WO2001035932A2

WO2001035932A2 - Sustained drug delivery from structural matrices

Info

Publication number: WO2001035932A2
Application number: PCT/US2000/031754
Authority: WO
Inventors: David J. Mooney; Lonnie D. Shea; Martin C. Peters; Elly Liao; Thomas P. Richardson
Original assignee: The Regents Of The University Of Michigan
Priority date: 1999-11-18
Filing date: 2000-11-17
Publication date: 2001-05-25
Also published as: WO2001035932A3; AU1622801A

Abstract

Disclosed are pre-fabrication methods for preparing particular 3-dimensional structural matrices containing proteins and/or drugs, the resultant compositions and in vitro and in vivo methods for the prolonged release of proteins and/or drugs in various biological environments. The pre-fabrication processes provide protein- and/or drug-matrix materials with both high incorporation efficiencies and control over sustained protein and/or drug release. The resultant matrices are thus particularly useful in vivo biodelivery embodiments, providing control over spatial delivery and differential release kinetics of multiple biological components.

Description

SUSTAINED DRUG DELIVERY FROM STRUCTURAL MATRICES

BACKGROUND OF THE INVENTION

The present application claims priority to U.S. provisional application Serial No. 60/166,191, filed November 18, 1999, the entire specification, claims and figures of which are incoφorated herein by reference without disclaimer. The U.S. Government may own rights in the present invention pursuant to Grant Numbers 1RO IDE 13004, DE07057 and AR40673 from the National Institutes of Health.

1. Field of the Invention

The present invention generally relates to the fields of porous polymer materials and their biological uses. More specifically, it concerns the fabrication and pre-fabrication of particular 3-dimensional structural matrices for controlled and prolonged release of proteins and drugs and methods of making and using such matrix compositions in vitro and in vivo. Particularly provided are matrix-protein and matrix-drug materials formulated to allow the in vitro and in vivo release of different proteins and drugs in a spatially controlled manner and/or with differentially controlled release kinetics.

2. Description of Related Art

Lost or deficient tissue function leads to millions of surgical procedures each year and a loss to the western economies of hundreds of billions of dollars. Tissue engineering has emerged as a potential means of growing new tissues and organs to treat such patients, and several approaches are currently under investigation to engineer structural tissues.

Improved biodegradable polymers and copolymers have recently been generated for use in the tissue engineering field. This has allowed developments in the generation of autologous and allogeneic tissues intended for use in transplantation. The role of biomaterials in the in vitro expansion of cultured cells is generally to serve as a vehicle to localize the cells of interest. Biomaterials can also be used in vivo to deliver biologically active substances. Biodegradable homopolymers and copolymers of lactic and glycolic acid, poly(lactic- co-glycolic acid) (PLGA; now also termed poly(lactide-co-glycolide) or PLG), have become attractive candidates for fabricating tissue engineering matrices due to their flexible and well defined physical properties and relative biocompatibility. The degradation products of these polymers are also natural metabolites and are readily removed from the body.

Several techniques have been used to fabricate polymers into porous matrices for tissue engineering applications, including solvent-casting/particulate leaching (SC/PL) (Mikos et al, 1994); phase separation (Lo et al, 1995); fiber extrusion and fabric forming processing (Cavallaso et al, 1994); and gas foaming (Mooney et al, 1996). However, the current techniques each suffer from their particular drawbacks.

The solvent-casting/particulate leaching and phase separation approaches require the use of organic solvents. Residues of organic solvents that remain in these polymers after processing may damage transplanted cells and nearby tissue and/or inactivate biologically active factors incoφorated into the polymer matrix for controlled release. Fiber forming typically requires high temperatures (above the transition temperature of polymer), and is not amenable to processing amoφhous polymers. The high temperatures used in such processes would likely denature any biologically active molecules incoφorated into the matrix.

The gas foaming method, as exemplified by Mooney et al. (1996), provides a technique to fabricate highly porous matrices from PLGA using a high pressure gas that avoids the use of organic solvents and high temperatures. However, the technique typically yields a closed pore structure, which is disadvantageous in many applications of cell transplantation. In addition, a solid skin of polymer results on the exterior surface of the foamed matrix and this may lead to mass transport limitations.

Therefore, there exists in the art a need for improved polymer materials for use in tissue engineering and biodelivery protocols. In terms of in vivo uses in particular, several other problems also need to be overcome. These include limitations such as the delivery of biological materials to an appropriate site or micro-location of the body, exposure of the materials to the appropriate cell types, efficient release of the biological materials, maintenance of an effective concentration of the released materials, prolonged and appropriate activity of the released materials. The development of biomatrices capable of providing for the controlled release of different biological agents from the same delivery vehicle, both spatially and time-wise, would be a particular advantage.

SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks inherent in the prior art by providing improved fabrication and pre-fabrication methods and polymer materials for use in protein and drug delivery protocols. The invention particularly provides pre- fabrication methods for preparing matrices with controlled structural properties in functional association with proteins and/or drugs, preferably three-dimensional structural matrices with controlled pore structures, including interconnected or open pore structures, in functional combination with proteins and/or drugs and combinations thereof. Preferred matrices of the invention provide for the release of different proteins and/or drugs in a spatially controlled manner and/or with differentially controlled release kinetics, thus allowing more effective control in vivo processes.

Applicants reserve the right to claim priority to U.S. Patent Application Serial

No. 09/402,119, filed September 20, 1999, which claims priority to PCT Application No. PCT/US98/06188 (WO 98/44027), filed March 31, 1998, which designates the United States and claims priority to U.S. Provisional Application Serial No. 60/042,198, filed March 31, 1997, the entire text and figures of which applications are incoφorated herein by reference without disclaimer, irrespective of reserving the right to claim priority.

The predesigned structural matrix compositions of the invention have advantages in the prolonged release of proteins and/or drugs, allowing cell exposure to proteins and/or drugs for extended time periods. The porosity and other physical properties of the preferred matrices are also controllable, allowing the number and type of cell populations that are exposed to the proteins and/or drugs to be regulated. As such, the matrix-protein and matrix- drug compositions of this invention represent an important advance.

A further advantage of the structural matrices of the present invention lies in the differential control provided over the release of proteins and/or drugs, including different proteins and/or drugs, incoφorated into the matrix. Both the spatial release and the kinetics of release are controllable using the techniques of the invention. Accordingly, different proteins and/or drugs may be released from different portions of the matrix, and different proteins and/or drugs may be released over different time periods. Equally, the same proteins and/or drugs may be released at differential times, such as in an initial burst, followed by a prolonged release. Control can be exercised over all such variables in a single protein-matrix or drug-matrix preparation, such that the same and different proteins and/or drugs are released from different functional portions of the matrix over a differentially controlled time span.

Control over the "time" of the first release, second, subsequent or series of release events and/or the duration of release is thus provided for matrices comprising a single protein or drug. Controlling the time(s) of release of the same protein or drug allows control or greater control over biological processes that include a regulated, cyclic or rhythmic element. Appropriately time-regulated biological pathways can thus be provided when absent, corrected when dysfunctional or fine-tuned when necessary or desired. These aspects of the invention also allow for overt intervention or artificial control, such as in the differentially- controlled release of immunogens as part of a primary and booster immunization protocol. The "time control" aspects of the invention allow control over latency and further provide for "contingent" delivery, such that a protein or drug is substantially maintained within the matrix under certain conditions and its release is then activated by subsequent exposure to one or more endogenous or exogenous agents.

For matrices comprising at least two different proteins and/or drugs, additional control is provided over the "sequence" of release of the different proteins and/or drugs.

Controlling the sequence(s) of release of two, three, four, five, or a plurality of different proteins and/or drugs is a particularly powerful biological tool made possible by the present invention. Biological processes that require the sequential action of different components can thus be provided, stimulated or recreated in a controlled manner. Regulating the release of different proteins or drugs that act sequentially can be used to provide quantitative and/or qualitative control over biological processes.

Thus, additive or even synergistic effects can result from the sequential action of two proteins or drugs in comparison to the action of a single protein or drug. The use of different proteins or drugs to be released at different times also allows for amplification or cascade effects, e.g., where the second or subsequent components enhance or amplify the effects of the first or earlier components. Alternatively, homeostatic control can be provided, wherein the second or subsequent protein or drug moderates or inhibits the effects of the first or earlier components.

According to this invention, the "drugs" of the proteins and/or drugs, as used herein, are not nucleic acids, DNA, RNA, plasmids, vectors, viral particles or other genetic material. This is notwithstanding the use of "drug delivery" to sometimes include the delivery of such nucleic acids. Therefore, unless otherwise specifically stated herein, neither proteins nor drugs include components of nucleic acids.

Preferred embodiments of the invention concern compositions, matrices, kits, admixtures for their preparation, methods and uses, wherein the matrices include pores formed by a gas foaming-particulate leaching process, and wherein the starting materials are microspheres pre-loaded with proteins and/or drugs. Porous polymer materials and matrices made from a combination of gas foaming (GF) and particulate leaching (PL) steps, i.e., "GF/PL processes", have two types of porosity: the first formed by the gas-foaming processing and the second formed by the action of particulate leaching.

Further preferred aspects of the invention are compositions, structural matrices, kits, admixtures for their preparation, methods and uses, wherein the structural matrices are comprised of at least two different forms of polymer, wherein the different polymers each comprise at least a first protein and/or drug. The proteins and/or drugs may be the same or different in each of the different forms of polymers. Where the protein(s) and/or drug(s) are the same in each different polymer, the different polymers will preferably be selected so that the release kinetics of the protein(s) and/or drug(s) are sufficiently different so as to provide control or added control over at least a first biological process. Given the range of biological processes that may be controlled or fine-tuned, the different release kinetics of the protein(s) and/or drug(s) may be grossly different or marginally different, so long as the timing difference is matched appropriately to the biological process to be controlled.

Where different protein(s) and/or drug(s) are present in at least two different polymers, the different polymers may or may not be selected so that the release kinetics of the protein(s) or drug(s) are different. Different protein(s) and/or drug(s) may thus be released at substantially the same time or at significantly different times, as desired.

Particularly preferred aspects of the invention are compositions, structural matrices, kits, admixtures for their preparation, methods and uses, wherein the matrices are comprised of two different forms of polymer, wherein the different polymers each comprise at least a first protein and/or drug, which may be the same or different according to the criteria set forth above, and wherein the different forms of polymer are particulate and microsphere forms of polymer processed by gas foaming/particulate leaching.

Accordingly, the invention provides a population or plurality of microspheres prefabricated with proteins and/or drugs, where the microspheres comprise at least two different polymers. The individual microspheres can themselves be mixtures of polymeric materials (heteropolymeric microspheres), or a mixture of microspheres can be employed in which each individual microsphere is comprised of a single polymer (homopolymeric microspheres). The same or different proteins and/or drugs can be associated with either each type of polymer, each type of microsphere, or both, so that the ultimate release kinetics can be controlled in a variety of different ways. The invention this includes admixtures, combinations and/or kits comprising at least a first leachable particulate material in combination with a population or plurality of microspheres pre-fabricated with proteins and/or drugs, wherein the microspheres comprise at least two different polymers.

The range of the foregoing preparative compositions, with the same or different polymers and the same or different proteins and/or drugs, are preferably fabricated so that at least a portion of the resultant structural matrix is comprised of a porous polymer that contains pores formed by gas foaming and pores formed by leaching out of a particulate from the polymer. At least some portions of such structural matrices may be comprised of a porous polymer that has a substantially uniform open pore structure. Structural matrices consisting essentially of a porous polymer that has an open pore structure are also provided.

One of the important aspects of the present invention is that the structural matrices, kits and methods of use involve structural matrices that include controlled pore structures. In certain embodiments, polymers with interconnected and open pore structures will be preferred. The combination of the two foregoing porosity types can be regulated by controlling the processing conditions and starting materials used. Thus, a range of porous polymeric materials can be generated, each having particular advantageous properties.

Compositions, matrices, kits and methods of use, wherein the structural matrices comprise at least a first matrix portion comprised of the porous polymer integrally connected to at least a second matrix portion comprised of an impermeable polymer are also included. Further provided are structural matrices that comprise at least a first matrix portion comprised of a porous polymeric material that has a substantially uniform open pore structure, wherein at least a second matrix portion is comprised of the same polymeric material in a form that lacks an open pore structure.

In preferred embodiments of the invention, the polymeric structures are formulated with proteins and/or drugs to yield matrix-protein and or matrix-drug preparations in which the proteins and/or drugs are generally physically immobilized within the polymer during the fabrication process or, preferably, during a pre-fabrication step.

For simplicity, control of sterility and to allow most effective incoφoration of proteins and/or drugs throughout the controlled, preferably open pore, structure of the matrix, the proteins and/or drugs are preferably formulated into the matrices of the invention during one or more pre-fabrication steps. An important advantage of incoφoration during pre- fabrication is that the subsequent release is actually controlled by the pre-fabrication materials and steps, /^'. e. , by controlling polymer formation, degradation and pore size, rather than later being a function only of desorption from the polymer surface.

The same or different proteins and/or drugs are preferably formulated into matrices in pre-fabrication steps using different forms of polymer, preferably different forms of poly(lactide-co-glycolide) polymer, and more preferably, particulate and microsphere forms of such polymers, and processed by gas foaming/particulate leaching. Resultant advantages include the control provided over the release of the same or different proteins and/or drugs according to the properties of the chosen polymers into which they are prefabricated.

The overall fabrication is thus generally achieved by incoφorating proteins and/or drugs within polymer particles, such as beads or microspheres, prior to adding the leachable particulate materials and executing the gas foaming-particulate leaching methodology.

Preferred fabrication means involve incoφorating the same or different proteins and/or drugs within different polymer particles, beads and/or microspheres.

In certain embodiments, closed pore polymer combinations can be directly used in sustained delivery embodiments, both in vitro and in vivo. The preparation of a closed pore matrix structure does not utilize any leachable particulate and can be simply achieved using a one step foaming process.

In preferred embodiments, e.g., to generate a matrix that facilitates cellular invasion, a matrix with an open pore structure will be employed. Open pore structure matrices are preferably created using a GF/PL process with a pre-fabrication step. In a preferred GF/PL pre-fabrication process, polymer particles, such as beads or microspheres already pre-loaded with proteins and/or drugs are mixed with a leachable particulate, foamed and leached. Other preferred pre-fabrication processes require the combined use of different types of polymer particles, beads or microspheres pre-loaded with the same or different proteins and/or drugs.

In the preferred pre-fabrication processes of the invention, the proteins and/or drugs are first incoφorated into polymer particles, preferably, beads or microspheres, to provide pre-loaded polymer particles, beads or microspheres. Preferably, a combination of two or more different polymers is used, optionally with the same or different proteins and/or drugs. Any microsphere fabrication process may be used, including atomization/extraction processes operated at cryogenic temperatures. The polymer particles, beads or microspheres pre-loaded with proteins and/or drugs are then admixed with the leachable particulate material(s) and the two component mixture is later subjected to gas-foaming particulate leaching. The protein- and/or drug-containing microspheres and leachable particulate are typically first molded, optionally with compression, to a desired size and shape, generally guided by the ultimate intended use.

