).

Example 6: Push-out Test for Interference Screw Rigid polyurethane (PU) foam (Sawbones, Pacific Research Laboratories, Vashon WA), with mechanical properties similar to osteoporotic cancellous bone, was used to characterize the push-out strength of polystyrene PS screws. The uniformity and consistent properties of the PU foam make it an ideal material for comparative testing of bones screws and other medical devices and instrument. ASTM F-1839 Standard was adopted to test the mechanical performance of the PS model bone screws. Fixtures were designed for mechanical testing of the screws (Figure 10). The PS screw was screwed into a predrilled hole on the sawbone testing block; the upper fixture acted as a pusher plate to push the screw out of the testing block into the hollow lower fixture. The failure mode of the PS interference screw has been evaluated by the push-out test. The maximum load at failure for the PS screws was 1200±100N (Figure 11), which compares favorably to the pull-out strength reported for commercially available biodegradable or titanium screws (400-900 N).

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. An implantable device comprising a gradient porous structure, wherein said structure has a gradient of pore size such that the size of pores located closer to the outer surface of the structure is smaller than the size of pores located further from the outer surface of the structure.

2. The device of claim 1, wherein the gradient porous structure is a permeable structure.

3. The device of claim 1, further comprising a hollow core, wherein the core can be filled with a bioactive agent.

4. The device of claim 1, wherein the gradient porous structure comprises a polymer selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), co-polyglycolic/lactic acid (PLGA), and any combinations thereof.

5. The device of claim 1, wherein the gradient porous structure comprises a composite of PLGA and calcium phosphate (CaP).

6. The device of claim 1, wherein the gradient porous structure comprises a composite of PLA and calcium phosphate (CaP).

7. The device of claim 3, wherein the bioactive agent is released or controlled released within a recipient.

8. The device of claim 3, wherein the bioactive agent comprises at least one agent selected from the group consisting of an antibiotic, a growth factor, a drug, a cell, and any combination thereof.

9. The device of claim 1, wherein the gradient porous structure degrades over a period of time within a recipient.

10. The device of claim 1, wherein the device promotes bone growth within a recipient.

11. A method of producing a structure having a gradient of pore size such that the size of pores located closer to the outer surface of the structure is smaller than the size of pores located further from the outer surface of the structure, said method comprising injection molding a biodegradable blend, wherein the injection molding comprises spatially controlled thermal conditioning to adjust phase size of the porous structure, thereby producing said device.

12. The method of claim 1 1 , wherein the thermal conditioning comprises a lower temperature at areas located closer to the outer surface of the structure and a higher temperature at areas located further from the outer surface of the structure.

13. The method of claim 1 1 , wherein the biodegradable blend comprises a biomaterial and a sacrificial polymer.

14. The method of claim 13, wherein said biomaterial is selected from the group consisting of of poly lactic acid (PLA), polygly colic acid (PGA), co-polyglycolic/lactic acid (PLGA), and any combination thereof.

15. The method of claim 13, wherein the biomaterial is a composite of PLGA and CaP.

16. The method of claim 13, wherein the sacrificial polymer is polystyrene.

17. A method of treating a bone defect in a mammal, said method comprising administering an implantable device into said mammal, wherein said device comprises a gradient porous structure, wherein said structure has a gradient of pore size such that the size of pores located closer to the outer surface of the structure is smaller than the size of pores located further from the outer surface of the structure.

18. The method of claim 17, wherein the gradient porous structure is a permeable structure.

19. The method of claim 17, wherein the device further comprises a hollow core, wherein the core can be filled with a bioactive agent.

20. The method of claim 19, wherein the bioactive agent is released or controlled released within the mammal.

21. The method of claim 19, wherein the bioactive agent comprises at least one agent selected from the group consisting of an antibiotic, a growth factor, a drug, a cell, and any combination thereof.

22. The method of claim 17, wherein the gradient porous structure degrades over a period of time within said mammal.

23. The method of claim 17, wherein the device promotes bone growth within said mammal.