WO2013116736A1 - Scaffolding biomaterials based on polymer-coated high-stiffness particles - Google Patents

Abstract

A particle can include a hard core, and one or more polymeric shells encapsulating the core. The hard core can include hydroxyapatite, tricalcium phosphate, calcium carbonate, titanium dioxide, or combinations thereof. The one or more polymeric shells includes a biocompatible polymer, such as polycaprolactone, poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) ( PLGA), derivatives thereof, salts thereof, or combinations thereof. The particle can be a nanosphere or a microsphere. One or more bioactive agents can be encapsulated in or located on the one or more polymeric shells. One or more growth factors can be encapsulated in or located on the one or more polymeric shells. The growth factors include insulin-like growth factor-I (IGF-I), a transforming growth factor, TGF-beta1, TGF-beta3, a bone morphogenetic protein, BMP-2, BMP-7, or combinations thereof.

Description

SCAFFOLDING BIOMATERIALS BASED ON POLYMER-COATED HIGH- STIFFNESS PARTICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application claims the benefit of U.S. Provisional Patent Application No. 61/594,568 filed February 3, 2012. These patent applications and patent are incorporated herein by specific reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under R01 AR056347 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Engineered particles, such as nanospheres and microspheres, continue to gain importance for their use in a wide variety of industries. Continued improvement and modifications to engineered particles is sought to enhance current applications and for adaptation to new end uses. The engineered particles can be configured for broad application to a wide variety of technologies or to be tailored for a specific end use. Biotechnology applications of engineered particles, such as for implantation of microsphere tissue scaffolds, can benefit from having a range of different types of properties. For example, tissue scaffolds can be engineered for implantation into hard or soft tissue areas or for replacement of hard or soft tissues as well as for the interfaces therebetween. Thus, there is still a need to develop improved engineered particles with different characteristics for improved use in different applications.

BRIEF DESCRIPTION OF THE FIGURES The foregoing and following information as well as other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:

Figures 1A-1B illustrate embodiments of an engineered particle having a hard core and one or more polymeric shells encapsulating the core.

Figure 2 illustrates embodiments of shapes that can be formed by sintering the engineered particles.

Figure 3A includes images of uncoated glass beads.

Figure 3B includes images of glass beads coated with PLGA.

Figure 4A shows a phase contrast image of uncoated glass beads.

Figure 4B shows a phase contrast image of glass beads coated with PLGA.

Figure 5 shows an engineered particle having a scratch on the surface of the polymer, where the arrow points to the scratch on the surface to visualize the polymeric coating.

Figure 6A shows a top view and Figure 6B shows a side view of a body formed from selective laser sintering of the engineered particles so that the polymeric shells meld together at sintered location, and where no sintering leaves loose particles that can be removed to form the holes, which provides the shape of a window frame.

Figures 7A and 7B show side views of implants with engineered particles with the polymeric shells melded together with methylene chloride with 3 hours of sintering (Figure 7 A) and 4 hours of sintering (Figure 7B).

Figure 8 shows PLGA coated glass beads sintered by sub-critical C02.

Figure 9A shows a scaffold of engineered particles melded together before compression.

Figure 9B shows a lateral view of the scaffold of Figure 9A after compression. Figure 9C shows cross sectional view after compression.

Figure 10 shows a graph of data for stress versus strain of a scaffold prepared from PLGA-PCL dual coated particles melded for 3 hours with methylene chloride. Figures 11A and 11B include images of 1 mm engineered particles having the hard core and polymeric shells with live: dead cells at one week, which images are color contrasted to show the cells.

Figure 12 includes images of 1 mm engineered particles having the hard core and polymeric shells with live: dead cells at 24 hours, which images are color contrasted to show the cells.

Figures 13A and 13B include images of 200 micron engineered particles having the hard core and polymeric shells with live: dead cells at one week, which images are color contrasted to show the cells.

Figures 14A and 14B include images of 200 micron engineered particles having the hard core and polymeric shells with live:dead cells at 24 hours, which images are color contrasted to show the cells.

Figure 15A includes an image of 200 micron engineered particles in a sintered scaffold with interstitial spaces between the particles.

Figure 15B includes an image of 1 mm engineered particles in a sintered scaffold with interstitial spaces between the particles.

Figure 16 includes a graph that illustrates the average elastic modulus of PCL coated 200 micron glass bead scaffolds compared to with 200 micron PCL microsphere scaffolds.

DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

Generally, the present invention is related to core and shell engineered particles (e.g., nanoparticles or microparticles) and implant tissue engineering scaffolds prepared from the particles as well as methods of making and using the same. The engineered particles can include a hard or rigid core with one or more polymeric coatings on the core. The polymeric coating can include various agents, such as therapeutic agents, biological agents, or the like. The implants can be prepared into any shape with the particles bound together selectively at discrete locations or as melded masses. The hard core can provide stiffness and rigidity to the implant, and the polymer coating can allow for enhanced bonding and implant formation without using high heat, and as such the implants can include bioactive agents that would otherwise be susceptible to degradation at elevated temperatures. Melding of the polymeric shell can associate adjacent particles so as to form implants in any suitable shape and porosity. Also, the particles can range in size from nano-sized to micron-sized particles. The particles can range in shapes from irregular shapes to uniform shapes, but often will be in the form of a sphere or generally spherical so as to be generally referenced as nanospheres or microspheres. While the present invention is generally described in the context of microspheres, the particles may not be spherical and may be microparticles or the like. Also, while the term "microspheres" refers to size in the scale of microns, the engineered particles may have sizes in the nanoparticle range and may be nanospheres or the like.

The implants formed from the core and shell particles can be considered to be bioengineering scaffolds as the implants can be sufficiently porous to allow for cellular migration and penetration therein in order for the implant to function as a cell culture scaffold in vivo. The porosity of the implant can be modulated as described herein. The hardness of the core of each particle makes these implants especially suitable for bone scaffolds for bone regeneration, and may even be useful for orthodontic scaffolds for tooth regeneration. While the uses of the core and shell particles are generally described in connection to tissue engineering, it is understood that the engineered particles of the present invention can be applied to other fields and can be included in other articles of manufacture.

Figure 1 shows an embodiment of an engineered particle 10 having a core 12 and a single polymeric shell 14. Figure 1A shows a particle 10a having a core 12a and a plurality of polymeric shells 14a, each shell 14a being of the same polymeric material or of different polymeric materials. Figure 2 shows different embodiments of an implant 20 (e.g., frame 20a, beveled frame 20b, and cube 20c) formed from the particles 22 of Figure 1-lA. While not shown, the implants 20, 20a, 20b, and 20c can be formed from one type of particle or a mixture of two or more different types of particles that have different parameters for a characteristic. This may include the different types of particles having different spatial distributions, such as in gradient distributions. For example, one type of particle with a first characteristic can be distributed in a body so that a first end has a majority of the particles with the first characteristic and an opposite second end has a majority of a different second type of particle. The first type and second type of particles can have gradient distributions with respect to each other, and with respect to the first and second ends of the body.

However, the particles can be prepared into any basic or complex shape with various types of surface and internal features. The shapes can have overhangs, undercuts, apertures, holes, pores, porous networks, conduits, channels, or other feature. The different shapes can be molded and/or selectively formed with any of a variety of manufacturing processes that can selectively sinter adjacent particles together by melding the polymeric shells of adjacent particles together.