The molded mixture of pre-loaded particles or microspheres is then subject to a high pressure gas atmosphere so that the gas dissolves in the polymer. Next, a thermodynamic instability is created, for example by reduction of the pressure, so that the dissolved gas nucleates and forms gas pores within the polymer. The gas pores cause expansion of the preloaded particles or microspheres and as they expand they fuse, creating a continuous polymer matrix containing the particulate material. Finally, the particulate material is leached from the polymer with a leaching agent creating a further porosity. Proteins and drugs are substantially unaffected by each of the foregoing processes, including the pre-fabrication step.

The polymer and particulate materials are selected so that the particulate can be leached with a leaching agent that does not significantly dissolve the polymer or otherwise significantly adversely impact either the polymeric material or the proteins and/or drugs admixed therewith. The mixture is preferably as uniform as possible and can be provided by any conventional means, by pre-loading microspheres with the proteins, drugs or combinations thereof.

Any polymer with which proteins and/or drugs can be mixed, or into which proteins and/or drugs can be incoφorated, into which gas can be dissolved and pores formed thereby, and in which a particulate can be incoφorated and leached therefrom can be used in the process. It is generally preferred, to facilitate dissolution of the gas, that the polymer be an amoφhous or predominantly amoφhous polymer. However, if it is desired to use a crystalline polymer, the crystallinity can be reduced to a level such that the gas can be dissolved therein and then the crystallinity restored after formation of the pores.

Depending upon the application of the materials, the polymer may be selected to be biodegradable or non-biodegradable. Biodegradable polymers will often be preferred. For the most preferred applications of the invention, the polymer is preferably biocompatible to the environment in which it is used, such as the human in vivo environment.

A preferred useful class of polymers for use in the invention are homopolymers and copolymers of lactic acid and glycolic acid, for example, poly-L-lactic acid (PLLA), poly- D,L-lactic acid (PDLLA), polyglycolic acid (PGA) and copolymers of D,L-lactide and glycolide (PLGA), particularly with 50% or more of the lactide in the copolymer. Polylactic- polyglycolic acid, known as PLGA, is now also termed poly(lactide-co-glycolide), or PLG, and such terms may be used interchangeably herein. Other useful polymers, for example, are aliphatic polyesters, such as polyhydroxybutyrate, poly- e -caprolactone. Further, polyanhydrides, polyphosphazines, polypeptides may be used.

As polymer composition and molecular weight have an effect on the porosity and mechanical properties of three dimensional matrices, altering the polymer composition allows for functional control. In the GF/PL processes, copolymers of PLGA have been shown to foam to a much greater extent than either homopolymer of PGA or PLLA. This is likely due to an increased gas dissolution in amoφhous polymers, as compared to crystalline polymers. An informed choice between copolymer and homopolymer can thus be made. Such a choice can be used to influence the delivery of the same or different protein(s) and/or drug(s), particularly to control the time at which meaningful release begins and the duration of release.

The molecular weight of the polymer also has an effect on scaffold porosity. Polymers with a high molecular weight (large i.v.) do not form scaffolds with as high porosity as the same polymers with a lower molecular weight. The longer polymer chains of the high molecular weight polymer likely entangle to a greater extent, thus providing a stronger resistance to expansion than the shorter polymer chains. Such can also be considered in choosing a polymeric matrix for use with the invention.

In certain preferred embodiments, advantageous pore formation is achieved by the use of a low molecular weight amoφhous copolymer of lactide and glycolide.

Advantages of the invention include high incoφoration efficiencies and sustained release of proteins and/or drugs, which release can be controlled in part through the microsphere fabrication process. Particular advantages include controlling the onset and/or duration of release of the same or different protein(s) and/or drug(s) and the resultant ability to control biological processes in vitro and in vivo.

Blends of different polymers may also be used, as may polymers that contain other agents, particularly those that affect the mechanical properties of the resulting matrix. For example, blends of different PLGA polymers that have distinct properties can be used to take advantage of the properties of each polymer. Also, other polymers can be blended with, e.g., PLGA polymers, particularly for modifying the mechanical properties thereof. For example, blends of PLGA polymers and alginate materials can provide a tougher matrix with greater elasticity and ability to withstand greater strain before breaking.

The present invention therefore contemplates the use of blends of polymers that result in matrices with better pliability and/or strength. Blends using materials that act as plasticizers, toughening agents or modifiers of other properties may be preferred for certain aspects of the invention. Such materials can either be polymers or smaller molecule agents that may act in a temporary manner and then diffuse from a matrix.

The leachable particulate for use in the invention will be any particulate material that can be leached from the polymer matrix with a leaching agent and that does not significantly adversely affect the polymer or the proteins and/or drugs in the admixture. Currently preferred are salts soluble in an aqueous medium, preferably water, and sugars and sugar alcohols soluble in aqueous media, preferably water, serum and/or biological tissue fluids. As salts, NaCl, Na citrate, Na tartrate, and KCl are useful particulate materials. Useful sugar and sugar alcohol particulates include trehalose, glucose, sucrose and mannitol. Other useful particulates leachable by dissolution include, for example, gelatin, heparin and heparin derivatives, collagen and alginate particulates.

It is also possible to use particulates that are leachable by organic solvents where the solvent does not adversely effect the polymer, protein and/or drug; however, this is not preferred since such would mitigate the advantage of lack of need for an organic solvent and lack of residue in the product. The use of organic solvents would also generally mean that the proteins and/or drugs should be added after matrix formulation.

In general, the size of any particulate will generally affect the size of the pores formed upon leaching of the particulate. Although not limiting of the invention, it is currently preferred that the particulate has an average size of from about 10 to about 500 microns. This size will correspond approximately to the size of the pores formed by the leaching thereof.

A gas is dissolved in the pre-loaded microsphere and particulate by subjecting the mixture to a pressurized atmosphere of a gas that is inert to the system and that will dissolve in the polymer under suitable conditions. Examples of suitable gases include CO₂, air, nitrogen, helium, argon and oxygen. Also, volatile liquids that provide a gas at the gas foaming temperature may be used, e.g., water. Other gases or volatile liquids that form gases known to be useful as blowing agents may also be used. These include, for example, fluorinated, including perfluorinated, hydrocarbons. Preferred for these are aliphatic or cycloaliphatic fluorinated hydrocarbons of up to 8 carbon atoms such as trifluoromethane, difluoromethane, difluoroethane, tetrafluoroethane, heptafluoroethane, perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorohexane, perfluoroheptane, pefluorooctane, perfluorocyclopentane, perfluorocyclohexane, hexafluoropropane and heptafluoropropane.

Sulfur hexafluoride may also be a useful blowing agent. Other known blowing agents include alkanes such as propane, butanes and pentanes; cycloalkanes and cycloalkenes such as cyclobutane, cyclopentene and cyclohexene; dialkyl ethers such as dimethyl ether, methyl ethyl ether and diethyl ether; cycloalkylene ethers such as furan; ketones such as acetone and methyl ethyl ketone; and carboxylates such as formic acid, acetic acid and propionic acid. All such agents may be used in these aspects of the invention.

The pressure is generally selected to facilitate dissolution of gas into the polymer and will, thus, depend upon the gas used, the polymer used and the temperature. Pressures of from about 600 to about 900 psi are generally useful for CO₂ and PLGA polymers, although this is not limiting on the invention. Gases at super- or sub-critical conditions can also be used. Furthermore, a volatile liquid that can be dissolved in the polymer and that forms a gas upon imposition of the thermodynamic instability can also be used. As an example, CO₂ can be dissolved in a mixture of poly[D,L-lactic-co-glycolic acid] polymer and NaCl particulate at a pressure of about 800 psi applied for about 48 h to allow saturation.

The specific gas used in foaming can be an important variable in the production of porous matrices for use herewith and the choice of gas used has an effect on the final scaffold structure. CO₂ produces highly porous matrices, whereas N₂ and He do not yield measurable pore formation. Although the mechanism underlying these results does not need to be known in order to practice the invention, the greater degree of foaming experienced with CO₂ as compared to both N2 and He may be the result of a specific interaction between CO₂ and the carbonyl groups of PLGA. Gas equilibration times and pressure release rates may also affect the porosity and stability of the matrices formed.

In order to initiate nucleation of the dissolved gas and growth of gas pores in the material, a thermodynamic instability is created. This phenomenon is described by Park et al. (1995; incoφorated herein by reference). Preferably, this is done by lowering the pressure of the gas atmosphere, for example, down to about atmospheric pressure over a short time period. The time period being, for example, from a few seconds to about 15 or 30 minutes or so. The gas phase separates from the polymer via pore nucleation and growth of the pores occurs through diffusion of gas into areas adjacent the nucleation sites. The pore growth in turn reduces the polymer density.

Other methods for creating the instability, such as raising the temperature, may be used, but, are not preferred due to ease of processing of the current methods. The pore structure and pore size of the gas pores formed will be a factor of, for example, the type of gas used; the amount of gas, which will depend upon temperature and initial and final pressure of the gas atmosphere applied; the solubility of the gas in the particular polymer; the rate and type of pore nucleation; and the diffusion rate of the gas through the polymer to the nuclei. These and other factors can be adjusted to provide gas pores of a suitable size. Sufficient gas should be dissolved to cause formation of a continuous polymer matrix when the polymer expands during gas pore growth.

As a result of the thermodynamic instability, pore nucleation and gas pore formation and expansion, the polymer containing the particulate material and proteins and/or drugs forms a continuous phase, i.e. matrix, around the gas pores.

The particulate is leached from the polymer with a leaching agent. Useful as leaching agent is any agent that will leach, e.g., dissolve and remove, the particulate from the polymer.

An aqueous-based leaching agent, particularly water, is preferred. Body fluids can also be used as both in situ and in vitro leaching agents. The methods are executed such that the leaching agent that leaches the particulate from the polymer does not leach or otherwise remove a substantial amount of the proteins and/or drugs from the polymer. However, as the preferred embodiments of the present invention involve the provision of proteins and/or drugs to target cells, loss of some material from the matrix during the leaching process will not be detrimental to practice of the invention.

Methods and uses of the invention where the particulate is not removed before implantation, but rather dissolves in the body to create the porosity, e.g., for cell invasion, are also provided. The dissolving particulates can be chosen to have a minimal effect on the surrounding tissue and to diffuse away. They may also be chosen to actually activate cell migration into the scaffolds, such that the particulate that leaches out modulates cellular invasion by controlling chemotaxis of cells to the site and such like.

In such embodiments, the same processes are used to fabricate the matrices, but leaching is not conducted before implantation. Rather, the solid material (containing polymer, proteins and/or drugs and particulate) is implanted. The particulate is then allowed to leach or dissolve in the body simply by exposure to body fluids, thus creating the porous structure. The criteria for the particulate in such processes are that it be biocompatible and soluble in aqueous solutions. Sugars are preferred, such as trehalose, sucrose, mannitol, glucose etc.

In such processes, there is further control over the kinetics of protein and/or drug release, as the release is controlled by the rate of dissolution of the particulate in the body following implantation. Rapidly dissolving particulates allow faster release, while slower dissolving particulates retard release. In addition, this allows virtually 100% efficiency of protein and/or drug delivery, as proteins and/or drugs are not lost in the particulate leaching step, which accounts for most of the loss during processing. Using a combination of two or more different polymers to prepare particulates with two or more different dissolution properties will thus allow the release of biological materials at two or more different rates. The same or different proteins and/or drugs can be sequentially released by such means. The preferred preparative methods of the present invention generally comprise incoφorating at least a first protein and/or drug within a polymeric structure in particle form, e.g., as beads or microspheres, admixing with the leachable particulate material, subjecting the admixture to a gas foaming process and leaching out the particulate material from the gas foamed admixture. Preferably, the same or different protein(s) and/or drug(s) are incoφorated within polymeric structures, particles, beads or microspheres comprised of different polymeric materials.

Such methods therefore generally comprise the steps of:

(a) preparing an admixture comprising a leachable particulate material and particles of a polymeric material capable of forming a polymeric structure, wherein the particles incoφorating at least a first protein and/or drug;

(b) subjecting the admixture to a gas foaming process to create a porous polymeric structure that comprises at least a first protein and/or drug and the leachable particulate material; and

(c) subjecting the porous polymeric structure to a leaching process that removes the leachable particulate material from the porous polymeric structure, thereby producing a polymeric structure of additional porosity that comprises at least a first protein and/or drug.

In certain preferred embodiments, the admixture will comprise at least a first protein and/or drug that is incoφorated within beads or microspheres capable of forming a polymeric structure and the leachable particulate material. In other preferred embodiments, the admixture will comprise the same or different proteins and/or drugs incoφorated within beads or microspheres of different polymeric composition. Certain microsphere populations themselves are thus included within the invention, even in the absence of a leachable particulate material. Such populations will comprise beads or microspheres of at least two different polymers suitable to undergo gas foaming- particulate leaching, wherein the beads or microspheres have incoφorated therein at least a first protein and/or drug. The proteins and/or drugs may be the same or different.

The invention also includes a wider variety of beads or microspheres when admixed or packaged in combination with at least a first leachable particulate material. Such compositions, admixtures and kits comprise at least a first leachable particulate material in combination with beads or microspheres of a polymer capable of forming a gas-foamed polymeric structure, wherein the beads or microspheres have incoφorated therein at least a first protein and/or drug. The compositions, admixtures and kits of course include those that comprise a leachable particulate material in combination with beads or microspheres made from at least two different such polymers, wherein the beads or microspheres have incoφorated therein at least a first protein and/or drug.

The methods of making may thus comprise:

(a) pre-fabricating a polymeric_particle that is capable of forming a polymeric structure, preferably a bead or microsphere, with at least a first protein and/or drug to prepare a polymeric particle that incoφorates at least a first protein and/or drug;

(b) preparing an admixture comprising a leachable particulate material in combination with the pre-fabricated polymeric particle, bead or microsphere that incoφorates at least a first protein and/or drug;

(c) subjecting the admixture to a gas foaming process to create a porous polymeric structure that comprises at least a first protein and/or drug and the leachable particulate material; and (d) subjecting the porous polymeric structure to a leaching process that removes the leachable particulate material from the porous polymeric structure, thereby producing a polymeric structure of additional porosity that comprises at least a first protein and/or drug.

Further methods of making comprise:

(a) pre-fabricating a first polymeric particle comprised of a first polymer and a second polymeric particle comprised of a second polymer, preferably beads or microspheres, with at least a first protein and/or drug to prepare first and second polymeric particles with two different polymer compositions that incoφorate at least a first protein and/or drug;

(b) preparing an admixture comprising a leachable particulate material in combination with the first and second pre-fabricated polymeric particles, beads or microspheres that incoφorate at least a first protein and/or drug;

(c) subjecting the admixture to a gas foaming process to create a porous polymeric structure with two different polymer compositions that comprises at least a first protein and/or drug and the leachable particulate material; and

(e) subjecting the porous polymeric structure to a leaching process that removes the leachable particulate material from the porous polymeric structure, thereby producing a polymeric structure with two different polymer compositions, and with additional overall porosity, which polymeric structure comprises at least a first protein and/or drug.

The preferred preparation methods, or "GF/PL processes", of the invention result in a suφrisingly effective combination of gas foamed and particulate leached porosity, with the particulate leached pores also being termed "macropores". The combined use of gas-foaming and particulate leaching, as disclosed herein, provides a controlled range of matrices with interconnected and open pore structures, the particular composition of which is dictated by the processing conditions and materials employed.

Interconnected and open pore structures are preferably prepared by using a mixture of polymer and leachable particulate wherein the amount of leachable particulate is at least about 50% by volume. A higher amount of leachable particulate can be used to obtain a fully interconnected structure, up to about 99%.

Overall, the process of the invention can provide materials with a total porosity of, for example, from above 0 to 97% or even higher. In certain embodiments, it will be preferable to use matrices with a total porosity of about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96% or about 97% or so.

The protein and/or drug-containing materials of the invention also generally exhibit much higher strength properties, e.g., tensile strength, as compared to previous materials. For example, preferred materials according to the invention have a tensile modulus in the range of about 850 kPa, and more preferably, up to and including about 1 100 kPa, or even higher. The preferred materials also exhibit improved compression resistance. For instance, preferred materials have a compression modulus of, for example, about 250 kPa, and more preferably, up to and including about 289 kPa, or even higher. Typical prior art materials exhibit a tensile modulus of only about 334 ± 52 kPa and a compression modulus of only about 159 ± 130 kPa.

Polysaccharides, such as alginates, modified to bind biological agents may also be used in the invention. Alginates modified so that they have controllable physical properties, such as sol-gel properties, and the like, are contemplated.

Alginates comprising at least one alginate chain section bonded to at least one molecule useful for cellular interaction (cell adhesion molecules, cell attachment peptides, proteoglycan attachment peptide sequences, proteoglycans, and polysaccharides exhibiting cell adhesion) are also envisioned. Particular examples are RGD peptides, fibronectin, vitronectin, Laminin A, Laminin Bl, Laminin B2, collagen 1 or thrombospondin. Various polypeptide or peptide growth factors or enzymes may also be used as the cellular interacting molecules.

The preparation and use of porous hydrogel materials formed by first creating gas pockets in the gel and then removing the gas to create a material with an open, interconnected pore structure is also included. Such matrices maintained their pore structure over extended time periods and have high mechanical integrity. U.S. Provisional Application Serial No. 60/128,681, filed April 09, 1999, is specifically incoφorated herein by reference without disclaimer for the puφoses of describing the preparation and use of such unique polymeric materials and matrices thereof.

Accordingly, at least a portion of the structural matrix may be a modified alginate matrix prepared by a method comprising:

(a) providing a solution of a hydrogel-forming material and a surfactant;

(b) mixing said solution in the presence of a gas to form a stable foam;

(c) exposing said stable foam to conditions or agents that result in gelling of the hydrogel-forming material and in the generation of gas bubbles therein; and

(d) exposing the hydrogel containing gas bubbles to a vacuum to release the gas and form the hydrogel material having macroporous open pore porosity.

Irrespective of the form of matrix, the protein(s) and/or drug(s) will preferably have a therapeutic use and/or effect, although marker proteins and diagnostic agents are also included. Human proteins and polypeptides will often be preferred. Exemplary proteins, polypeptides and/or drugs are those that stimulate growth or proliferation of cells, such as bone progenitor cells; that stimulate wound healing fibroblasts, granulation tissue fibroblasts and/or repair cells; that stimulate an antigenic or immunogenic response by cells of the immune system, such as antigen presenting cells; and cytotoxic or apoptosis-inducing proteins, polypeptides and/or drugs that induce cell death in a target cell.

Particular examples include a transcription or elongation factor, cell cycle control protein, kinase, phosphatase, DNA repair protein, oncogene, tumor suppressor, cytotoxin, angiogenic protein, anti-angiogenic protein, apoptosis-inducing agent, anti-apoptosis agent, immune response stimulating protein, cell surface receptor, accessory signaling molecule, transport protein, enzyme, anti-bacterial, anti-microbial, anti-parasitic or anti-viral protein or polypeptide.

Further examples include a hormone, neurotransmitter, growth factor, growth factor receptor, hormone receptor, neurotransmitter receptor, adhesion ligand, interferon, interleukin, chemokine, cytokine, colony stimulating factor and chemotactic factor protein.

Particular examples are growth hormone (GH) proteins and polypeptides; parathyroid hormone (PTH) proteins and polypeptides, such as PTH1-34 polypeptides; bone moφhogenetic protein (BMP) proteins and polypeptides, such as BMP-2A, BMP-2B, BMP- 3, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8; TGF- , TGF-βl, TGF-β2 and latent TGFβ binding protein (LTBP) proteins and polypeptides; activin/inhibin proteins and polypeptides; fibroblast growth factor (FGF); granulocyte/macrophage colony stimulating factor (GMCSF); epidermal growth factor (EGF); platelet derived growth factor (PDGF); vascular endothelial cell growth factor (VEGF); an angiopoietin; insulin-like growth factor (IGF) and leukemia inhibitory factor (LIF). Further examples are estrogen, progesterone and testosterone.

Extracellular matrix components, molecules, ligands and peptides are further suitable for use in the invention. Suitable examples include, but are not limited to, fibrin, collagen, fibronectin, vitronectin, hyaluronic acid and RGD-containing peptides or polypeptides.

As used herein, the term "drug" means a therapeutic agent other than nucleic acids and genetic material such as DNA, RNA, plasmids, vectors and the like. As the term "proteins and/or drugs" is employed, the term "drug" is typically used herein to refer to a therapeutic agent other than a protein, polypeptide or peptide, although peptidomimetics are more conveniently referred to as "drugs". Accordingly, a "drug" is a non-nucleic acid, non- proteinaceous therapeutic agent.

The "drugs" for use in the invention include all such drugs that are physiologically or pharmacologically active or rendered physiologically or pharmacologically active upon delivery to an animal or human. "Pro-drugs" or drug derivatives may thus be used, where the pro-drug or drug derivative is converted to an active form within the , e.g., through the action of body enzyme-assisted transformation, pH, specific organ activities, and such like, or through the application of at least one more exogenous agent(s). The "exogenous agent" that activates a drug derivative may also be provided in the matrices of the invention, preferably, for sequential release at a controlled point.

The drugs may be physiologically or pharmacologically active at a point "local" to the delivery of the matrix-drug formulation or device or may be "systemically" active upon delivery, thus producing a physiological or pharmacological response at one or more sites distant or remote from the point of application of the matrix-drug formulation or device. All drugs approved for human or veterinary use, or undergoing clinical trials for approval, may be used with the present invention.

Such drugs include drugs acting on the central nervous system, such as hypnotics and sedatives, e.g. , pentobarbital sodium, phenobarbital, secobarbital, thiopental and amides and ureas exemplified by diethylisovaleramide and alpha-bromoisovaleryl urea; heterocyclic hypnotics, such as dioxopiperidines and glutarimides; hypnotics and sedative alcohols, such as carbomal, naphthoxyethanol, methylparaphenol; hypnotic and sedative urethans, disulfanes and the like; psychic energizers, such as isocarboxazid, nialamide, phenelzine, imipramine, tranylcypromine and pargylene; tranquilizers, such as chloropromazine, promazine, fluphenazine reseφine, deseφidine, meprobamate and benzodiazepines, such as chlordiazepoxide; anticonvulsants, such as primidone, diphenylhydantoin, ethotoin, pheneturide and ethosuximide; muscle relaxants and anti-Parkinson's agents such as mephenesin, methocarbomal, trihexylphenidyl, biperiden and levo-dopa, also known as L- dopa and L-alpha-3-4 dihydroxyphe-nylalanine.

Further exemplary drugs for use with the invention include analgesics, such as moφhine, codeine, meperidine, naloφhine and the like; antipyretics and anti-inflammatory agents such as aspirin, salicylamide and sodium salicylamide; local anesthetics, such as procaine, lidocaine, naepaine, piperocaine, tetracaine and dibucaine; antispasmodics and antiulcer agents, such as atropine, scopolamine, methscopolamine, oxyphenonium, papaverine, prostaglandins, such as prostaglandin E, prostaglandin F and prostaglandin A.

In terms of anti-microbial drugs, suitable examples include penicillin, tetracycline, oxytetracycline, chlorotetracycline, chloramphenicol, sulfonamides and the like; antimalarials, such as 4-aminoquinolines, 8-aminoquinolines and pyrimethamine. Antiparasitic agents, such as bephenium hydroxynaphthoate and dichlorophen, dapsone and such like may also be used. Suitable anti- viral agents include acyclovir and gancyclovir, and anti-HIV agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, nucleoside analog reverse transcriptase inhibitors, protease inhibitors and the like. Suitable examples include AZT (Zidovudine, Retrovir™), Amprenavir, Lamivudine, Zidovudine, Indinavir, Efavirenz, Lamivudine, Saquinavir, Zalcitabine, Hydroxyurea, Ritonavir, Adefovir Dipivoxil, Delavirdine, Didanosine, Nelfinavir, Nevirapine, Stavudine and Abacavir.

Exemplary hormonal drugs include prednisolone, hydrocortisone, cortisol and triamcinolone; androgenic, estrogenic and progestational steroids, e.g., methyltestosterone, fluoximesterone, 17B-estradiol, ethinyl estradiol, 17a-hydroxyprogesterone acetate,

19-noφrogesterone and norethindrone. Suitable sympathemimetic drugs include amphetamines, ephinephrine, epinephrine and such like.

Cardiovascularly-active drugs include, but are not limited to, procainamide, amyl nitrite, nitroglycerin, dipyridamole, sodium nitrate, isosorbide dinitrate and the like. Diuretic drugs may also be used, such as chlorothiazide, flumethiazide and the like. For implantation of the matrix-drug formulation or device into, or proximal to a tumor, anti-neoplastic or chemotherapeutic agents may be used. Suitable chemotherapeutic agents include anti-metabolites, such as cytosine arabinoside, fluorouracil, methotrexate or aminopterin; anthracyclines; mitomycin C; vinca alkaloids; antibiotics; demecolcine; etoposide; mithramycin; anti-tumor alkylating agents, such as chlorambucil or melphalan; DNA synthesis inhibitors, such as daunorubicin, doxorubicin, adriamycin, and the like.

Drugs for restoring normoglycemia or otherwise treating diabetics are further suitable examples, including hypoglycemic therapeutic agents, such as insulins, protamine zinc insulin suspensions, globin zinc insulin, isophane insulin suspension, and other extended insulin suspensions; sulfonylureas, such as tolbutamide, acetohexamide, tolazamide and chloφropamide, the biguanides and the like. Prandin™ may also be used.

In addition to drugs of the pharmacological sense, the term "drug", as used herein, also encompasses nutritional agents, such as vitamins, minerals, amino acids, fats and such like, so that "drugs" extend to any necessary or beneficial substance for administration-to a human or animal. Moreover, the term "drug", as used herein, further encompasses reparative or even cosmetic agents and formulations comprised within surgically and cosmetically acceptable preparations. Such drug-matrix formulations are applicable for surgical and cosmetic intervention in connection with skin diseases, disorders and burns; and in connection with internal and external traumas, reconstructive surgery and the like.

The admixtures, compositions, matrices, kits, methods and uses of the invention include those wherein at least a first and second, third, fourth, fifth, sixth, etc, protein, polypeptide and/or drug is present, up to and including a plurality of proteins, polypeptides and/or drugs. Functional biochemical reaction points and pathways can thus be recreated by the invention.

The compositions, matrices and kits of the invention also comprise populations of biological cells, both in vitro and in vivo. Portions of the proteins, polypeptides and/or drugs may be taken up by the cells comprised within such compositions either before or after transplantation to an animal or human, or during both stages. The biological cells for combination with the present invention include, by way of example, bone progenitor cells, fibroblasts, endothelial cells, endothelial cell precursors, stem cells, macrophages, fibroblasts, vascular cells, osteoblasts, chondroblasts and osteoclasts.

The present invention also provides kits that comprise any of the admixtures, compositions or matrix-protein, matrix-polypeptide or matrix-drug compositions in accordance herewith, optionally in at least a first suitable container. Implantable medical devices comprising protein-matrix or drug-matrix compositions in accordance herewith are also provided in bioimplantable forms.

The compositions, matrices, kits and devices of the invention have various uses, such as in the controlled release, including the controlled sequential release, of proteins, polypeptides and/or drugs; in providing proteins, polypeptides and/or drugs to cells, both simultaneously and sequentially; in culturing cells and recombinant cells (that express nucleic acid segments); and in stimulating cells within tissue sites of animals and humans.

Uses of the compositions, matrices, kits and devices thus extend to the manufacture of medicaments for all aspects of protein therapy and drug delivery, such as stimulating bone tissue growth; promoting wound healing, tissue regeneration and organ regeneration; generating immune responses; killing invading pathogens, aberrant, malignant and virally- infected cells; controlling the reproductive system, such as in fertility treatment or in birth control interventions; and in cell transplantation, tissue engineering and guided tissue regeneration.

The methods and uses of the invention include the controlled release of proteins, polypeptides and/or drugs, allowing the release of at least a first protein, polypeptide and/or drug from a matrix composition that comprises at least a first protein, polypeptide and/or drug in association with a structural matrix that comprises at least a portion fabricated from a porous polymer that contains pores formed by gas foaming and pores formed by leaching out of a particulate from the polymer.

Such methods and uses further include the controlled, differential or sequential release of proteins, polypeptides and/or drugs, thus allowing the release of the same protein, polypeptide and/or drugs at different times, the release of different proteins, polypeptides and/or drugs at the same time, and the release of different proteins, polypeptides and/or drugs at different times. The same or different proteins, polypeptides and/or drugs are released from a structural matrix composition in such controlled manners by fabricating the structural matrix with at least two different polymers, preferably each fabricated to from an overall porous polymer matrix that contains pores formed by gas foaming and pores formed by leaching out of a particulate from polymers.

The release of the proteins, polypeptides and/or drugs from the matrix in all embodiments may be controlled by controlling the rate of degradation or dissolution the structural matrix, by controlling diffusion through the pores in the structural matrix, by desoφtion from the structural matrix, or combinations thereof, most preferably wherein different polymers make up the overall matrix.

Methods and uses for providing at least a first protein, polypeptide and/or drug to a cell are provided, comprising contacting a cell with a composition of the invention in a manner effective to release at least a first protein, polypeptide and/or drug from the structural matrix composition, preferably wherein the structural matrix composition is fabricated from two different polymers. The cell may be located in a tissue site of an animal or human, wherein the composition is provided thereto.

In vitro culture methods and uses are also provided, which comprise growing cells in contact with a therapeutic structural matrix composition of the invention. The cells may be separated from the therapeutic structural matrix composition and used in vitro and/or provided to an animal. The cells may also be maintained in contact with the therapeutic structural matrix composition, which may be provided to an animal or patient. Methods and uses are further provided comprising contacting a tissue site of an animal or human with a structural matrix composition of the invention in a manner effective to provide at least a first protein, polypeptide and/or drug to cells within the tissue site.