The particle-based scaffold can be prepared into substantially any shape by preparing a mold to have the desired shape or by using point specific laser melding. For example, the particle-based scaffold can be prepared into the shapes of rods, plates, spheres, wrappings, patches, plugs, depots, sheets, cubes, blocks, bones, bone portions, cartilage, cartilage portions, implants, orthopedic implants, orthopedic screws, orthopedic rods, orthopedic plates, and the like. Also, the particle-based scaffolds can be prepared into shapes to help facilitate the transitions between tissues, such as between bone to tendon, bone to cartilage, tendon to muscle, dentin to enamel, skin layers, disparate layers, and the like. The particle-based scaffolds can also be shaped as bandages, plugs, or the like for wound healing.

In one embodiment, the mean particle size of the particles used to prepare the scaffolds can have a range between about less than 1 μιη to about greater than 1 mm, more preferably about 100 μιη to about 300 μιη, and most preferably from about 180 to about 240 μιη. An example of particle size is about 180 μιη to about 220 μιη.

In one embodiment, the core size of the particles can vary in order to vary stiffness or hardness of the implants. Size of the core particle can be in a range of about 5 μιη to about 1200 μιη, more preferably from about 50 μιη to about 500 μιη, and most preferably from about 100 μιη to about 200 μιη. In some instances, the core of the particle can be larger than about 1200 μιη.

In one embodiment, the shell thickness can be varied to a thinner size in order to facilitate particle-particle melding such that the thin shell can result in an increase in stiffness or hardness as there is less polymer. The range of the thinner shell coating can be from about 0.025 μιη to 125 μιη, more preferably from about 0.05 μιη to about 75 μιη, and most preferably from about 0.25 μιη to about 50 μιη. Also, a range of the ratio of the coating thickness/core diameter can be between about 0.005 and 0.25, more preferably between about 0.01 to about 0.15, and most preferably between about 0.05 and about 0.1.

In one embodiment, the shell thickness can be varied to a thicker size in order to facilitate particle-particle melding while retaining bioactive agent in the shell, such that the thick shell can result in a decrease in stiffness or hardness as well as increase in bioactive agent loading potential as there is more polymer. The range of the thicker shell coating can be from about 1.5 μιη to about 750 μιη, more preferably from about 3 μιη to about 600 μιη, and most preferably from about 4.5 μιη to about 550 μιη. Also, the range of the ratio of the coating thickness/core diameter can be between about 0.3 and 1.5, more preferably between about 0.6 to about 1.2, and most preferably between about 0.9 and about 1.1.

In one embodiment, the invention can include a new material, based on high- stiffness particles (e.g., nanospheres or microspheres) having a hard core (e.g., hydroxyapatite, bioactive glass) that are coated with a polymeric material (e.g., poly(lactic-co-glycolic acid)). These hard core and polymeric shell engineered particles can be sintered together to form articles that include a singly type of particles as well as in articles with other types of particles in order to create a macroscopic implant that can be used as bioengineering scaffolding biomaterial. Accordingly, the hard core and polymeric shell engineered particles in an implant may include: the same polymer material and high-stiffness core material; different polymers; different high-stiffness core materials; and/or may be a polymeric particles without the high-stiffness material inside as a solid particle or multi-layered or multi-shelled particles. The polymeric shells of each engineered particle can be formed of a single type of polymer or different types of polymers, and each individual shell can be a single type of polymer (e.g., homo polymer or co-polymer) or mixture of different polymers. The scaffolding biomaterials may be made into different shapes based on the mold that the particles are placed into with the polymeric shells melded together, which may include one or more different types of engineered particles. Particles with different types of properties, such as different hard cores and/or different polymeric shells) can be selectively arranged in a mold in order to prepare an article with different properties at different locations. Also, point-specific laser sintering can be performed in order to prepare complex shapes by selectively melding the shells of certain adjacent particles together. Interfaces between polymeric shells that can meld together can be used at locations of an article that are solid, while polymeric shells that do not meld together (e.g., under a certain melding process) can be used in locations of an article where the particles are later removed or retained as free- flowing or movable particle powders or retained as a particles in a matrix of another matrix material. Particles that are on an internal portion of the article may not be melded together so that they can be removed from the article prior to implantation.

The present invention provides the surprising and unexpected result that it is indeed possible to use a polymeric material to coat hard particles of hydroxyapatite or bioactive glass. Moreover, it is now possible to form an implant from particles that have hard cores with properties provided by hydroxyapatite and/or bioactive glass. For example, the properties of hydroxyapatite and/or bioactive glass can be useful for implantation into bone for bone repair or bone regeneration. Particles of hydroxyapatite and bioactive glass are commercially available, and can be made into "composite" materials of particulate hydroxyapatite with polymers, with or without surface modification of hydroxyapatite nanoparticles. However, hydroxyapatite requires extremely high temperatures for sintering into a three dimensional structure, and thereby is not suitable for containing many bioactive agents or biological materials. The implants of the present invention provide for surprising and unexpected results of having a stiff implant scaffold that includes bioactive agents, such as proteomic growth factors, that can be released.

Now, however, individual high-stiffness engineering particles (e.g., hydroxyapatite, bioactive glass core with polymer shell) can each receive their own individual polymer coating with or without a bioactive material and can be sintered or melded into complex shapes for use as bioengineering implants with suitable porosity. Examples of the core material can include bioactive glass and hydroxyapatite; however, other hard materials that are biocompatible can be used, such as biocompatible metals or other ceramics or hard plastics. The high-stiffness microparticles can be configured as particles that are individually coated with a polymeric coating so that the individual engineered particles can be used as particle, such as in a particle powder. The polymeric shell is not a matrix polymer that encapsulates multiple cores. Each engineered particle can have a single core with its own unique shell.

The high-stiffness particles themselves are stiffer that conventional polymeric particles used for implants, and correspondingly the macroscopic implant scaffolding biomaterial prepared is also stiffer that traditional polymeric particle-based implants. However, the particles and resulting implant have properties of polymeric materials in that they can be melded at low or room temperature with solvents and may be prepared to include bioactive materials, such as nucleic acids or proteins, which are not denatured or degraded by the scaffold manufacturing process. Moreover, the polymeric shell coating allows for controlled release of the bioactive materials from the scaffold once implanted.

It has now been found that a particle made of a polymer-coated hydroxyapatite particle (i.e., a single sphere of hydroxyapatite, with a polymeric shell of one or more polymeric coatings) is significantly stiffer than either a particle of equivalent size and shape made of either the polymer alone or a composite of the polymer and particulate hydroxyapatite in a bulk polymer matrix. As used herein, the shell has substantially the same of the core or more spherical and rounded with only one particle therein, where a matrix is a bulk polymer with a plurality of particles therein. This means that an implant scaffolding biomaterial made of the hard core and polymeric shell engineered particles is stiffer as well. Therefore, an implant scaffolding biomaterial intended for bone regeneration can be made stiffer than a material made of either the pure polymer particles or the composite particles, and can be made into a specific shape (e.g., to replace a segmental defect).