In all in vivo methods and uses of the invention, the "animal" may be a human or non- human animal. Therefore, the phrases "biocompatible" and "pharmaceutically or pharmacologically acceptable", as used herein, refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. As veterinary and clinical uses are equally included within the invention, the biocompatible polymers, compositions and matrices are "veterinarily" as well as pharmaceutically acceptable. Non-human animals to be treated by the invention particularly include primates, valuable or valued domestic household, sport or farm animals, and laboratory animals, such as mice, rats, guinea pigs, rabbits and the like.

Target cells include bone progenitor cells (e.g., stem cells, macrophages, granulation tissue fibroblasts, vascular cells, osteoblasts, chondroblasts and osteoclasts) located within bone progenitor tissue sites or bone fracture sites; repair cells or fibroblasts located within wound tissue sites, such as sites of connective tissue injury or organ damage; immune and antigen presenting cells; aberrant, malignant and infected cells; and cells of the reproductive system.

Methods and uses for stimulating bone progenitor cells located within a bone progenitor tissue site of an animal or human comprise contacting the tissue site with at least a first osteotropic protein, polypeptide and/or drug-structural matrix composition of the invention in a manner effective to provide at least a first osteotropic protein, polypeptide and/or drug to the cells. The cells are stimulated to promote bone tissue growth, e.g., in a bone cavity site that is the result of dental or periodontal surgery or the removal of an osteosarcoma. Fibroblast stimulation methods and uses comprise contacting a wound tissue site of an animal or human with at least a first therapeutic protein, polypeptide and/or drug- structural matrix composition of the invention in a manner effective to provide at least a first therapeutic protein, polypeptide and/or drug to the fibroblast cells. This stimulates the fibroblast cells to promote wound healing.

Methods and uses for promoting wound healing comprise applying a biocompatible structural matrix containing at least a first therapeutic protein, polypeptide and/or drug to a wound site in an animal or human so that repair cells in the wound site promote wound healing.

Further methods and uses are for providing at least a first immunogenic protein, polypeptide and/or drug to immune cells or antigen presenting cells within a tissue site of an animal or human, comprising contacting the tissue site with an immunogenic protein, polypeptide and/or drug-structural matrix composition of the invention in a manner effective to provide at least a first immunogenic protein, polypeptide and/or drug to immune cells or antigen presenting cells within the tissue site.

These lead to immunization methods and uses that comprise contacting a tissue site of an animal or human with an immunogenic protein, polypeptide and/or drug-structural matrix composition of the invention in a manner effective to provide at least a first immunogenic protein, polypeptide and/or drug to immune or antigen presenting cells in the tissue site, thereby causing the immune or antigen presenting cells to stimulate an antigenic or immunogenic response in the animal or human.

The immunogenic protein, polypeptide and/or drug-structural matrix composition may comprise a plurality of immunogenic proteins, polypeptides and/or drugs obtained from one or more pathogenic organisms. The immunogenic protein, polypeptide and/or drug- structural matrix composition may release the same immunogenic protein(s), polypeptide(s) and/or drug(s) at different times, preferably, in an initial (priming) burst, followed later by a subsequent (booster) deliver. Vaccinations, such as hepatitis B, may be given in such a manner.

Anti-microbial, antibiotic, anti-viral and cytotoxic methods and uses for killing invading pathogens and/or treating diseased cells in an animal or human comprise contacting a tissue site of an animal or human with an anti-microbial, antibiotic, anti-viral or cytotoxic protein, polypeptide and/or drug-structural matrix composition of the invention in a manner effective to express at least a first anti-microbial, antibiotic, anti-viral or cytotoxic protein, polypeptide and/or drug in diseased cells within the tissue site. Invading pathogens are treated thereby, including where the anti-microbial, antibiotic and anti-viral proteins, polypeptides and/or drugs have direct effects on the invading pathogen and where such proteins, polypeptides and or drugs stimulate the host immune or defense system. Cancer cells and virally-infected cells are also treated thereby, including where the cytotoxic proteins, polypeptides and or drugs have direct cytotoxic effects, induce apoptosis and/or inhibit proliferation of the diseased cells.

Methods and uses for transplanting cells into an animal or human comprise applying to a tissue site of an animal or human a cell-therapeutic protein, polypeptide and/or drug- structural matrix combination of the invention. The cells of the cell-therapeutic protein, polypeptide and/or drug-structural matrix combination may be recombinant cells that also comprise a therapeutic gene(s) applied to the matrix.

Tissue engineering methods and uses for animals and humans comprise contacting a tissue site of an animal or human with a therapeutic protein, polypeptide and/or drug- structural matrix composition of the invention in a manner effective to both provide at least a first therapeutic protein, polypeptide and/or drug to cells within the tissue site and to provide a matrix for tissue growth. Guided tissue regeneration comprises contacting a regenerating tissue site of an animal or human with a therapeutic protein, polypeptide and/or drug- structural matrix composition of the invention in a manner effective to both provide at least a first therapeutic protein, polypeptide and/or drug to cells within the regenerating tissue site and to provide a matrix to guide tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of illustrative embodiments and the detailed examples presented herein.

FIG. 1 : A graph comparing mechanical properties (tensile strength) of SC/PL and GF/PL matrices.

FIG. 2: Shows the release profile of radiolabeled growth factor from the polymer matrix according to Example 2.

FIG. 3: Shows the cumulative VEGF release over time for the matrix according to Example 3.

FIG. 4: The effect of gas type on porosity of matrices. 85:15 PLGA (i.v. = 1.4 dL/g) disks were equilibrated for 1 hour in 850 psi gas prior to pressure release. The time for pressure release was 2.5 minutes.

FIG. 5: The effect of pressure release rate on porosity of PLGA matrices. 85:15 PLGA (i.v. = 1.4) disks were foamed for 1 hour in CO₂, with a pressure release time of 1 to 10 minutes.

FIG. 6: Porosity of matrices fabricated from different polymers. Polymers were exposed to 850 psi CO₂ for 24 hours with pressure release of 2.5 minutes.

FIG. 7: The effect of molecular weight on porosity of PLGA matrices. Matrices of 85:15 PLGA with varied intrinsic viscosity were foamed for 24 hours in 850 psi CO₂ with a pressure release time of 2.5 minutes. FIG. 8A: Porosity of matrices with varied equilibration times. 85:15 PLGA (i.v. = 1.4) and NaCl disks were foamed in 850 psi CO₂ for time ranging from 1-48 hours. The pressure release time was 2.5 minutes.

FIG. 8B: The elastic modulus of polymer/NaCl scaffolds fabricated with different equilibration times. 85: 15 PLGA (i.v. = 1.4)/NaCl disks were foamed in 850 psi CO₂ for 1- 12 hours with 2.5 minute pressure release.

FIG. 9A: The effect of polymer composition on porosity of polymer/NaCl scaffolds. Different copolymers of PLGA, PGA, and PLLA with NaCl were foamed for 24 hours in 850 psi CO₂ with a pressure release time of 2.5 minutes.

FIG. 9B: The elastic modulus of matrices formed with different polymer compositions. Different copolymer ratios of PLGA with NaCl were foamed for 24 hours in 850 psi CO₂ with 2.5 minute pressure release.

FIG. 10. Release kinetics of DNA from a biodegradable matrix of PLGA fabricated from microspheres pre-loaded with DNA (D; open square) or from a matrix fabricated by mixing the same PLGA polymer with DNA (•; closed circle).

FIG. 1 1. Release profile of VEGF and PDGF from a structural matrix comprised of

PLG (85:15) particle and PLG (75:25) microsphere forms of poly(lactide-co-glycolide), processed by gas foaming/particulate leaching. Shown is the cumulative release of VEGF

(closed square) pre-loaded into the bulk PLG matrix and PDGF (closed triangle) pre-loaded into PLG microspheres.

FIG. 12. Blood Vessel Density resulting from implantation of the VEGF-PDGF matrices of FIG. 11 into the subcutaneous space in the back of Lewis rats. After 2 and 4 weeks, blank, dual implants or those containing VEGF or PDGF alone were removed, fixed and the blood vessel number quantified and normalized, ns, not statistically significant; *, statistically significant, p<0.5 relative to the 2-week blank; **, statistically significant, p<0.5 relative to the 4-week blank.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The following embodiment is provided as a representative, non-limiting, example of the development of the invention. Discs comprised of polymer (e.g., poly[D,L-lactic-co- glycolic acid]) and NaCl particles were compression molded at room temperature, and subsequently allowed to equilibrate with high pressure CO₂ gas (800 psi). Creation of a thermodynamic instability led to the nucleation and growth of gas pores in the polymer particles, and the formation of a continuous polymer matrix. The NaCl particles were subsequently leached to yield macropores, and a macropore structure.

The overall porosity and level of pore connectivity was regulated by the ratio of polymer:salt particles. Both the compressive modulus (159 ± 130 kPa for SC/PL vs. 289 ± 25 kPa for GF/PL) and tensile modulus (334 ± 52 kPa for SC/PL vs. 1100 ± 236 kPa for GF/PL) of matrices formed with this approach were significantly greater than those formed with a standard solvent casting/particulate leaching process. The potential of these matrices for engineering new tissue was demonstrated by finding that smooth muscle cells readily adhered and proliferated on these matrices, forming new, high density tissues (3xl0⁷ cells/ml) in culture.

This process, a combination of high pressure gas foaming and particulate leaching techniques, allows one to fabricate matrices from biodegradable polymers with a well controlled porosity and pore structure. The inventors developed this process into the even more advantageous aspects of the present invention.

The present invention therefore particularly concerns fabricating three-dimensional matrices from microspheres that are loaded with proteins and/or drugs that are to be delivered from the matrix. Incoφorating proteins and/or drugs within a polymeric structure in particle form, e.g., as beads or microspheres, or blended with other polymers or molecules, is therefore an important aspect of the invention. This is exemplified by the incoφoration of proteins and/or drugs, optionally with other biological factors, into microspheres of poly (lactide-co-glycolide) utilizing an atomization/extraction process operated at cryogenic temperatures. Three-dimensional matrices are then fabricated using gas a foaming/particulate leaching process.

These approaches provide high incorporation efficiencies and a sustained release of functional biological factors, particularly proteins and/or drugs. Release of the proteins and/or drugs can be controlled in part through the microsphere fabrication process. Importantly, the microspheres can be formed from polymers or copolymers (e.g., PLGA) that degrade at different rates, or combinations of microspheres can be employed to give defined matrix regions that degrade at a rate different to the polymer or copolymer utilized to form the bulk of the matrix. Polymers or copolymers such as PLGA may also be combined with alginates or modified alginates to achieve similar differential control.

Such systems provide an additional level of control over the protein and/or drug release kinetics from the matrices. This gives additional control over their bioactivity, as proteins and/or drugs contained within the microsphere-derived polymeric structure can be designed to provide a controlled release effect therefrom in addition to the release kinetics provided by the matrix. The release in this situation will likely be controlled by either disassociation of the proteins and/or drugs from the bead, release from the PLGA, or both. Thus, a high degree of control over release kinetics is provided over a potentially wide range.

The data of Example 6 demonstrate the successful application of these aspects of the invention in the sequential release of two different drugs from a structural matrix formulated comprised of two forms of poly(lactide-co-glycolide), particulate and microsphere, wherein the matrix is processed by gas foaming/particulate leaching. Using vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) to exemplify the use of different factors, distinct release kinetics were achieved. Implantation of the matrix in vivo results in a rapid increase of a mature, vascular network in the matrix, relative to control matrices delivering only one of either VEGF or PDGF. The results shown in Example 6 are the first demonstration of the ability to deliver two distinct macromolecules from a structural matrix. The present invention therefore has important applications in veterinary and clinical intervention in biological processes requiring sequential release of molecules, including guided organ development, regeneration, and wound healing; cancer/HIV treatments and vaccinations; birth control interventions and antibiotic regimens.

The fabrication of multiple proteins and/or drugs into a matrix (in multiple types of the described particles and/or in polymer comprising the bulk of matrix) that will release the proteins and/or drugs at varying times will thus be useful to create cascades of different proteins, or waves of release of the same protein (e.g., for use in immunizations). Incoφoration of proteins and/or drugs into such particles (e.g., alginate beads) may also be more suitable for maintaining long-term bioactivity than immobilization directly in the polymer comprising the bulk of foamed matrix.

For example, the present invention provides matrices fabricated from combinations of polymers and bioactive proteins and or drugs to provide spatial and temporal control of release. This includes matrices fabricated from microspheres composed of different PLG copolymers, with each copolymer loaded with a different protein, drug and/or bioactive factor. Variations in the PLG co-polymer affect the polymer degradation and thus the time course of release. Also, the microsphere fabrication rate can be varied to control the release of the proteins and/or drugs. The entire variety of porous and solid outer wall-bounded matrices may be constructed by these processes. Thus, the spatial and temporal control over release provided by these fabrication methods is significant, meaning that the protein- and/or drug-matrices can be used in the treatment of a wide variety of disorders and injuries that occur in a number of tissues.

Nondegradable polymers, or matrices comprised partly of nondegradable polymers, are intended for use in situations in which permanent implants, or portions thereof, are desired. Additionally, some matrix materials are not degraded, but are remodeled. For example, hydroxyapatite is not degraded, but is used by osteoblasts and used to remodel new bone. Such materials can be combined with the GF/PL matrix-protein and/or matrix-drug compositions of the present invention, or used in conjunction therewith, e.g., in various implantable devices.

The materials prepared by the process of the invention thus exhibit a wide range of utilities. They may be applied to any use that requires a porous polymeric material, particularly with an open pore structure. The materials are particularly applicable for uses wherein organic solvent residue is not tolerable, e.g., in applications wherein biocompatibility is desired.

The problem with organic solvents is that residue remains in these polymers after processing may damage the transplanted cells and nearby tissue. Further, exposure to organic solvents would inactivate many biologically active factors. These disadvantages can be minimized or eliminated with the matrix materials of the invention because the growth factor can be incoφorated directly into the polymer matrix to obtain a better release.

The materials of the present invention are therefore useful as matrices in which cells are compatible and grow to achieve their intended function, such as in tissue replacement, eventually replacing the matrix depending on its biodegradability. Furthermore, the materials can be used to provide matrices already bound to cells, which may then be surgically implanted into a body. The materials can also be used as a sustained release drug delivery system, as wound healing matrix materials, as matrices for in vitro cell culture studies or uses similar thereto. The stable structure of the materials of the invention provides ideal cell culture conditions.

The materials of the invention prepared by the GF/PL process generally further have applications similar to those of materials prepared by the SC/PL and phase separation techniques, for example, in a variety of cell transplantation applications, including for hepatocytes (Mooney et al, (1994); Mooney et al, (1995), chondrocytes and osteoblasts. Ishaug et al, (1994). However, the materials of the invention have better mechanical properties and avoid the problem of organic solvent residue that may damage transplanted or migrating cells and nearby tissue and/or inactivate biologically active factors.

Smooth muscle cells readily adhere to the matrix material of the invention and create three-dimensional tissues within these porous structures; thus, they provide a suitable environment for cell proliferation. In vitro studies indicate concentrated cell growth around the periphery of the matrix. This is likely due to O₂ diffusion limitations to the cells at the center of the matrix because of the thickness (3.4 mm) of the sponge.