In one embodiment, the microparticles can be sintered in a manner that results in interstitial spaces between the polymeric shells so as to create or maintain packing pores. As such, the implant is not completely solid from side to side because there are spaces formed between the shells. The shells do not melt to form a solid matrix. The sintering can be used to control porosity and the degree of melding of the adjacent particles. That is, the particles are sintered together so that the polymeric shells of adjacent particles partially meld together to leave interstitial spaces between the melded particles and to leave general porosity. One example of sintering can be with solvent liquid or solvent vapor sintering, where the liquid or vapor of a solvent is flowed through the implant in order to meld the polymeric shells of adjacent particles to each other and to leave interstitial spaces therebetween. Some examples of the solvents that can be used in liquid or vapor sintering can include ethanol, ethanol-acetone, dense-phase carbon dioxide, chloroform, methylene chloride, or the like. The vapor melding can sinter the microparticles at a faster rate than composite particles made of the polymer with nanoparticulate hydroxyapatite. Such a sintering process is usable for core and shell microparticles as well as a mixture of core and shell microparticles with regular polymeric microparticles.

In one embodiment, the polymer used to form the shells of the microparticles can include an active agent, such as a drug, growth factor, or the like. The shells can be configured for controlled release of the active agent. As such, one or more shell layers can include the active agent, and one or more outer shell layers can be over the active agent-containing layers to control diffusion and ultimate release from the microparticles. As opposed to a scaffold made of only hydroxyapatite or bioactive glass, the polymeric component makes controlled release of bioactive molecules straightforward. This enables a high- stiffness implant scaffolding biomaterial with controlled release of active agents.

In one embodiment, two or more types of engineered particles can be used and prepared to have a corresponding uniform distribution and or relative gradient between the different types of particles. That is, the two or more different types of particles can be arranged in a gradient format with respect to each other, and then sintered into an implant having the particle gradients. This allows the sintering of particles in a gradient based design where the high-stiffness material is not desired on one side of the scaffolding biomaterial. This can be useful for scaffolding biomaterial intended for osteochondral (bone + cartilage) tissue regeneration, where the present invention allows for polymeric coated hard particles and polymer-only particles to be sintered together in a gradient design.

While the present invention has been described with respect to hard implants that are useful for bone regeneration, the invention may also be used for regeneration of any hard tissue, such as tooth regeneration. The therapeutic method can use biostable polymeric materials or biodegradable polymeric materials. When biodegradable, the resulting bone, tooth, or other hard tissue that is regenerated can retain the hard cores therein. The hard cores in the regenerated tissue can provide structural support. In one embodiment, the implant scaffold includes a biomaterial that is made of only one type of particle that has a hard core material and a polymeric shell material.

In one embodiment, the implant scaffold has a substantially homogenous distribution of a first type of particle that has a first core material and a second core material. Optionally, the implant can have substantially homogenous distributions of any type of particle, whether a core and shell particle or a uniform polymeric particle. In one aspect, the implant can be devoid of having a gradient distribution of the first type of particle. That is, the implant is substantially uniform with respect to the first type of particle and possibly substantially uniform with respect to any type of particle included therein.

In one embodiment, the implant prepared from the hard core and polymeric shell particle has a stiffness in the mega-Pascal (MPa) range. In another embodiment, the implant prepared from the hard core and polymeric shell microparticle has a stiffness in the giga-Pascal (GPa) range. In one example, the implant can have a stiffness of about 1 MPa to about 200 MPa when the implant is dry or substantially without water or other solvent of the polymeric shell material, or from 2.5 MPa to about 150 MPa, or from 5 MPa to about 125 MPa, or from 7.5 MPa to about 100 MPa. In one example, the implant can have thin polymeric shells and have a stiffness of about 1 GPa to about 20 GPa when the implant is dry or substantially without water or other solvent of the polymeric shell material, or from 3 GPa to about 15 GPa, or from 5 GPa to about 12 GPa, or from 7.5 GPa to about 10 GPa. In one embodiment, the average moduli of elasticity of the scaffolds can have a range between about 0.5 MPa to about 75 MPa, more preferably about 1 MPa to about 50 MPa, and most preferably from about 5 MPa to about 10 MPa.

In one embodiment, the implant is prepared using laser sintering. The laser beam is focused so as to heat at least a portion of the polymeric shell thereof in order to cause melding of the adjacent particles in the path of the laser in order to link adjacent particles together. The use of laser sintering can allow for different layers to be sintered with point specific melding. Laser sintering allows for specific points or specific regions within a bulk of particles in a mold or elsewhere, such that a specific and unique shape can be formed, such as having overhanging features and under-hanging features. In one example, laser sintering can be used to prepare a shape that has gaps or holes extending through otherwise solid portions. During the laser sintering protocol, the portions that are not desired to be sintered and that are retained as flowable particles (e.g., not sintered or melded together) can be removed by allowing the un-melded or free particles to flow out. A specific example includes preparing an implant with features similar to as shown in the implant that appears like a window pane. Thus, laser sintering with point specific melding can be used to create complex shapes with complex holes or other features that are not capable of being molded.

In one embodiment, the laser sintering can be computer-controlled. That is, a CAD program, used on a computer, can be used to design an implant, and the computer can control the laser to sinter the microparticles into the shape provided by the CAD program. In one aspect, a CT image of a subject can be used to create a template for an implant to fit within a defect of a subject, such as a bond defect, and the laser sintering process can then custom sinter the microparticles into an implant to fit within the subjects-specific defect.

In one embodiment, the laser sintering protocol can be used for controlling macroporosity or general holes, conduits or apertures as well as recesses that extend into or through the sintered body. Microporosity is the small pores or interstitial space between adjacent particles and macroporosity is a hole, conduit, or aperture that extends into and through the body of the sintered body. One area can be laser sintered with small macropores while another can be larger macropores. For example, this selective macroporosity can be useful for preparing a mandibular condyle, which has one portion with regular porosity from interstitial space between particles and the other from laser- created macropores. The scaffolds can have a macropore, hole, conduit or aperture sizes ranging from about 40 μιη to about 1650 μιη. For cartilage tissue engineering, the macropore hole, conduit or aperture sizes can be from about 70 μιη to about 120 μιη. In bone tissue engineering, macropore hole, conduit or aperture sizes as small as about 50 μιη or larger can be used. Optimal macropore hole, conduit or aperture sizes may be within a range of about 100 μιη to about 600 μιη.

In one embodiment, the implant can be prepared using one of solvent sintering, liquid solvent sintering, vapor solvent sintering, C02 sintering, or laser sintering. In one aspect, general heat sintering, such as performed in an oven with a heater, is generally avoided or not used to prepare the implants due to general heating deactivating or degrading bioactive agents. However, general heat in an oven can be used when the particles are devoid of a bioactive agent that denatures or degrades under heat. Heat from a laser, however, is suitable in many instances. When no heat can be used, solvent liquid or solvent vapor or C02 sintering can be performed.

In one embodiment, the hard core, such as bioactive glass or hydroxyapatite, can be spray coated with a polymeric solution to prepare one or more shells to the particle. The polymeric solution can include a bioactive agent, and the spraying can be done at low or room temperature in order to retain activity of the bioactive agent. The spray coating can be via traditional spray coating of particles, and may include inject coating.

In one embodiment, the coating protocol is accomplished by a circulating fluidized bed (CFB), and can provide for discrete coated particles. This is distinct from methods where numerous particles are encapsulated within a given polymer, e.g., the encapsulation of numerous hydroxyapatite nanoparticles inside of a given polymer particle. In the present invention, each polymer coating encapsulates exactly one stiff particle, and a result is a powder of polymer coated particles. The CFB technique can produce discrete coated particles, such as coated microspheres, where each particle only has one hard core.

In one embodiment, the sintering of microparticles into an implant can be accomplished by any of the following methods: heat sintering (increase the temperature), which is less desirable when bioactive agents or other temperature sensitive components are included; C02 sintering (high pressure C02, with or without temperature increase, which depresses melting point of the polymer and thus allows the polymer to melt together at a lower temperature than accomplished with heat sintering); solvent sintering (immersion in a liquid solvent such as acetone, ethanol, methylene chloride, or a combination of the above), solvent vapor sintering (same as the previous one, but vapors instead of immersion); and SLS, which leads to 2nd level porosity.

In one embodiment, the present invention provides a process to make a particle- based scaffold that is stiffer than polymeric particle-based scaffolds and that includes bioactive agents such as growth factors. The core of the particles can include hydroxyapatite, tricalcium phosphate, calcium carbonate, titanium dioxide, or the like, which core is encapsulated inside of the polymer shells. A stiffer implant can be obtained by a hard core (e.g., sphere) coated with an outer layer that is polymeric and capable of melding and formation of three dimensional implants. Accordingly, the individual sphere units that are stiffer result in the bulk implant material also being stiffer. Mechanical testing data show that we have an increase in bulk material stiffness of about two orders of magnitude relative to bulk materials with the polymer particles alone. For example, with the biomaterial scaffold based on sintered PCL-coated particles of glass, compressive moduli in hydrated conditions at 37 degrees Celsius were approximately 3.7 MPa or about 10 MPa, compared to scaffolds made exclusively of PCL particles, with moduli under these same testing conditions of about 50 kPa. In comparison, some PLGA microsphere scaffold can have values between 142-308 kPa. The present invention can provide scaffolds with about two orders of magnitude higher stiffness for the coating-based scaffolds.

The particles when packed together in a mold present a common stacking observed with sphere-shaped objects, and with particles of similar diameter this may correspond to a void space or porosity (1st level of porosity or microporosity) of approximately 40%, depending on the extent to which the particles are sintered (in an extreme case, the microporosity would be 0%> if sintered excessively to the point of being melted together completely into a solid with no pores), but typically approximately 40%. This value is the 1st level of porosity (microporosity), which can range from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 60% and from about 40% to about 50%. In one embodiment, the mean theoretical porosities of the scaffolds can have a range between about 10% to about 95%, more preferably about 40% to about 60%, and most preferably from about 45% to about 50%. An example of porosity is about 44.9% to about 49%>.

The selective layer sintering (SLS), such as through laser sintering for point specific sintering, allows for the particles to be sintered together without the need for complete packing, instead allowing "struts" of sintered particles to be connected to each other, and the distance between these struts on a larger length scale presents a 2nd level of porosity or macroporosity. This can be used to form solids that have recesses, holes, macropores, or apertures therein or extending therethrough.

In one embodiment, the present invention provides a method of coating a stiff particle with a softer polymeric material, and the individual particles are then linked together by virtue of the polymer coating melding with the polymer coatings of the other coated particles.

In one embodiment, the stiffer core may be a bioceramic such as a bioactive glass (any one of a wide variety of possible formulations) or hydroxyapatite, or any calcium- based bioceramic, or the like. In one aspect, the polymer coating may be a polymer such as polycaprolactone, PGA, PLA, or PLGA, or any other polymer capable of being merged (sintered/melted/dissolved). The shell allows for growth factors to be encapsulated therein. The particles can release a growth factor encapsulated in the polymer. Example growth factors might be insulin-like growth factor-I (IGF-I), any of the transforming growth factors (e.g., TGF-betal, TGF-beta3), and any of the bone morphogenetic proteins (e.g., BMP -2, BMP-7). The particles may also contain and thereby release natural extracellular matrix materials such as chondroitin sulfate, hyaluronic acid, collagen, or the like.

In one embodiment, different types of particles (e.g., different polymer coating, core material, released growth factor or material, etc.) can be sintered together, either in a controlled fashion as in particle gradients that are described in the incorporated patent applications or simply mixed together, e.g., homogeneously.

In one embodiment, the hard core and polymeric shell coated particles can be used in a free-flowing composition. That is, the particles can be affirmatively not sintered together. As such, the particles can be used as a powder, or be introduced into a liquid solution or paste. Also, the particles can be included in liquid suspensions, gels, pastes, and other formats. The particles may also be included in solids, such as when in a solid polymer matrix with or without pores.

A tissue engineering scaffold prepared with the hard core and shell particles can be configured for growing cells, and can include a plurality of biocompatible particles linked together to form a three-dimensional matrix with interstitial spaces or pores between adjacent particles. The matrix can include a plurality of pores for growing cells. The biocompatible particles can include only a single type of particle, or can include first and second sets of particles. The first set of particles can have a first characteristic (e.g., hard core and polymeric shell), and a first predetermined spatial distribution with respect to the three-dimensional matrix. The second set of particles can have a second characteristic (e.g., only polymeric without a hard core) that is different from the first characteristic, and a second predetermined spatial distribution that is different from the first predetermined spatial distribution with respect to the three-dimensional matrix. Additional characteristics can include composition, polymer, particle size, particle size distribution, type of bioactive agent, type of bioactive agent combination, bioactive agent concentration, amount of bioactive agent, rate of bioactive agent release, mechanical strength, flexibility, rigidity, color, radiotranslucency, radiopaqueness, or the like. The scaffold includes at least one hard core and polymeric shell engineered particle as described herein.

In one embodiment, the scaffold can include a first bioactive agent contained in or disposed on the particles (e.g., hard core and polymeric shell particles). The scaffold can be configured to release the first bioactive agent so as to create a first desired spatial and temporal concentration gradient of the first bioactive agent. Optionally, a second set of different particles (e.g., different hard core and polymer shell or only polymeric) can be substantially devoid of the first bioactive agent, or can include a second bioactive agent. When the second bioactive agent is contained in or disposed on the second set of particles, the scaffold can be configured to release the second bioactive agent so as to create a second desired spatial and/or temporal concentration gradient of the second bioactive agent that is different from the first desired spatial and/or temporal concentration gradient of the first bioactive agent.

In one embodiment, the particles can be melded together by only a portion of a polymeric shell of a particle merging with only a portion of at least one adjacent polymeric shell of a particle. Methods of melding particles together are described herein.

In one embodiment, the bioactive agent contained in a particle can be a growth factor for growing the cells. However, the particles can include any type of bioactive agent. Accordingly, the first characteristic can be a first bioactive agent contained in or disposed on the particles, and the second characteristic can be a second bioactive agent contained in or disposed on the particles. For example, the first bioactive agent can be an osteogenic factor and the second bioactive agent can be a chondrogenic factor.

In one embodiment, the scaffold can include a medium sufficient for growing cells disposed in the pores. The medium can be a cell culture medium. Additionally, the medium can be a body fluid or tissue.

In one embodiment, the scaffold can include a plurality of cells attached to the plurality of particles and growing within the pores. Such cells can include a single type of cell, or a first cell type associated with the first set of particles and a second cell type associated with the second set of particles.