Another useful application for the polymer matrices of the invention is for guided tissue regeneration (GTR). This application is based on the premise that progenitor cells responsible for tissue regeneration reside in the underlying healthy tissue and can be induced to migrate into a defect and regenerate the lost tissue. An important feature of matrices for GTR is the transport of cells into the matrix, a property that is dictated by the pore size distribution and pore continuity, i.e., interconnectivity. The present matrices allow the desired cells to invade the matrix while preventing access to other cell types.

Materials of the invention, particularly polymer sponges made of poly(lactic acid)

PLA, poly(glycolic acid) (PGA), or poly(lactic-co-glycolic acid) (PLGA), having an impermeable layer on one side can provide this selective permeability feature. The impermeable layer is composed of the same polymers but without the extent of porosity, and a variety of methods can be used to couple the impermeable layer to the polymeric sponge.

An impermeable layer can be created on one side of the sponge by one of the following techniques, preferably performed before gas foaming of the material. The sponge can be pressed into shape on a layer of PGA at a temperature greater than the melting temperature for PGA. The melted PGA will be able to adhere to the sponge thus forming a thin layer. This layer is impermeable because the foaming process and the leaching process have a negligible effect on pure PGA. An impermeable layer of PLGA can also be created on the sponge by pressing the sponge onto a layer of PLGA. Spraying a solution of PLA in chloroform onto one side of the sponge can also create an impermeable layer. Further, it is possible to use the same polymer material and alter the amount of leachable particulate in each section so that one section forms an open pore structure and one does not.

Also, by using different polymers, materials wherein one section foams, and the impermeable layer section does not, can be provided. Although PLGA does foam following release of pressure from the bomb, an impermeable skin forms on the thin layer of PLGA that remains intact during the leaching process. Alternatively, following the foaming and leaching process, the polymeric sponge can be dipped in either melted PGA or in a solution of PLGA in chloroform. These procedures can be used to create a sponge that has a porosity of greater than 95% with an impermeable side.

Similar methods can be applied to analogous materials, as discussed above, to provide other sponge materials according to the invention useful for GTR applications.

The PLGA matrices also can provide a suitable substrate for bone formation. An important feature of a matrix for replacement of bony tissues is its ability to provide an appropriate environment for tissue development and matrix mineralization. The ability of the GF/PL matrices to allow cell adhesion and tissue formation was assessed in vitro by seeding and culturing MC3T3-E1 cells, an osteogenic cell line, on PLGA scaffolds with techniques previously optimized for other cell types (Kim et al, 1998). Cells adhered to the GF/PL matrix, proliferated, and began secreting extracellular matrix proteins, and by 4 weeks in culture patches of mineralization could be observed. A new tissue with large areas of mineralization was formed by 6 weeks. There was no observed change in the size and shape of the matrices over this time period suggesting they had sufficient mechanical properties to control gross formation of engineered bone tissue.

An important feature of the matrix for use in guided tissue regeneration is the ability of cells to migrate into the matrix. Studies confirm that cells readily migrated into and throughout the matrix in vitro. This was expected as previous studies with these types of matrices demonstrated fibrovascular ingrowth in vivo at a rate of 0.1 - 0.3 mm/day (Mooney et l, l99A). Another potential application of these sponge materials for GTR is for the treatment of periodontal disease. Periodontal disease is characterized by the loss of attachment of the periodontal ligament to the alveolar bone. The epithelial cells of the gingiva begin to grow into the site where the periodontal ligament was attached. A sponge of the matrix material according to the invention with an impermeable side could be used to prevent the downgrowth of epithelial cells while allowing the appropriate cells to occupy the porous sponge thereby regenerating the periodontal ligament. Further guidance as to such application is provided by Shea et al. (Biodegradable Polymer Matrices in Dental Tissue Engineering).

For other applications in which cells are seeded or otherwise incoφorated and grown within the inventive matrices, incoφoration and growth of the cells can be facilitated in a manner known in the art. Examples of such methods are provided in U.S. Patent Nos. 5,041,138; 5,567,612; 5,696,175 and 5,709,854; all of which are incoφorated herein by reference.

The following examples are included to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art will, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1 MATRIX PROCESSING

Pellets of an 85:15 copolymer of D,L-lactide and glycolide (PLGA) were purchased from Boehringer Ingelheim (Henley, Montvale, NJ, USA), and utilized to fabricate polymer matrices in all studies. The intrinsic viscosity of the polymer was about 1.3-1.7. Polymer pellets were ground using a Tekmar grinder (Bel-Art Products, Pequannock, NJ, USA), and sieved to obtain particles ranging from 106 to 250 μm.

In certain studies the polymer particles were mixed with sodium chloride particles (Mallinckrodt, Paris, KY, USA). The salt particles were sieved to yield a range of sizes, and the weight ratio of NaC PLGA masses ranged from 0 to 50. In all cases, the total mass of PLGA and NaCl was held constant at 0.8 g.

The mixtures of PLGA and NaCl were loaded into a KBr die (1.35 cm in diameter; Aldrich Chemical Co., Milwaukee, WI, USA), and compressed at 1500 psi for 1 minute using a Carver Laboratory Press (Fred S. Carver, Inc., Menominee Falls, WI, USA) to yield solid discs (thickness=3.4 mm). The samples were then exposed to high pressure CO₂ gas (800 psi) for 48 hours to saturate the polymer with gas. A thermodynamic instability was then created by decreasing the gas pressure to ambient pressure. This led to the nucleation and growth of CO₂ pores within the polymer matrices. The NaCl particles were subsequently removed from the matrices by leaching the matrices in ddH₂O for 48 hours. All processing steps were performed at ambient temperature.

Porous sponges were also fabricated using a previously described solvent casting- particulate leaching technique. (Mikos et al, 1994).) In this process, PLGA was dissolved in chloroform (Mallinckrodt; Paris, KY, USA) to yield a solution of 10% (w:v), and 0.12 ml of this solution was loaded into Teflon cylinders (diameter 0.5 cm;, Cole Parmer) packed with 0.4 g of sodium chloride particles sieved to a size between 250 and 500 mm. Following solvent evaporation, polymer films with entrapped salt particles (3 mm thick) were carefully removed from the molds. The salt was removed by immersing films in distilled water for 48 hrs.

1. Characterization

The porosity of samples was initially determined by gross measurements and weights after processing using the following equation: Eqn. 1 : porosity(%) = 1 -[(weight/volume) / (density of polymer)] x 100

The samples were imaged using a scanning electron microscope (ISI-DS 130, Topcon Technologies, Pleasanton, CA, USA). The samples were gold coated using a Sputter Coater (Desk II, Denton Vacuum, Cherry Hill, NJ, USA), and the microscope was operated at 10 kV to image the samples. Polaroid 55 film was used for the photomicrographs.

Compression and tensile testing were performed on an MTS Bionix 100 (Sintech, Research Triangle Park, NC, USA). Samples were cut into lxl cm squares for compression testing. For tensile testing, the samples (lxl cm) were attached to cardboard using epoxy glue. A 7 mm slot was cut into the center of the card board and the sample was centered, then glued to standardize the gage length. Compression and tensile tests were performed with a constant strain rate (lmm/min). The moduli were determined from the slopes in the elastic portion of the stress-strain diagram.

Thermogravimetric analysis was utilized to determine the amount of salt residue that remained in the sponge after leaching. Matrices were heated from 150°C to 300°C at a constant rate of 10°C/min, and the residual mass was monitored.

2. Cell Studies

Smooth muscle cells (SMC) were used in all studies. SMCs were isolated and cultured using a modification of the techniques described in Rothman et al. (Rothman et al. , 1992). In brief, the cells were isolated from aortas of 300-350 g adult male Lewis rats (Charles River Laboratories, Wilmington, MA, USA) using an enzymatic dissociation.

After fat, adventitia, and connective tissue surrounding the arteries were removed by blunt dissection, the SM tissue was cut into multiple small pieces and placed into a spinner flask containing an enzymatic dissociation buffer at 37°C. This buffer contains 0.125 mg/mL elastase (Sigma Chemical Co., St. Louis, MO, USA), 1.0 mg/mL collagenase (CLS type I,

204 units/mg, Worthington Biochemical Coφ., Freehold, NJ, USA), 0.250 mg/mL soybean trypsin inhibitor (type 1-S, Sigma), and 2.0 mg/mL crystallized bovine serum albumin (BSA, Gibco/Life Technologies, Gaithersburg, MD, USA). After 90 minutes of incubation, the suspension was filtered through a 100 5m Nitex filter (Tetko, Inc., Briarcliff Manor, NY) and centrifuged at 200 g for 5 minutes. The pellet was resuspended in Medium 199 (Sigma) supplemented with 20% (v/v) fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), and 50 units/mL penicillin-streptomycin (Gibco).

The cells were cultured on tissue culture plastic in a humidified 5% CO₂ atmosphere with the medium (Medium 199, 10%(v/v) fetal bovine serum, 50 units/mL penicillin- streptomycin) changed every other day. Cells at passage 17 were used in these studies.

The matrices were seeded with SMCs by placing a 40 mL cell suspension containing

3.14 x 10⁷ cells/mL on top of each matrix and allowing the cell suspension to absorb into the matrix. Matrices were contained in tissue culture dishes and incubated at 37 DC for ~36 hours. Next, the polymer matrices were cultured for two weeks and placed in a spinner flask

(100 mL, Bellco Glass, Inc., Vineland, NJ, USA) stirred at 40 RPM.

The number of cells in the matrices was determined by measuring the DNA content in enzyme-digested triplicate samples using Hoechst 33258 dye and a fiuorometer (Hoefer DyNA Quant 200, Pharmacia Biotech, Uppsala, Sweden) as previously described.

For scanning electron microscopic examination, samples were fixed in 1% glutaraldehyde and 0.1% formaldehyde for 30 minutes and 24 hours, respectively, dehydrated in a graded series of ethanol/water solutions, dried, and then sputter-coated with gold. A scanning electron microscope (ISI-DS 130, Topcon Technologies) was operated at 10 kV to image samples. Histological sections were prepared by fixing cell-polymer matrices (10% formalin), dehydrating, embedding, sectioning and staining with hematoxylin and eosin or VerhoefUs using standard techniques. 3. Integrity and Porosity/Pore Structure of Foamed Matrices

Photomicrographs showed that gas foaming, alone, of solid polymer discs led to the formation of highly porous matrices. However, these matrices had a non-porous skin on the external surfaces and the pores were largely closed, as expected from previous studies (Mooney et al, 1996). In contrast, gas-foaming and subsequent leaching of discs containing a high percentage (95%) of large (250<d<425 μm) NaCl particles, according to the invention, led to the formation of highly porous, open pore matrices with no evidence of an external, non-porous skin.

The pore structure observed in cross-sections of these matrices was similar to that observed in cross-sections of matrices formed with a SC/PL technique. However, the pore structure of matrices formed from the SC/PL process is often not uniform throughout the matrix due to evaporation of the organic solvent and subsequent increase in the polymer concentration of the remaining solution entrapped within the salt bed.

For example, the surface of these matrices that is adjacent to the glass coverslip during processing is shown in photomicrographs to be typically less porous than the remainder of the matrix. In contrast, the pore structure of gas foamed-particulate leached (GF/PL) matrices was uniform throughout the matrix and on the exterior surfaces.

TGA analysis of matrices indicated that negligible amounts of NaCl remained after leaching. There was a trace of a white residue left in the dish. To confirm that the gas foaming was responsible for the formation of stable matrices, control samples were compression molded, but not foamed. Leaching of the NaCl from these matrices led to complete breakdown of the matrices.

The ratio of NaC PLGA and the size of NaCl particles in GF/PL matrices were next varied to determine the range of porosity and pore structure that could be obtained with this process (Table 1). The gross porosity of these matrices increased from 85.1% ± 2.3 to 96.5 % ± 0.5 as the ratio of NaC PLGA was similarly increased. At constant NaCl (95%), the increase in salt particle diameter had very little effect on the overall porosity. However, photomicrographs showed that as the salt diameter was increased, the pore size increased in parallel.

The stability of the matrices was next assessed using compressive and tensile mechanical tests. In general, the GF/PL matrices exhibited improved mechanical properties as compared to the SC/PL matrices (FIG. 1). The average compression moduli were 159 ± 130 kPa and 289 ± 25 kPa for the SC/PL and GF/PL matrices, respectively. The average tensile moduli were 334 ± 52 kPa for the SC/PL matrices and 1100 ± 236 kPa for the GF/PL matrices (Table 2). This data represents a 80% increase in compression strength and a 300% increase in tensile strength.

TABLE 1

Gross Porosity of Sponges

NaCl Concentration Diameter(μm)

(%) 106-250 250-425 >425

80 85.1 ± 2.3 —

90 87.3 ± 1.9 91.5 ± 1.4 —

95 93.9 ± 0.9 94.6 ± 0.9 95.0 ± 0.8

97 96.5 ± 0.5 —

TABLE 2

Mechanical Properties

Tensile Test

Method Compressive Modulus (kPa) Modulus (kPa) Elong ation @ break (%)

Solvent/leach 159 ± 130 334 ± 52 17.5 ± 1.9

Foam/leach 289 ± 25 1100 ± 236 12.0 ± 1.3

4. Tissue Development : on Synthetic Matrices

The ability of the GF/PL matrices to allow cell adhesion and tissue formation was next assessed in an in vitro study. Photomicrographs show that SMCs adhered to the GF/PL matrix and covered the available surface area following seeding. A significant increase in cell number was shown after 2 weeks in culture. The average cell density was 1.71 x 10⁷ cells/mL and 3.05 x 10⁷ cells/mL at 0 and 2 weeks, respectively. This is a 43.8% increase in cell density. The cells filled the pores of the matrix and created a new three-dimensional tissue within the synthetic matrix. However, most of the cell growth occurred around the periphery of the matrix in a relatively uniform manner, and a low cell concentration was observed in the center of the matrices at 2 weeks. There was no observed change in the size and shape of the matrices over this time period.

EXAMPLE 2 GROWTH FACTOR RELEASE FROM FOAMED MATRICES

1. Methods

1251-labelled vascular endothelial growth factor (VEGF) was first added to a solution of 1% sodium alginate, and then beads of this solution were gelled by injecting droplets into a aqueous solution containing calcium chloride. The alginate beads (approximately 3 mm in diameter) were collected, rinsed, and lyophilized. The lyophilized beads were mixed with 85:15 PLGA and NaCl particles and the mixture compression molded and processed with the gas foaming/particulate leaching process as previously described.

Following salt leaching and drying, the matrices were placed in serum free tissue culture medium and maintained at 37°C. Medium samples were taken periodically, and analyzed for the content of 125I-VEGF (released from PLGA matrices). The released growth factor was normalized to the total incoφorated growth factor.

2. Results An initial burst of approximately 20% of the incoφorated growth factor was noted in the first day, and a sustained release of growth factor was noted for the remaining 20 days of the study (FIG. 2). EXAMPLE 3 GROWTH FACTOR DELIVERY

One factor that may facilitate the development of tissues on the matrices is the delivery of growth factors into the local environment. The incoφoration and release of growth factors from these matrices was assessed in vitro using 1251-labeled vascular endothelial growth factor (VEGF). A substantial fraction of the drug was released during the particulate leaching process; however, the remaining drug was released in a sustained manner during the 21 days of the study. FIG. 3 shows the cumulative VEGF release over time for this matrix.