In one embodiment, the scaffold can include a first end and an opposite second end. Accordingly, the first set of particles can have a first bioactive agent, and the first end can have a majority of particles of the first set. Correspondingly, the second set of particles can have no bioactive agent or a second bioactive agent that is different from the first bioactive agent, and the second end having a majority of particles of the second set.

In one embodiment, the present invention can include a method of generating or regenerating tissue in an animal, such as a human. The method can include providing an endoprosthesis having the hard core and polymeric shell microparticles for growing cells. The endoprosthesis can have a plurality of biocompatible particles linked together so as to form a three-dimensional matrix having a plurality of pores defined by and disposed between the particles. Accordingly, the endoprosthesis can include a particle-based scaffold. The plurality of particles can have a surface area sufficient for growing cells within the plurality of pores. The biocompatible particles can be characterized as described herein. Additionally, the method of generating or regenerating tissue can include implanting the endoprosthesis in the animal such that cells grow on the particles and within the pores. This process can be used to grow specific types of cells for growth of tissue, bone, cartilage, or the like.

In one embodiment, the method of generating or regenerating tissue can include any one of the following: introducing a cell culture media into the pores; introducing cells into the pores; and/or culturing the cells such that the cells attach to the particles and grow within the pores.

In one embodiment, the three-dimensional implants can be used for the following: osteochondral defect repair (in the presence of growth factors with or without cells) and tissue engineering; axonal regeneration; study of chemotaxis in three-dimensions; directed angiogenesis; regeneration of other interfacial tissues such as muscle-bone, skin layers; temporal and spatial control of release of inflammatory and/or immune system modulators in regenerative medicine applications; and any application requiring a biocompatible, biodegradable material with spatial and temporal control over material composition, bioactive signal release, and porosity.

In one embodiment, the shells of the particles can be prepared from substantially any polymer, such as biocompatible, biostable, bioerodable, and/or biodegradable polymers. Examples of such biocompatible polymeric materials can include a suitable hydrogel, hydrophilic polymer, hydrophobic polymer, biostable polymers, biodegradable polymers, bioabsorbable polymers, and monomers thereof. Examples of such polymers can include nylons, poly(alpha-hydroxy esters), polylactic acids, polylactides, poly-L- lactide, poly-DL-lactide, poly-L-lactide-co-DL-lactide, polyglycolic acids, polyglycolide, polylactic-co-glycolic acids, polyglycolide-co-lactide, polyglycolide-co-DL-lactide, polyglycolide-co-L-lactide, polyanhydrides, polyanhydride-co-imides, polyesters, polyorthoesters, polycaprolactones, polyesters, polyanhydrides, polyphosphazenes, poly(phosphoesters), polyester amides, polyester urethanes, polycarbonates, polytrimethylene carbonates, polyglycolide-co-trimethylene carbonates, poly(PBA- carbonates), polyfumarates, polypropylene fumarate, poly(p-dioxanone), polyhydroxyalkanoates, polyamino acids, poly-L-tyrosines, poly(beta-hydroxybutyrate), polyhydroxybutyrate-hydroxyvaleric acids, polyethylenes, polypropylenes, polyaliphatics, polyvinylalcohols, polyvinylacetates, hydrophobic/hydrophilic copolymers, alkylvinylalcohol copolymers, ethylenevinylalcohol copolymers (EVAL), propylenevinylalcohol copolymers, polyvinylpyrrolidone (PVP), poly(L-lysine), poly(lactic acid-co-lysine), poly(lactic acid-graft-lysine), polyanhydrides (such as poly(fatty acid dimer), poly(fumaric acid), poly(sebacic acid), poly(carboxyphenoxy propane), poly(carboxyphenoxy hexane), poly(anhydride-co-imides), poly(amides), poly(iminocarbonates), poly(urethanes), poly(organophasphazenes), poly(phosphates), poly(ethylene vinyl acetate) and other acyl substituted cellulose acetates and derivatives thereof, poly(amino acids), poly(acrylates), polyacetals, poly(cyanoacrylates), poly(styrenes), poly(vinyl chloride), poly(vinyl fluoride), poly(vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, combinations thereof, polymers having monomers thereof, or the like. In certain preferred aspects, the nano-particles include hydroxypropyl cellulose (HPC), N-isopropylacrylamide (NIPA), polyethylene glycol, polyvinyl alcohol (PVA), polyethylenimine, chitosan, chitin, dextran sulfate, heparin, chondroitin sulfate, gelatin, etc. and their derivatives, co-polymers, and mixtures thereof.

The methods of making the scaffolds from the particles can be changed to include a solvent or solvent system (i.e., media or media system) that is liquid or vapor and that is compatible with the particular polymer of the particle. That is, the solvent or solvent system can be selected to meld the particles together as described herein in either liquid or vapor format. Vapor melding can be especially advantageous to control melding properties. Examples of some solvents can include hexane, benzene, toluene, diethyl ether, chloroform, ethyl acetate, 1,4-dioxane, tetrahydrofuran, dichloromethane, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, acetic acid, n-butanol, 2-butanol, 3- butanol, t-butyl alcohol, carbon tetrachloride, chlorobenzene, isopropanol, n-propanol, ethanol, methanol, formic acid, water, cyclohexane, 1,2-dichloroethane, diethyl ether, diethylene glycol, diglyme, dimethyl ether, dioxane, ethylene glycol, glycerin, heptane, hexamethylphosphoramide, hesamethylphosphorous triamide, hexane, nitromethane, pentane, petroleum ether, propanol, pyridine, o-xylene, m-xylene, p-xylene, and the like. Carbon dioxide can also be used as a solvent or media to meld the particles together. Also, solvents known for particular polymers can be used or combined with the solvents described herein.

The implant scaffolds can be prepared to contain and release substantially any therapeutic agent from the polymeric shells of the microparticles. Examples of some pharmaceutics agents that be useful in scaffolds for use in a body lumen, such as a blood vessel can include: anti-proliferative/antimitotic agents including natural products such as vinca alkaloids (i.e. vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e. etoposide, teniposide), antibiotics (dactinomycin (actinomycin D) daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin, enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents such as G(GP) Ilb/IIIa inhibitors and vitronectin receptor antagonists; anti-proliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nirtosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); anti-proliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs (fluorouracil, floxuridine, and cytarabine), purine analogs and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine {cladribine}); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones (i.e. estrogen); anti-coagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory; antisecretory (breveldin); anti-inflammatory: such as adrenocortical steroids (Cortisol, cortisone, fludrocortisone, prednisone, prednisolone, 6-alpha- methylprednisolone, triamcinolone, betamethasone, and dexamethasone), non-steroidal agents (salicylic acid derivatives e.g., aspirin; para-aminophenol derivatives i.e. acetaminophen; indole and indene acetic acids (indomethacin, sulindac, and etodalac), heteroaryl acetic acids (tolmetin, diclofenac, and ketorolac), arylpropionic acids (ibuprofen and derivatives), anthranilic acids (mefenamic acid, and meclofenamic acid), enolic acids (piroxicam, tenoxicam, phenylbutazone, and oxyphenthatrazone), nabumetone, gold compounds (auranofm, aurothioglucose, gold sodium thiomalate); immunosuppressives: (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), everolimus, azathioprine, mycophenolate mofetil); angiogenic agents: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF); angiotensin receptor blockers; nitric oxide donors; antisense oligionucleotides and combinations thereof; cell cycle inhibitors, mTOR inhibitors, and growth factor receptor signal transduction kinase inhibitors; retenoids; cyclin/CDK inhibitors; HMG co-enzyme reductase inhibitors (statins); and protease inhibitors; beta-2 agonists (e.g. salbutamol, terbutaline, clenbuterol, salmeterol, formoterol); steroids such glycocorticosteroids, preferably anti-inflammatory drugs (e.g. Ciclesonide, Mometasone, Flunisolide, Triamcinolone, Beclomethasone, Budesonide, Fluticasone); anticholinergic drugs (e.g. ipratropium, tiotropium, oxitropium); leukotriene antagonists (e.g. zafirlukast, montelukast, pranlukast); xantines (e.g. aminophylline, theobromine, theophylline); Mast cell stabilizers (e.g. cromoglicate, nedocromil); inhibitors of leukotriene synthesis (e.g. azelastina, oxatomide ketotifen); mucolytics (e.g. N-acetylcysteine, carbocysteine); antibiotics, (e.g. Aminoglycosides such as, amikacin, gentamicin, kanamycin, neomycin, netilmicin streptomycin, tobramycin; Carbacephem such as loracarbef, Carbapenems such as ertapenem, imipenem/cilastatin meropenem; Cephalosporins-first generation—such as cefadroxil, cefaxolin, cephalexin; Cephalosporins-second generation—such as cefaclor, cefamandole, defoxitin, cefproxil, cefuroxime; Cephalosporins-third generation-cefixime, cefdinir, ceftaxidime, defotaxime, cefpodoxime, ceftriaxone; Cephalosporins— fourth generation— such as maxipime; Glycopeptides such as vancomycin, teicoplanin; Macrolides such as azithromycin, clarithromycin, Dirithromycin, Erythromycin, troleandomycin; Monobactam such as aztreonam; Penicillins such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin, Piperacillin, Ticarcillin; Polypeptides such as bacitracin, colistin, polymyxin B; Quinolones such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, Trovafloxacin; Sulfonamides such as Mafenide, Prontosil, Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim-Sulfamethoxazole Co-trimoxazole (TMP-SMX); Tetracyclines such as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline; Others such as Chloramphenicol, Clindamycin, Ethambutol, Fosfomycin, Furazolidone, Isoniazid, Linezolid, Metronidazole, Nitrofurantoin, Pyrazinamide, Quinupristin/Dalfopristin, Rifampin, Spectinomycin); pain relievers in general such as analgesic and antiinflammatory drugs, including steroids (e.g. hydrocortisone, cortisone acetate, prednisone, prednisolone, methylpredniso lone, dexamethasone, betamethasone, triamcino lone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone); and non-steroid antiinflammatory drugs (e.g. Salicylates such as aspirin, amoxiprin, benorilate, coline magnesium salicylate, diflunisal, faislamine, methyl salicylate, salicyl salicylate); Arylalkanoic acids such as diclofenac, aceclofenac, acematicin, etodolac, indometacin, ketorolac, nabumetone, sulindac tolmetin; 2- Arylpropionic acids (profens) such as ibuprofen, carprofen, fenbufen, fenoprofen, flurbiprofen, ketoprofen, loxoprofen, naproxen, tiaprofenic acid; N-arylanthranilic acids (fenamic acids) such as mefenamic acid, meclofenamic acid, tolfenamic acid; Pyrazolidine derivatives such as phenylbutazone, azapropazone, metamizole, oxyphenbutazone; Oxicams such as piroxicam, meloxicam, tenoxicam; Coxib such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib (withdrawn from market), valdecoxib (withdrawn from market); Sulphonanilides such as nimesulide; others such as licofelone, omega-3 fatty acids; cardiovascular drugs such as glycosides (e.g. strophantin, digoxin, digitoxin, proscillaridine A); respiratory drugs; antiasthma agents; bronchodilators (adrenergics: albuterol, bitolterol, epinephrine, fenoterol, formoterol, isoetharine, isoproterenol, metaproterenol, pirbuterol, procaterol, salmeterol, terbutaline); anticancer agents (e.g. cyclophosphamide, doxorubicine, vincristine, methotrexate); alkaloids (i.e. ergot alkaloids) or triptans such as sumatriptan, rizatriptan, naratriptan, zolmitriptan, eletriptan and almotriptan, than can be used against migraine; drugs (i.e. sulfonylurea) used against diabetes and related dysfunctions (e.g. metformin, chlorpropamide, glibenclamide, glicliazide, glimepiride, tolazamide, acarbose, pioglitazone, nateglinide, sitagliptin); sedative and hypnotic drugs (e.g. Barbiturates such as secobarbital, pentobarbital, amobarbital; uncategorized sedatives such as eszopiclone, ramelteon, methaqualone, ethchlorvynol, chloral hydrate, meprobamate, glutethimide, methyprylon); psychic energizers; appetite inhibitors (e.g. amphetamine); antiarthritis drugs (NSAIDs); antimalaria drugs (e.g. quinine, quinidine, mefloquine, halofantrine, primaquine, cloroquine, amodiaquine); antiepileptic drugs and anticonvulsant drugs such as Barbiturates, (e.g. Barbexaclone, Metharbital, Methylphenobarbital, Phenobarbital, Primidone), Succinimides (e.g. Ethosuximide, Mesuximide, Phensuximide), Benzodiazepines, Carboxamides (e.g. Carbamazepine, Oxcarbazepine, Rufinamide) Fatty acid derivatives (e.g. Valpromide, Valnoctamide); Carboxilyc acids (e.g. Valproic acid, Tiagabine); Gaba analogs (e.g. Gabapentin, Pregabalin, Progabide, Vigabatrin); Topiramate, Ureas (e.g. Phenacemide, Pheneturide), Carbamates (e.g. emylcamate Felbamate, Meprobamate); Pyrrolidines (e.g. Levetiracetam Nefiracetam, Seletracetam); Sulfa drugs (e.g. Acetazolamide, Ethoxzolamide, Sultiame, Zonisamide) Beclamide; Paraldehyde, Potassium bromide; antithrombotic drugs such as Vitamin K antagonist (e.g. Acenocoumarol, Dicumarol, Phenprocoumon, Phenindione, Warfarin); Platelet aggregation inhibitors (e.g. antithrombin III, Bemiparin, Deltaparin, Danaparoid, Enoxaparin, Heparin, Nadroparin, Pamaparin, Reviparin, Tinzaparin); Other platelet aggregation inhibitors (e.g. Abciximab, Acetylsalicylic acid, Aloxiprin, Ditazole, Clopidogrel, Dipyridamole, Epoprostenol, Eptifibatide, Indobufen, Prasugrel, Ticlopidine, Tirofiban, Treprostinil, Trifusal); Enzymes (e.g. Alteplase, Ancrod, Anistreplase, Fibrinolysin, Streptokinase, Tenecteplase, Urokinase); Direct thrombin inhibitors (e.g. Argatroban, Bivalirudin, Lepirudin, Melagatran, Ximelagratan); other antithrombotics (e.g. Dabigatran, Defibrotide, Dermatan sulfate, Fondaparinux, Rivaroxaban); antihypertensive drugs such as Diuretics (e.g. Bumetanide, Furosemide, Torsemide, Chlortalidone, Hydroclorothiazide, Chlorothiazide, Indapamide, metolaxone, Amiloride, Triamterene); Antiadrenergics (e.g. atenolol, metoprolol, oxprenolol, pindolol, propranolol, doxazosin, prazosin, teraxosin, labetalol); Calcium channel blockers (e.g. Amlodipine, felodipine, dsradipine, nifedipine, nimodipine, diltiazem, verapamil); Ace inhibitors (e.g. captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, benzapril); Angiotensin II receptor antagonists (e.g. candesartan, irbesartan, losartan, telmisartan, valsartan); Aldosterone antagonist such as spironolactone; centrally acting adrenergic drugs (e.g. clonidine, guanabenz, methyldopa); antiarrhythmic drug of Class I that interfere with the sodium channel (e.g. quinidine, procainamide, disodyramide, lidocaine, mexiletine, tocamide, phenyloin, encamide, flecamide, moricizine, propafenone), Class II that are beta blockers (e.g. esmolol, propranolol, metoprolol); Class III that affect potassium efflux (e.g. amiodarone, azimilide, bretylium, clorilium, dofetilide, tedisamil, ibutilide, sematilide, sotalol); Class IV that affect the AV node (e.g. verapamil, diltiazem); Class V unknown mechanisms (e.g. adenoide, digoxin); antioxidant drugs such as Vitamin A, vitamin C, vitamin E, Coenzime Q10, melanonin, carotenoid terpenoids, non carotenoid terpenoids, flavonoid polyphenolic; antidepressants (e.g. mirtazapine, trazodone); antipsychotic drugs (e.g. fluphenazine, haloperidol, thiotixene, trifluoroperazine, loxapine, perphenazine, clozapine, quetiapine, risperidone, olanzapine); anxyolitics (Benzodiazepines such as diazepam, clonazepam, alprazolam, temazepam, chlordiazepoxide, flunitrazepam, lorazepam, clorazepam; Imidaxopyridines such as Zolpidem, alpidem; Pyrazolopyrimidines such as zaleplon); antiemetic drugs such as Serotonine receptor antagonists (dolasetron, granisetron, ondansetron), dopamine antagonists (domperidone, droperidol, haloperidol, chlorpromazine, promethazine, metoclopramide) antihystamines (cyclizine, diphenydramine, dimenhydrinate, meclizine, promethazine, hydroxyzine); antiinfectives; antihystamines (e.g. mepyramine, antazoline, diphenihydramine, carbinoxamine, doxylamine, clemastine, dimethydrinate, cyclizine, chlorcyclizine, hydroxyzine, meclizine, promethazine, cyprotheptadine, azatidine, ketotifen, acrivastina, loratadine, terfenadine, cetrizidinem, azelastine, levocabastine, olopatadine, levocetrizine, desloratadine, fexofenadine, cromoglicate nedocromil, thiperamide, impromidine); antifungus (e.g. Nystatin, amphotericin B, natamycin, rimocidin, filipin, pimaricin, miconazole, ketoconazole, clotrimazole, econazole, mebendazole, bifonazole, oxiconazole, sertaconazole, sulconazole, tiaconazole, fluconazole, itraconazole, posaconazole, voriconazole, terbinafme, amorolfme, butenafme, anidulafungin, caspofungin, flucytosine, griseofulvin, fluocinonide) and antiviral drugs such as Anti-herpesvirus agents (e.g. Aciclovir, Cidofovir, Docosanol, Famciclovir, Fomivirsen, Foscarnet, Ganciclovir, Idoxuridine, Penciclovir, Trifluridine, Tromantadine, Valaciclovir, Valganciclovir, Vidarabine); Anti-influenza agents (Amantadine, Oseltamivir, Peramivir, Rimantadine, Zanamivir); Antiretroviral drugs (abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, zidovudine, adeforvir, tenofovir, efavirenz, delavirdine, nevirapine, amprenavir, atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfmavir, ritonavir, saquinavir, tipranavir); other antiviral agents (Enfuvirtide, Fomivirsen, Imiquimod, Inosine, Interferon, Podophyllotoxin, Ribavirin, Viramidine); drugs against neurological dysfunctions such as Parkinson's disease (e.g. dopamine agonists, L-dopa, Carbidopa, benzerazide, bromocriptine, pergolide, pramipexole, ropinipole, apomorphine, lisuride); drugs for the treatment of alcoholism (e.g. antabuse, naltrexone, vivitrol), and other addiction forms; vasodilators for the treatment of erectile dysfunction (e.g. Sildenafil, vardenafil, tadalafil), muscle relaxants (e.g. benzodiazepines, methocarbamol, baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, dantrolene, metaxalone, orphenadrine, tizanidine); muscle contractors; opioids; stimulating drugs (e.g. amphetamine, cocaina, caffeine, nicotine); tranquillizers; antibiotics such as macrolides; aminoglycosides; fluoroquinolones and beta-lactames; vaccines; cytokines; growth factors; hormones including birth-control drugs; sympathomimetic drugs (e.g. amphetamine, benzylpiperazine, cathinone, chlorphentermine, clobenzolex, cocaine, cyclop entamine, ephedrine, fenfluramine, methylone, methylphenidate, Pemoline, phendimetrazine, phentermine, phenylephrine, propylhexedrine, pseudoephedrine, sibutramine, symephrine); diuretics; lipid regulator agents; antiandrogen agents (e.g. bicalutamide, cyproterone, flutamide, nilutamide); antiparasitics; blood thinners (e.g. warfarin); neoplastic drugs; antineoplastic drugs (e.g. chlorambucil, chloromethine, cyclophosphamide, melphalan, carmustine, fotemustine, lomustine, carboplatin, busulfan, dacarbazine, procarbazine, thioTEPA, uramustine, mechloretamine, methotrexate, cladribine, clofarabine, fludarabine, mercaptopurine, fluorouracil, vinblastine, vincristine, daunorubicin, epirubicin, bleomycin, hydroxyurea, alemtuzumar, cetuximab, aminolevulinic acid, altretamine, amsacrine, anagrelide, pentostatin, tretinoin); hypoglicaemics; nutritive and integrator agents; growth integrators; antienteric drugs; vaccines; antibodies; diagnosis and radio-opaque agents; or mixtures of the above mentioned drugs (e.g. combinations for the treatment of asthma containing steroids and beta-agonists); or any other biologically active agent such as nucleic acids, DNA, R A, siRNA, polypeptides, antibodies, and the like. Growth factors and adhesion peptides can be useful for tissue development within a subject and can be included in the particles.