EXAMPLE 4 MATRIX CHARACTERIZATION

1. Matrix Fabrication

Pellets of poly L-lactic acid [PLLA], a 50:50 copolymer of D,L-lactide and glycolide (50:50 PLGA) with intrinsic viscosity (i.v. of 0.2 dL/g), a 75:25 PLGA copolymer (i.v. = 1.3), and an 85:15 PLGA copolymer (i.v. = 1.4) were obtained from Boehringer Ingelheim (Henley, Montvale, NJ, USA). PGA, 50:50 PLGA (i.v. = .8) and 85: 15 PLGA (iv = .63) were purchased from Medisorb (Cincinnati, OH, USA). 85:15 PLGA (i.v. = 3.63) was obtained from Purasorb (Lincolnshire, IL, USA).

The solid polymer (PLLA, PLGA, PGA) was ground (after freezing with liquid nitrogen) using a Scienceware Micro-Mill (Bel-Art Products, Pequannock, NJ, USA) and sieved to a diameter of 106-250 5m. NaCl, obtained from Fisher Scientific (Pittsburgh, PA, USA), was sieved to a diameter of 250-425 5m for use in certain studies. Solid polymer disks were formed by placing 150 mg polymer (PGA, 50:50 PLGA, 75:25 PLGA, 85:15 PLGA, and PLLA) into a round stainless steel KBr die with diameter 1.35 cm (Aldrich Chemical Co., Milwaukee, WI, USA) and compressing for 60 seconds at 1500 psi in a Carver Laboratory Press (Fred S. Carver, Inc., Menominee Falls, WI, USA). This method yields solid disks to be foamed. All samples were fabricated in triplicate. The disks were foamed in a high pressure vessel using CO₂, N₂, or He at 850 psi. After the disks were equilibrated (1-48 hours) with the gas, the pressure was reduced to ambient. The resulting thermodynamic instability caused nucleation and growth of gas pores within the polymer matrix. 85:15 solid polymer disks (i.v. = 1.4) were foamed for 1 hour in CO₂ and the pressure was released at different rates (1, 2.5, 5, 10 minutes) to determine if the rate of pressure release affects the final structure of the sponges. All processing steps were performed at ambient temperature.

Polymer/NaCl disks were fabricated in a similar way using 40 mg polymer and 760 mg NaCl, compressed into disks. Following foaming, the disks were placed in distilled water in order to remove the NaCl. This leaching solution was changed several times over the course of about 18 hours. The disks were considered to be completely leeched when the leeching solution did not give a precipitate with AgNO3. If CI- is present in solution, it precipitates with Ag+ to form a white precipitate. The failure of this precipitate to form indicated that the NaCl is completely removed from the scaffolds. The disks were then air dried overnight, measured and weighed, and stored in a dessicator under vacuum. The polymer disks were measured and weighed immediately following foaming, then stored in a dessicator under vacuum.

2. Characterization

In order to calculate the porosity of the foamed disks, a boley gauge was used to measure the diameter and thickness of each disk. The disks were weighed on a Mettler balance and the following equation was used: (d = polymer density, g = disk wt, cm3 = calculated disk volume).

porosity = 100 [l-(g/cm3)/d]

Several of the samples were imaged using a scanning electron microscope (ISI-DS

130, Topcon Technologies, Pleasanton, CA, USA). The samples were gold coated using a Sputter Coater (Desk II, Denton Vacuum, Cherry Hill, NJ, USA) and the microscope was operated at 10 kV to image the samples. Polaroid 55 film was used for the photomicrographs .

Compression testing was performed on an MTS Bionix 100 (Sintech, Research Triangle Park, NC, USA). Only polymer/NaCl disks were used in compression tests because the solid polymer disks foamed to irregular shapes. A constant strain rate of 1 mm/min was used, and moduli were determined from the stress-strain curves.

3. Foaming solid polymer disks In the first series of studies, solid polymer disks were foamed to investigate the role of the gas type, pressure release rate, and polymer composition and molecular weight on the porosity of polymer matrices. 85:15 PLGA matrices were foamed for 1 hour with several different gases (CO₂, N₂, He). Significant porosity resulted from foaming with CO₂ as compared to N₂ and He. The "prefoam" porosity refers to the calculated porosity following disk preparation, but prior to high pressure equilibration (FIG. 4). Visualization of matrices foamed with CO₂ revealed a highly porous matrix consisting largely of closed pores.

In the next study, the rate of release of pressure was varied from 1 to 10 minutes total time. The porosity of the matrices was relatively constant regardless of pressure release rate, except in the case of a very rapid release, when the gas froze within the chamber. This led to a small decrease in the matrix porosity (FIG. 5).

The effect of the polymer composition was investigated by using different copolymer ratios of PLGA (pure PGA, 50:50, 75:25, 85:15 PLGA and pure PLLA). Neither PGA nor PLLA foamed appreciably. The copolymers all foamed to a porosity greater than 90% (FIG. 6). In fact, the 75:25 copolymer foamed so extensively that it did not maintain its integrity in the pressure release/gas expansion phase and literally fell apart. Hence, no porosity value could be calculated for that sample.

In order to study the effect of polymer molecular weight on pore formation, disks of

85:15 PLGA with intrinsic viscosity (i.v.) ranging from .63 to 3.59 dL/g were foamed in 850 psi CO2 for 24 hours with a pressure release of 2.5 minutes. The high i.v. PLGA led to matrices with relatively low porosity, whereas the lower i.v. PLGA resulted in much higher porosity (FIG. 7).

4. Foaming polymer/NaCl disks

In the second series of studies, NaCl was incoφorated into the polymer disk for the puφose of creating an open pore structure. Different variables (equilibration time and polymer composition) were studied in order to determine their effects on the structure and stability of the scaffolds. The results of the first series of studies led us to use CO₂ as the foaming gas, and a pressure release time of 2.5 minutes in this series of studies. Examination of a typical matrix formed by foaming 85:15 PLGA with NaCl in CO₂ shows a highly porous structure with largely open, interconnected pores.

In the first study, the equilibration time was varied from 1 to 48 hours. The porosity of the matrices was relatively constant for equilibration times greater than 6 hours, but decreased for equilibration times less than 6 hours (FIG. 8A). Matrices fabricated with various equilibration times were subsequently tested to determine if the equilibration time affected their mechanical properties. Even though maximal porosity was achieved with 6 hours of gas equilibration, a stronger scaffold was produced with longer equilibration times (FIG. 8B).

The polymer composition was next varied to determine if results similar to those in the first series of studies would be obtained. Copolymers of PLGA led to a much greater porosity than did the homopolymers PGA and PLLA (FIG. 9A). Both the PLLA and PGA disks disintegrated in the leaching process, indicating that little, if any, foaming had occurred. Even though all PLGA copolymers led to matrices with similar porosities, the matrices fabricated from PLGA with higher lactic acid content were more rigid (FIG. 9B). EXAMPLE 5 MATRIX PREFABRICATION

Controlled open pore matrices can also be fabricated by gas foaming/particulate leaching processes applied to polymer particles pre-loaded with biological materials. The following data exemplify an aspect of such processes, where nucleic acids are incoφorated into microspheres of poly(lactide-co-glycolide) utilizing an atomization/extraction process operated at cryogenic temperatures. The three-dimensional matrix was then fabricated using a gas foaming/particulate leaching process. These approaches provide high incoφoration efficiencies and sustained release, which can be controlled in part through the microsphere fabrication process.

Plasmid DNA was incoφorated into microspheres composed of poly(lactide-co- glycolide) (PLGA) using an atomization and extraction process operated at cryogenic temperatures. The plasmid was dissolved in a Hepes/Mannitol buffer for stability during the lyophilization process. The plasmid solution was passed through a nitrogen atomizer into a vessel containing liquid nitrogen and lyophilized. The lyophilized plasmid was next mixed with a solution of PLGA in chloroform. The plasmid in polymer mixture was mixed and passed through an atomizer into a vessel containing frozen ethanol overlaid with liquid nitrogen. The vessel was then placed at -80°C, allowing the microspheres to harden as the melting ethanol extracted the chloroform. Microspheres incoφorating DNA were then isolated by filtering and dried in the lyophilizer.

Open pore matrices containing plasmid DNA were subsequently fabricated with a gas foaming/particulate leaching process. Microspheres incoφorating DNA were mixed with sodium chloride, compressed into a disc, and placed in a pressure vessel with a high-pressure gas. Release of the pressure caused the polymer microspheres to expand. Collisions between adjacent microspheres cause them to fuse, thereby producing interconnected structural matrices. Pores were formed within the matrix by leaching out the salt, leaving a matrix with an open pore structure. In situations where an open pore structure is not desired, such as where cellular invasion is not necessary, a closed pore polymer structure can be generated and used, e.g., for sustained DNA delivery. Studies showed that plasmid DNA trapped within biodegradable matrices fabricated from pre-loaded microspheres is gradually released from the matrix. The release kinetics of DNA from various copolymers of poly(lactide-co-glycolide) for the microsphere-loaded foamed matrices are shown in FIG. 10. Release kinetics are also provided for matrices formed by the foaming of an admixture of lyophilized plasmid, polymer, and salt (as in the previous examples). The release kinetics are a function of the processing conditions.

FIG. 10 shows a sustained release of plasmid from the microsphere-loaded matrices for up to 14 days. Release from other foamed sponges of comparable composition but fabricated from a plasmid-polymer admixture occurred in less than 1 day. Gel electrophoresis was performed for the DNA released at different times from the matrices prepared from the microsphere-preloaded particles. At all times of release (0.16, 1, 3, 7, and 14 days), the DNA was not degraded, as evidenced by bands appearing on the gel at the appropriate locations.

EXAMPLE 6 DIFFERENTIAL RELEASE OF TWO DIFFERENT DRUGS

The present example demonstrates the successful application of this system in the sequential release of two different drugs. A structural matrix was formulated comprised of two forms of poly(lactide-co-glycolide), particulate and microsphere, and the matrix was then processed by gas foaming/particulate leaching. The resultant matrix was capable of the sustained release of two different model macromolecules, as exemplified by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), each with distinct kinetics. Importantly, implantation of the matrix in vivo resulted in a rapid increase of a mature, vascular network in the matrix, relative to control matrices delivering only one factor.

1. Materials PLG formulations, Resomer RG858 (85:15, i.v. 1.5dl/g) and Resomer RG756 (75:25, i.v. 0.8dl/g), were purchased from Boehringer Ingleheim (Petersburg, VA). Poly(vinyl alcohol), ethyl acetate, sodium chloride, and Sigmatcote were purchased from Sigma, (St. Louis, MO). Platelet-derived growth factor and vascular endothelial growth factor were purchased from Intergen (Purchased, NY), and ¹²⁵I-PDGF and ^l25I-VEGF was purchased from New England Nuclear (Boston, MA). Alginate was from ProNova (Oslo, Norway).

2. Methods

Structural matrices were formed by the combination of PLG (85:15) particles and PLG (75:25) microspheres, gas foamed as described in the previous examples. VEGF was included in the bulk PLG matrix and PDGF was pre-loaded into PLG microspheres. Two types of sponges were fabricated: i) VEGF (2μg) and ¹²⁵I-VEGF (0.5μCi) incoφorated in 85:15 PLG, combined with 75:25 microspheres containing PDGF (3μg); and ii) Cold VEGF (2μg) incoφorated into 85:15 PLG, combined with 75:25 microspheres containing PDGF (3μg) and ¹²⁵I-PDGF (0.5μCi). VEGF was dissolved in 1% alginate (medium viscosity, high mannuronic acid content; total per matrix 5%w/w) solution in PBS. The VEGF solution was added to a total of 40mg of polymer (20mg 85:15 particulate PLG; sieved to 106μm < d < 250μm; and 20mg 75:25 microsphere PLG). The mixture was flash frozen in liquid nitrogen and lyophilized until dry.

Sodium chloride (760mg; sieved to dimensions of 250μm < d < 425μm) was mixed with the PLG and compression molded for lmin at 1500psi using a Carver Press to final dimensions of 3mm in thickness by 13mm in diameter. The matrices were placed in a pressure chamber and equilibrated for 24h at 800psi in ambient CO₂ for gas foaming. The release valve was opened and pressure was decreased to Opsi over the course of 2min, causing the PLG particles and microspheres to fuse into a continuous matrix. Matrices were leached in PBS (containing lOmM CaCl₂) for 24h at room temperature to remove the NaCl poragen.

To quantify the release kinetics of the incoφorated VEGF and PDGF, the matrices were placed in 5ml of PBS (containing lOmM CaCl₂) and samples were aliquoted periodically and counted using a gamma counter. Specifically, 4ml of PBS were removed for analysis and the remaining 1ml was discarded, and replaced with fresh PBS (5ml). Sample aliquots were taken every few hours for the first day, and every day for the first week, to monitor the initial burst of release described for PLG matrices. After the completion of the release study, total incoφoration of VEGF and PDGF were determined by analyzing counts from the leach, the total released and the remaining factors in the matrix.

Blood vessel density was quantified and histology was performed on matrices that were implanted into Lewis rats (male, 8-10weeks old). Matrices were implanted in the subcutaneous space in the back. For each timepoint of 2 and 4 weeks, 4 animals per condition, each receiving 4 implants (blank, PDGF, VEGF, dual) were implanted per animal. Implants were removed at the appropriate time and fixed in 3.7% formaldehyde overnight and subjected to hematoxylin/eosin staining using standard procedures for histology. Blood vessel number was quantified using a microscope (400x) by counting the number of blood vessels in each tissue section and normalizing to the area of the section.

3. Release Kinetics

The release kinetics of VEGF and PDGF demonstrated marked differences. VEGF, released from the bulk matrix, demonstrated a characteristic burst followed by a sustained profile (FIG. 11). Cumulative VEGF release reached 60% by 7 weeks, indicating a sustained release profile. PDGF, however, showed strikingly distinct release kinetics. PDGF, released from microspheres included in the matrix, did not exhibit a burst, but rather a slow, sustained release profile over the entire incubation period. Interestingly, inclusion of the PDGF into microspheres prior to mixing with the particulate PLG in the matrix resulted in less than 10% of the PDGF being released from the matrix over the time course evaluated. This indicates that the formulation of PLG matrices using both particulate and microsphere forms provides a mechanism to deliver multiple proteins from the same delivery vehicle with markedly distinct release profiles.

4. In Vivo Blood Vessel Formation

The matrices were evaluated for their ability to direct blood vessel formation in vivo. Matrices containing VEGF, PDGF, or both, were implanted subcutaneously into Lewis rats. Implants were removed after 2 weeks (n=4) and 4 weeks (n=4), and analyzed for blood vessels density and compared to control matrices with no growth factors.