In one embodiment, the present invention may be used in connection with a diverse type of eukaryotic host cells from a diverse set of species of the plant and animal kingdoms. Preferably, the host cells are from mammalian species including cells from humans, other primates, horses, pigs, and mice. For example, cells can be stem cells of any kind (e.g., umbilical cord or placenta derived, dental pulp derived, marrow-derived, adipose derived, induced stem cells, or cells of embryonic or amniotic origin), PER.C6 cells, HT-29 cells, LNCaP-FGC cells A549 cells, MDA-MB453 cells, HepG2 cells, THP- 1 cells, miMCD-3 cells, HEK 293 cells, HeLaS3 cells, MCF.sub.7 cells, Cos-7 cells, CHO cells and CHO derivatives, CHO-K.sup.l cells, BxPC-3 cells, DU145 cells, Jurkat cells, PC-3 cells, Capan-1 cells, HuVEC cells, HuASMC cells, HKB-11 human differentiated stem cells such as osteoblasts and adipocytes from hMSC; human adherent cells such as SH-SY5Y, IMR32, LAN5, HeLa, MCF10A, 293T, and SK-BR3; primary cells such as HUVEC, HUASMC, and hMSC; and other species such as 3T3 NIH, 3T3 LI, ES-D3, C2C12, H9c2 and the like. Additionally, any species of plant may be used. Any of these cells can be included with the particles when using the carbon dioxide process (e.g., single step) for preparing the scaffolds.