Matrices with no growth factors contained relatively few blood vessels after 2 weeks, and those containing PDGF alone showed vessels with thicker basement membranes. VEGF alone resulted numerous blood vessels, but retaining the characteristic immaturity marked by a thin basement membrane. When VEGF was delivered followed by PDGF, numerous vessels were induced to grow into the matrix, and these vessels showed a thick basement membrane, indicating a mature phenotype. Similar results were obtained after 4 weeks of implantation. Blank matrices contained small blood vessels and were relatively immature, whereas matrices with PDGF resulted in larger and more mature vasculature. VEGF alone resembled the 2 week timepoint in that the vessels induced to grow into the matrix were immature and had little substantial basement membrane. Dual release matrices, delivering both VEGF and PDGF, were consistent with the 2 week timepoint as well, in which large, mature blood vessels were found throughout the matrix.

The blood vessel index was determined to quantify the number of blood vessels within the matrices. After 2 weeks post-implantation, matrices containing VEGF (either VEGF alone or in conjunction with PDGF) contained a statistically significant higher number of blood vessels compared to the 2 week blank matrices (FIG. 12). In contrast, PDGF alone did not result in a significant difference. After 4 weeks, only the dual release matrices contained a statistically significant increase in the number of blood vessels, relative to controls. VEGF alone showed a decrease and PDGF alone resulted in an increase, though both vessel density indices for each condition were insignificant.

EXAMPLE 7 POLYSACCHARIDE AND ALGINATE MATRICES 1. Modified Alginates

Each of U.S. Provisional Application Serial No. 60/026,362, filed September 19, 1996; U.S. Provisional Application Serial No. 60/026,467, filed September 19, 1996; U.S.

Provisional Application Serial No. 60/041,565, filed March 21, 1997 and PCT Application Serial PCT/US97/16890, filed September 19, 1997 are specifically incoφorated herein by reference without disclaimer for the puφoses of describing the preparation and use of further unique polymeric materials and matrices thereof.

In particular, the foregoing applications, specifically incoφorated herein by reference, teach the preparation and use of polysaccharides modified to bind biological agents covalently and also provide mechanisms for dissolution of the covalent bond to effect release of the biological agents of interest. Particularly disclosed are alginates modified so that they have controllable physical properties, such as sol-gel properties, and the like.

More particularly, the foregoing incoφorated applications describe modified alginates that comprise at least one alginate chain section to which is bonded at least one molecule useful for cellular interaction. Preferred bonding mechanisms are those utilizing one or more uronic acid residues on the alginate chain section. As described in each of the foregoing applications, the biomolecules useful for cellular interaction are exemplified by cell adhesion molecules, cell attachment peptides, proteoglycan attachment peptide sequences, proteoglycans, and polysaccharides exhibiting cell adhesion. Particular examples are RGD peptides, fibronectin, vitronectin, Laminin A, Laminin B 1 , Laminin B2, collagen 1 or thrombospondin. Various polypeptide or peptide growth factors or enzymes may also be used as the cellular interacting molecules.

In certain embodiments, the alginate backbone of the modified alginate composition may comprise an oligomeric block unit of D-mannuronate, L-guluronate, or various combinations thereof. Alginates with naturally occurring alginate chain sections are also suitable for use in the modified alginate compositions.

In general, the alginate chain sections will have a molecular weight of about 30,000, about 50,000 or up to about 100,000 or more.

As described in each of the foregoing applications, specifically incoφorated herein by reference, the modified alginate compositions are suitable for formulation into an injectable composition or solution for use as a cell transplantation matrix. All such matrices can be used in combination with genes or DNA to prepare the DNA-matrix formulations of the present invention. The modified alginate compositions form a network for viable cells to multiply, and are thus effective cell transplantation matrices. The modified alginate components of such matrices may be considered to be "hydrogels".

In certain embodiments, the modified alginates may contain at least one alginate chain section bonded to a polymeric backbone section and/or at least one alginate chain section cross-linked to another alginate chain section on the same or a different molecule. Thus, the polymers may comprise polymeric backbone sections and various side chains bonded to the backbone, optionally through a biological linker. Appropriate linkers are those involving amino acids, amino aldehydes, amino alcohols, or derivatized groups such as hydrazine, hydrazide, or semicarbazide. Appropriate backbone sections are therefore various natural and synthetic polymers, such as peptides and polypeptides, poly(vinyl alcohol), poly(ethylene oxide), and poly(uronic acid) in addition to the alginate-based polymers.

As described in each of the foregoing U.S. and PCT patent applications, each incoφorated herein by reference, biodegradable linkers may be provided to form cleavable bonds between the backbone section and the side chain. Exemplary biodegradable bonds are those wherein a linker is bonded to the polymeric backbone section by an ester, imine, hydrozone or semicarbazone group.

Also provided by the foregoing incoφorated applications are alginate materials that comprise alginate chains with covalently bonded cross-linking between the chains. This provides alginate material that is cross-linked to the extent such that it resumes essentially its original shape after compression. In certain aspects, the alginate material has sol-gel properties. The alginate material may additionally be gelled by the action of a divalent cation.

In the cross-linked, form-retaining alginate materials, the alginate chains may be cross-linked with a polyfunctional cross-linking agent having at least two functional groups that are covalently bonded to readable groups within the alginate chains, such as carboxylic or uronic acid groups. These bifunctional cross-linking agents may therefore comprise at least two nitrogen-containing functional groups, as exemplified by containing at least two imine, hydrozide or semicarbazide functional groups, or combinations thereof. In certain embodiments, the cross-linking agents will be lysine or an alkyl ester thereof.

The flexible, cross-linked alginates can generally vary between forms in which 1-75 mole% of the carboxylic or uronic acid groups in the alginate chains are cross-linked. About

1, about 5, about 10, about 20, about 50 and about 75 mole% cross-linking provides useful polymers.

The various cross-linked alginate materials can thus be formulated in a viscous liquid form or in a swellable gel form. As mentioned above, these alginate materials may also be fabricated in non-swellable, compression-resistant forms having "shape memory properties". Any of these various alginate liquids, gels or shape-memory gels may also be bonded to other biomolecules, particularly any of the foregoing molecules useful for cellular interactions.

In the DNA-focused methods of the present invention, the genes or DNA may be linked to any one or more of the foregoing modified alginate compositions via a covalent bond, and preferably, via a biodegradable or releasable bond. Equally, as with any other structural matrix, genetic material may simply be physically and functionally associated with a matrix, and there is no particular requirement for covalent bonding.

2. Porous Alginate Hydrogels U.S. Provisional Application Serial No. 60/128,681, filed April 09, 1999, is specifically incoφorated herein by reference without disclaimer for the puφoses of describing the preparation and use of further unique polymeric materials and matrices thereof. In particular, this application teaches the preparation and use of porous hydrogel materials formed by first creating gas pockets in the gel and then removing the gas to create a material with an open, interconnected pore structure that is maintained over extended time periods and has high mechanical integrity. Two important aspects for the preparation of porous hydrogel materials are the gas bubble formation and subsequent stabilization. For embodiments using BSA surfactant and a bicarbonate gas-generating component, a certain ratio of BSA to bicarbonate solution is necessary to develop a foamy solution. Stabilization of the gas bubbles is dependent on the viscosity of the starting alginate solution and the concentration of BSA. A low viscosity solution cannot stabilize entrapped gas bubbles, while too high of a viscosity leads to a gel that is so strong that the gas bubbles cannot be readily removed during the vacuum step. In addition, the BSA serves to stabilize the gas bubbles in the alginate solution, and it is important to have the appropriate BSA concentration to enable formation of a stable foam containing gas bubbles.

Specifically, the following conditions resulted in the formation of open, interconnected porous hydrogels.

1. 3 w: w % alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.24g of the BSA solution and 0.12g of the bicarbonate solution to yield a foamy solution.

2. 3 w:w % alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.24g of the BSA solution and 0.24g of the bicarbonate solution to yield a foamy solution.

3. 4 w:w % alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.32g of the BSA solution and 0.16g of the bicarbonate solution to yield a foamy solution.

4. 4 w:w % alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.34g of the BSA solution and 0.34g of the bicarbonate solution to yield a foamy solution. 5. 5 w:w % alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.4g of the BSA solution and 0.2g of the bicarbonate solution to yield a foamy solution.

6. 5 w:w % alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.4g of the BSA solution and 0.4g of the bicarbonate solution to yield a foamy solution.

7. 5 w:w % alginate, l.OM bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.4g of the BSA solution and

0.2g of the bicarbonate solution to yield a foamy solution.

In sum, the following ranges of conditions were found preferable for forming interconnected pore structures by this embodiment:

Starting solutions of 3, 4 and 5 w:w % (weight % based on weight of water) alginate with 1.5% BSA and l.OM to 2.0M bicarbonate solutions lead to the development of foamy solutions. The weight ratio of BSA to the bicarbonate is preferably from 2:1 to 1 :1. Their amounts used depend on the concentration of the alginate solution.

Propylene glycol alginate was also used as a surfactant in order to replace the protein BSA. Equal amounts of alginate and propylene glycol alginate were dissolved in dd water to yield a 3 % w:w solution. 2 g of this solution was mixed with 0.12 g of a bicarbonate solution to yield a foamy solution.

All other surfactants tested, such as Pluronics F108 and F68, yielded a foamy solution and led to stable interconnected porous hydrogels. Using 2 g of 8% w:w alginate and 0.12 g of 2.0M bicarbonate, 10% w:w solution of F 108 yielded a foamy solution when added in amounts of 0.12 g and 0.06 g of the F108 solution, although use of 0.03 g of the F108 solution did not result in a stable and sufficient foamy solution. Also, the composition of the gelling solution was modified. The 0.1 M CaCl₂ containing 10 vol% acetic acid of the prior art did not lead a fast enough gelling of the alginate. The beads appeared sticky and beads tended to fuse together when in contact with other beads. The concentration of the CaCl₂ was raised to 0.5 M.

An indication of the porosity of the beads formed was obtained by observing beads suspended in an aqueous solution. Beads prior to exposure to vacuum appeared opaque and floated on the surface (indicating a low density as one would expect from the large amount of entrapped gas). Following exposure to vacuum, the beads appeared clear and sank to the bottom of the solution (indicating an increased density due to replacement of the gas with the more dense aqueous solution).

The porosity of beads formed was visually examined to confirm their porosity. Following isolation of beads from the gelling solution, a large number of gas bubbles could be observed within the alginate matrices. Following removal of the gas bubbles, an open porous structure was observed.

The interconnected pore structure of the matrices was assessed by seeding a solution of suspended cells onto porous alginate beads, and subsequently visualizing these cells using a MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay. The porous beads took up cells, and the distribution of dyed cells allowed confirmation of the amount of interconnected pores in the matrix (large pores (greater than approximately 10 microns in diameter) were present).

To determine whether the pore structure remained stable over time, porous beads were allowed to remain in an aqueous solution for varying periods of time (1 day to 2 weeks) and subsequently analyzed for porosity by seeding cells. The incoφoration and distribution of cells, and thus the matrix porosity, was unchanged following storage.

The pore structure remains intact in vivo, as shown by transplantation of porous alginate beads into subcutaneous pockets of rats. Invasion of macrophages and fibroblasts was noted throughout the matrices at one week, with more cells being present and greater new collagen deposition in the beads by the invading cells by two weeks. The beads maintained their original shape and dimensions, indicating that their mechanical properties were sufficient to withstand the compressional forces exerted in vivo.

The process to fabricate porous alginate matrices has been scaled up to allow large quantities of these materials to be produced. To incoφorate air bubbles in large volumes of the initial solution, the solution is vigorously mixed in a high speed mixer (e.g., Sunbeam hand mixer, Model 2485). A syringe pump is used to generate large numbers of reproducibly sized beads in a semi-automatic fashion.

It is desirable for a variety of biomedical applications to prepare materials that are porous but also show degradation after implantation. As mammals do not carry the appropriate enzyme necessary to optimally degrade alginate at physiological conditions, the use of alginates with an average molecular weight low enough to allow the excretion of the material through the kidneys is an attractive alternative. This molecular weight is usually considered to lay around 50 kD.

Thus, the controlled degradation of alginates was performed to generate materials with a molecular weight below 50 kD, utilizing acid hydrolysis (solution), heat treatment (solution and bulk) and γ-irradiation (solution and bulk). Acid Hydrolysis: Alginate solutions (2 % (w:w)) were refluxed in 0.3 M HC1 for various times. Heat treatment: Alginate (solution and bulk) were autoclaved (1.034 bar, 121 °C) to generate alginate with lower average molecular weights. Samples were autoclaved for 1 h, 2 h and 2.5 h, respectively, γ-irradiation: Alginate could be degraded through gamma irradiation at a variety of conditions (irradiation of alginate solutions (2 and 3%) was first used). Based on its ease of use, gamma irradiation at 5.0 Mrad for 2.83 h was used to generate alginate fragments.

High molecular weight alginate could be broken down into lower molecular weight fragments using each of the above methods. Each method provided conditions resulting in alginates with molecular weights below 50kD (as determined by GPC measurements). In addition, all alginate fragments still form gels in the presence of calcium ions.

Porous alginate beads were formed from alginate fragments. 8% w:w alginate, 2.0M bicarbonate and 1.5% BSA were used as the starting solutions. 2 g of the alginate solution were mixed with 0.24 g of the BSA solution and 0.12 g of the bicarbonate solution to yield a foamy solution.

The porosity of the beads formed from alginate fragments was confirmed by environmental scanning electron microscopy (ESEM). The interconnected pore structure of the alginate beads formed from alginate fragments (M_N=8920, M_w= 16800) was assessed by cell seeding and MTT visualization. All beads showed a high degree of cell incoφoration with a uniform cell distribution throughout the entire bead. The pore structure remained intact in vivo, as shown by transplantation into subcutaneous pockets of rats, when the porous beads maintained their original shape and dimensions and allowed cell invasion, indicating that their mechanical properties were sufficient to withstand the compressional forces exerted in vivo.

EXAMPLE 8 DNA RELEASE FROM DNA-ALGINATE MATRICES

As generally described above, alginate hydrogels are biocompatible, have gentle gelling properties and can be delivered in a minimally invasive manner. Varying the type of alginate (e.g., ratio of mannuronic acid to gul uronic acid) along with the fabrication process (e.g., source and amount of calcium, shape) allows control over the release kinetics.

Alginate matrices were fabricated generally as described above. In particular, alginate hydrogel matrices containing plasmid DNA were fabricated by ionically crosslinking the gel with calcium. Alginate discs were prepared by mixing an alginate solution with plasmid DNA and a super-saturated solution of CaSO₄. The gel was cast between glass plates and allowed to gel. Discs were cut from the slab. Alginate beads were formed by dropping alginate/DNA mixtures into a bath of CaCl₂. DNA release studies were performed by subsequently placing the alginate/DNA gels into a known volume of PBS buffer. The DNA released from the gel was quantified by measuring the concentration of DNA in the PBS solution over time using the Hoechst Dye binding assay.

Continuous release of plasmid DNA (>160 days) from the alginate matrices has been demonstrated. Release kinetics of DNA from alginate hydrogel matrices extends past 30 days up to 160 days. Virtually all of the plasmid DNA released from beads of alginate is structurally intact when analyzed by electrophoresis.

All of the compositions, methods and apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions, methods and apparatus of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and apparatus, and in the steps or in the sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incoφorated herein by reference without disclaimer.

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Claims

CLAIMS:

1. An admixture comprising at least a first protein or drug, a population comprising beads or microspheres of a polymer capable of forming a gas-foamed polymeric structure, and at least a first leachable particulate material; wherein said at least a first protein or drug is incoφorated within said beads or microspheres.

2. The admixture of claim 1, wherein at least a first protein is incoφorated within said beads or microspheres.

3. The admixture of claim 1 or 2, wherein at least a first drug is incoφorated within said beads or microspheres.

4. The admixture of any preceding claim, wherein at least a first protein and at least a first drug are incoφorated within said beads or microspheres.

5. The admixture of any preceding claim, wherein at least a second protein or drug is further incoφorated within said beads or microspheres.

6. The admixture of any preceding claim, wherein a plurality of proteins or drugs is incoφorated within said beads or microspheres.

7. The admixture of any preceding claim, wherein said population comprises at least a first sub-population of beads or microspheres of at least a first polymer and at least a second sub-population of beads or microspheres of at least a second, distinct type of polymer.

8. The admixture of claim 7, wherein said at least a first and second sub-populations of beads or microspheres each incoφorate a different protein or drug.

9. The admixture of claim 7, wherein said at least a first and second sub-populations of beads or microspheres each incoφorate the same protein or drug.

10. The admixture of claim 7, wherein said population comprises a plurality of sub- populations of beads or microspheres, each made of a distinct polymer and each incoφorating a distinct type of protein or drug.

11. The admixture of any preceding claim, wherein at least a portion of said beads or microspheres are of a biodegradable polymer.

12. The admixture of any preceding claim, wherein at least a portion of said beads or microspheres are of a lactic acid polymer, glycolic acid polymer or lactic acid/glycolic acid copolymer polymer.

13. The admixture of any preceding claim, wherein at least a portion of said beads or microspheres are of a lactic acid/glycolic acid (PLGA) copolymer.

14. The admixture of any preceding claim, wherein said admixture comprises a water- soluble leachable particulate material.

15. The admixture of claim 14, wherein said leachable particulate material is a salt, sugar or sugar alcohol.

16. The admixture of claim 14, wherein said leachable particulate material is NaCl, trehalose, glucose, sucrose or mannitol.

17. A kit comprising an admixture in accordance with any preceding claim.

18. A composition comprising a structural matrix in association with at least a first protein or drug, wherein at least a portion of said structural matrix is comprised of a porous polymer that contains pores formed by gas foaming and pores formed by leaching out of a particulate from the polymer.

19. The composition of claim 18, wherein at least a portion of said structural matrix is comprised of a porous polymer that has an open pore structure.

20. The composition of claim 18 or 19, wherein at least a portion of said structural matrix is comprised of a porous polymer that has an interconnected pore structure.

21. The composition of any one of claims 18 through 20, wherein said structural matrix consists essentially of a porous polymer that has an open pore structure.

22. The composition of any one of claims 18 through 20, wherein said structural matrix comprises at least a first matrix portion comprised of said porous polymer integrally connected to at least a second matrix portion comprised of an impermeable polymer.

23. The composition of claim 22, wherein said at least a first matrix portion is comprised of a porous polymeric material that has a substantially uniform open pore structure, and wherein said at least a second matrix portion is comprised of the same polymeric material in a form that lacks an open pore structure.

24. The composition of any one of claims 18 through 23, wherein at least a portion of said structural matrix is a biodegradable matrix.

25. The composition of any one of claims 18 through 24, wherein at least a portion of said structural matrix is comprised of a lactic acid polymer, glycolic acid polymer or lactic acid/glycolic acid copolymer matrix.

26. The composition of any one of claims 18 through 25, wherein at least a portion of said structural matrix is comprised of a lactic acid/glycolic acid (PLGA) copolymer matrix.

27. The composition of any one of claims 18 through 26, wherein said structural matrix comprises at least a first protein.

28. The composition of any one of claims 18 through 27, wherein said structural matrix comprises at least a first drug.

29. The composition of any one of claims 18 through 28, wherein said structural matrix comprises at least a first protein and at least a first drug.

30. The composition of any one of claims 18 through 29, wherein said structural matrix further comprises at least a second protein or drug.

31. The composition of any one of claims 18 through 30, wherein said structural matrix further comprises a plurality of proteins or drugs.

32. The composition of any one of claims 18 through 31, wherein said structural matrix comprises at least a first matrix portion comprised of at least a first polymer and at least a second matrix portion comprised of at least a second, distinct type of polymer.

33. The composition of claim 32, wherein said at least a first and second matrix portions each incoφorate a different protein or drug.

34. The composition of claim 32, wherein said at least a first and second matrix portions each incoφorate the same protein or drug.

35. The composition of claim 32, wherein said structural matrix comprises a plurality of matrix portions, each comprised of a distinct polymer and each incoφorating a distinct type of protein or drug.

36. The composition of any one of claims 18 through 35, wherein said at least a first protein or drug stimulates a bone progenitor cell, wound healing fibroblast, granulation tissue fibroblast, repair cell or cell of the immune system upon contact with said cell.

37. The composition of any one of claims 18 through 35, wherein said at least a first protein or drug is a cytotoxic, apoptosis-inducing, anti-bacterial, anti-microbial, anti-parasitic or anti-viral protein or drug that induces cell death upon contact with an aberrant or infected cell or invading pathogen.

38. The composition of any one of claims 18 through 37, wherein said at least a first protein or drug functions as, or modulates the function of, a transcription or elongation factor, cell cycle control protein, kinase, phosphatase, DNA repair protein, oncogene, tumor suppressor, angiogenic protein, anti -angiogenic protein, immune response stimulating protein, cell surface receptor, accessory signaling molecule, transport protein, enzyme, hormone, neurotransmitter, growth factor, growth factor receptor, interferon, interleukin, chemokine, cytokine, colony stimulating factor or chemotactic factor.

39. The composition of any one of claims 18 through 38, further comprising a population of cells.

40. The composition of any one of claims 18 through 39, further comprising a biological tissue fluid.

41. The composition of any one of claims 18 through 40, prepared by a process that comprises leaching out the particulate material from a composition comprising a gas foamed polymeric material, at least a first protein or drug and a leachable particulate material.

42. The composition of any one of claims 18 through 40, prepared by a process that comprises:

(a) incoφorating at least a first protein or drug within beads or microspheres of a polymer capable of forming a gas-foamed polymeric structure;

(b) admixing the protein-containing or drug-containing beads or microspheres with a leachable particulate material;

(c) subjecting the admixture to a gas foaming process to create a porous polymeric structure that comprises said at least a first protein or drug and said leachable particulate material; and

(d) leaching said leachable particulate material from said porous polymeric structure, thereby producing a polymeric structure of additional porosity that comprises said at least a first protein or drug.

43. The composition of claim 42, wherein said leaching process is conducted in vitro by subjecting said porous polymeric material to a leaching agent.

44. The composition of claim 42, wherein said leaching process is conducted in vivo by exposing said porous polymeric material to body fluids.

45. A composition in accordance with any one of claims 18 through 44 for use in the controlled release of at least a first protein or drug.

46. A composition in accordance with any one of claims 18 through 44 for use in providing at least a first protein or drug to a cell.

47. A composition in accordance with any one of claims 18 through 44 for use in culturing cells in contact with at least a first protein or drug.

'48. A composition in accordance with any one of claims 18 through 44 for use in transferring at least a first protein or drug to cells within a tissue site of an animal.

49. A composition in accordance with any one of claims 18 through 44 for use in therapy.

50. Use of a composition in accordance with any one of claims 18 through 49 in the manufacture of a medicament for treating a medical condition by protein or drug delivery.

51. Use according to claim 50, wherein said medicament is for use in stimulating bone tissue growth.

52. Use according to claim 50, wherein said medicament is for use in promoting wound healing, tissue regeneration or organ regeneration.

53. Use according to claim 50, wherein said medicament is for use in generating an immune response.

54. Use according to claim 50, wherein said medicament is for use in killing aberrant, malignant or virally-infected cells or invading pathogens.

55. Use according to claim 50, wherein said medicament is for use in controlling the reproductive system.

56. Use according to claim 50, wherein said medicament is for use in cell transplantation, tissue engineering or guided tissue regeneration.

57. A kit comprising a composition in accordance with any one of claims 18 through 49 in at least a first suitable container.

58. An implantable medical device comprising a composition in accordance with any one of claims 18 through 49 in a bioimplantable form.

59. A method of making a structural matrix incoφorating at least a first protein or drug, comprising subjecting a population comprising protein-containing or drug-containing polymer beads or microspheres to a gas foaming, particulate leaching process to produce a structural matrix that incoφorates said at least a first protein or drug.

60. The method of claim 59, comprising incoφorating said at least a first protein or drug within a population comprising beads or microspheres of a polymer capable of forming a gas-foamed polymeric structure and subjecting the protein-containing or drug-containing beads or microspheres to a gas foaming, particulate leaching process.

61. The method of claim 60, comprising the steps of:

(a) incoφorating said at least a first protein or drug within a population comprising beads or microspheres of a polymer capable of forming a gas- foamed polymeric structure;

62. The method of claim 61, wherein said gas foaming process comprises subjecting said admixture to an elevated pressure atmosphere of an inert gas in a manner effective to dissolve said gas into said polymer, and subjecting the gas-dissolved polymer to thermodynamic instability in a manner effective to cause nucleation and growth of gas pores sufficient to produce a continuous polymer matrix that comprises said at least a first protein or drug and said leachable particulate material.

63. The method of claim 62, wherein said thermodynamic instability is created by reducing said elevated pressure atmosphere.

64. The method of any one of claims 61 through 63, wherein said population of beads or microspheres comprises at least a first sub-population of beads or microspheres of at least a first polymer and at least a second sub-population of beads or microspheres of at least a second, distinct type of polymer.

65. The method of claim 64, wherein said at least a first and second sub-populations of beads or microspheres each incoφorate a different protein or drug.

66. The method of claim 64, wherein said at least a first and second sub-populations of beads or microspheres each incoφorate the same protein or drug.

67. The method of claim 64, wherein said population comprises a plurality of sub- populations of beads or microspheres, each made of a distinct polymer and each incoφorating a distinct type of protein or drug.

68. The method of any one of claims 61 through 67, wherein said leachable particulate material is a water-soluble leachable particulate material.

69. The method of claim 68, wherein said leachable particulate material is a salt, sugar or sugar alcohol.

70. The method of claim 69, wherein said leachable particulate material is NaCl, trehalose, glucose, sucrose or mannitol.

71. The method of any one of claims 61 through 70, wherein said leaching is conducted in vitro by contacting said porous polymeric material with a leaching agent.

72. The method of any one of claims 61 through 70, wherein said leaching is conducted in vivo by exposing said porous polymeric material to body fluids.

73. A structural matrix that incoφorates at least a first protein or drug prepared by a process in accordance with any one of claims 59 through 72.

74. A method for the controlled release of at least a first protein or drug, comprising allowing the release of at least a first protein or drug from a composition in accordance with any one of claims 18 through 44.

75. The method of claim 74, wherein the release of said at least a first protein or drug from the structural matrix of said composition is controlled by controlling the rate of degradation or dissolution of said structural matrix.

76. The method of claim 74, wherein the release of said at least a first protein or drug from the structural matrix of said composition is controlled by controlling diffusion through the pores in said structural matrix.

77. A method for providing at least a first protein or drug to a cell, comprising contacting said cell with a composition in accordance with any one of claims 18 through 44 in a manner effective to release at least a first protein or drug from the structural matrix of said composition.

78. The method of claim 77, wherein said at least a first protein or drug is released from the structural matrix of said composition by degradation of said structural matrix, dissolution of said structural matrix or by diffusing through pores in said structural matrix.

79. The method of claim 77, wherein said at least a first protein or drug is released from the structural matrix of said composition by desoφtion from said structural matrix.

80. The method of claim 77, wherein said cell is located in a tissue site of an animal and said composition is provided to said tissue site of said animal.

81. A method for culturing cells, comprising growing cells in contact with a composition in accordance with any one of claims 18 through 44; wherein said composition comprises at least a first protein or drug beneficial to cell culture.

82. The method of claim 81, wherein said cells are separated from the matrix of said composition and provided to an animal.

83. The method of claim 81, wherein said cells are maintained in contact with the matrix of said composition and wherein said composition is provided to an animal.

84. A method for providing at least a first protein or drug to cells within a tissue site of an animal, comprising contacting said tissue site with a composition in accordance with any one of claims 18 through 44.

85. The method of claim 84, wherein said at least a first protein or drug stimulates cell growth or proliferation.

86. The method of claim 84, wherein said at least a first protein or drug exerts a cytotoxic or apoptotic effect on said cells.

87. The method of claim 84, wherein said at least a first protein or drug exerts an antigenic or immunogenic effect in said cells

88. The method of claim 84, wherein said cells within said tissue site are bone progenitor cells located within a bone progenitor tissue site or bone fracture site of said animal.

89. The method of claim 88, wherein said bone progenitor cells are stem cells, macrophages, granulation tissue fibroblasts, vascular cells, osteoblasts, chondroblasts or osteoclasts.

90. The method of claim 84, wherein said cells within said tissue site are repair cells or fibroblasts located within a wound tissue site, a site of connective tissue injury or a site of organ damage.

91. The method of claim 84, wherein said cells within said tissue site are antigen presenting cells or T cells.

92. The method of claim 84, wherein said cells within said tissue site are cells of the reproductive system.

93. The method of claim 84, wherein said cells within said tissue site are aberrant, malignant or infected host cells or invading pathogenic cells.

94. A method for stimulating bone progenitor cells located within a bone progenitor tissue site of an animal, comprising contacting said tissue site with a composition in accordance with any one of claims 18 tlirough 44; wherein said composition comprises at least a first osteotropic protein or drug.

95. A method for stimulating fibroblasts within a wound tissue site of an animal, comprising contacting said tissue site with a composition in accordance with any one of claims 18 through 44; wherein said composition comprises at least a first stimulatory protein or drug.

96. A method for generating at least a first immune response in an animal, comprising contacting a tissue site of an animal with a composition in accordance with any one of claims 18 through 44; wherein said composition comprises at least a first immunogenic protein or drug.

97. The method of claim 96, wherein said composition comprises a plurality of immunogenic proteins obtained from at least a first pathogenic organism.

98. A method for treating diseased cells in an animal, comprising contacting a tissue site of an animal with a composition in accordance with any one of claims 18 through 44; wherein said composition comprises at least a first cytotoxic protein drug.

99. The method of claim 98, wherein said diseased cells are cancer cells.

100. The method of claim 98, wherein said diseased cells are virally-infected cells.

101. A method for transplanting cells into an animal, comprising applying to a tissue site of an animal a composition in accordance with any one of claims 18 through 44; wherein said composition further comprises a cell population.

102. A method for tissue engineering in an animal, comprising contacting a tissue site of an animal with a composition in accordance with any one of claims 18 through 44; wherein said composition comprises at least a first therapeutic protein or drug and provides a matrix for tissue growth.

103. A method for guided tissue regeneration in an animal, comprising contacting a regenerating tissue site of an animal with a composition in accordance with any one of claims 18 through 44; wherein said composition comprises at least a first therapeutic protein or drug and provides a matrix to guide tissue regeneration.