Fabrication of polymer coated glass beads

A circulating fluidized bed coating process was used to fabricate spheres with a stiff core and a soft outer layer of polymer. Soda lime glass beads of 1 -1.18 mm size (-16 + 18 mesh size, MO-SCI, Rolla, MO) were used as the stiffer core material. A 5% w/v solution of Poly(D,L-lactic-co-glycolic acid) (PLGA) (50:50 lactic acid:glycolic acid, acid end group, MW ~ 42,000-44,000 Da) of intrinsic viscosity 0.34-0.36 dL/g or polycaprolactone (PCL) (ester end group of intrinsic viscosity 1 -1.3 dL/g) dissolved in methylene chloride were used to coat the beads. The coating was carried out using a UniGlatt® fluidized-bed coater. Prior to coating, the beads were fluidized in the chamber for 10 minutes until the outlet temperature of the coater reached 40°C. The process parameters for the PLGA coating were as follows: atomization pressure 1.5 bar; inlet temperature 45-50°C; outlet temperature 40-42°C; fluidizing air setting 2.2-3.0 kPa; relative humidity 3% and spraying rate 7 g/minute. The process parameters for the PCL coating were as follows: atomization pressure 1.5 bar; inlet temperature 40-45°C; outlet temperature 38-40°C; fluidizing air setting 2.3-2.6 kPa; relative humidity 3% and spraying rate 2.5 g/minute. The coating process was continuously monitored through the glass window of the coating device to ensure a smooth fluidization of bed. At intervals, the coated beads were fluidized in air for 10 minutes to remove the residual organic solvent and to avoid clustering of the beads. The weight of uncoated glass beads and the coated beads were measured. A total of 8 g of PLGA and 18 g of PCL were coated per 1000 g of glass beads. Photographs and microscopic images of beads before and after coating were taken. The polymer coated beads appeared white/opaque in color when compared to the uncoated transparent glass beads. (Figs. 3A-3B and 4A-4B). Figure 3A includes images of uncoated glass beads, and Figure 3B includes images of glass beads coated with PLGA. Figure 4A shows a phase contrast image of uncoated glass beads, and Figure 4B shows a phase contrast image of glass beads coated with PLGA. A scratch was created on the surface of the beads using a scalpel blade and imaged to visualize the coating as shown in Fig. 5, where the arrow points to the scratch on the surface to visualize the coating.

Solvent vapor sintering

The PCL coated glass beads were packed in cylindrical polypropylene molds of 7 mm diameter to a height of 10 mm. These molds were exposed to 50 ml of methylene chloride vapors for 3 hrs and 4 hrs in a tightly closed container. The scaffolds were removed from molds and placed in a fume hood overnight to remove the residual vapors. The control scaffold made of PCL 200 micron particles are sintered by C02 exposure to an absolute pressure of 690 psi (47.6 bar) at 45 °C for a period of 4 hours followed by depressurization at a rate of -0.2 psi/s for 1 hour.

Selective laser sintering

Using an SLS system (Sinterstation 2500plus, DTM, USA) that was modified to accommodate a limited amount of material, settings were tuned in order to achieve the desired laser sintering. The coated beads were placed into the build chamber and the two feed chambers, then the chamber was closed and warmed to 38 oC with the build heater at 18% power and at an inner/outer power ratio of 0.5, and with the feed chamber heaters at 10% power. The chamber was then opened and custom-made heat shields were inserted, and the chamber continued to heat for about 20 additional minutes to a set point temperature of 48 oC. The laser power was set at 25 W, with an overlap of 0.3 mm and spacing set at 0.1 mm. The build chamber was set to drop 1.4 mm per layer, and the feed pistons were set to raise 2.8 mm per layer. A part (scaffold) was sintered that had a "window frame" shape, with a 4 cm x 4 cm square, with four 1 cm x 1 cm "windows" and 0.5 cm wide struts. A build height (part thickness) of 5 mm was achieved as shown in Figs. 6A-6B. In other similar runs, thicknesses of up to 1.0 cm were achieved, limited only by the available amount of coated beads. Figure 6A shows a view of a "window pane" formed from the engineered particles and shape sintered by SLS, with a 4 x 4 cm outer dimension, 1 x 1 cm windows, and 0.5 cm struts (e.g., frame). Figure 6B shows a side view of the window pane of Figure 6A to demonstrate a thickness of 0.5 cm, providing evidence of not only selective laser sintering, but also layering of selective laser sintering.

C02 sintering

Scaffolds were fabricated by exposure to sub-critical levels of C02 in a custom- designed stainless steel vessel having a pressure safety rating of 60 bar. Specific amounts of PLGA coated glass beads were loaded into a Teflon molds and exposed to C02. The beads were exposed to a C02 absolute saturation pressure of 700 bar at 45 °C for 4 hours followed by depressurization at the rate of 0.101 psi/s for 1 hour.

Heat sintering

The PLGA and PCL coated beads were packed in molds and sintered by heat at

56°C for 4 hours. Three-dimensional scaffolds could be fabricated by sintering the outer polymer layer of each beads by all of these methods as shown in Figures 7A-7B and 8. Figure 7A shows PCL coated glass beads sintered by methylene chloride vapors at 3 hours of sintering and Figure 7B shows 4 hours of sintering. Figure 8 shows PLGA coated glass beads sintered by sub-critical C02.

Mechanical characterization of 3 dimensional scaffolds sintered using methylene chloride vapors

The compressive modulus of 3 dimensional scaffolds was performed using a uniaxial testing apparatus (Instron Model 5848, Canton, MA) with a 50 N load cell. Tare- loaded (0.05 N) constructs were compressed at a rate of 1 mm/minute in a dry state and immersed in phosphate buffered saline (PBS) at 37°C. Moduli of elasticity were calculated from the initial linear regions of the stress-strain curves. (Table 1) The beads remained sintered together after compression to 80% of its initial height. (Figs. 9A-9C). Figure 9A shows a scaffold before compression. Figure 9B shows a lateral view of the scaffold of Figure 9A after compression. Figure 9C shows cross sectional view after compression.

Table 1. Compressive elastic modulus of polymer coated beads

Bead Testing Modulus Modulus (Pa)

Type condition (Pa) 4 hour sintering

3 hour

sintering

PCL Dry state Range (2 - Range (1 - 2 x coated glass (RT) (n=3) 5 x 107) 108)

beads Mean (2.5 Mean (1.6 + 0.53 x 107) x 108)

PCL Wet state Range (1 - 9 x coated glass (37°C, PBS) 106) beads (n=6) Mean (3.66 + 2.9 x 106)

PCL Wet state

microspheres (37°C, PBS) Mean (45.6 ±

200 (n=5) 26.3 x 103)

microns

Figure 10 shows a graph of stress-strain data obtained for PLGA-PCL dual coated microspheres melded with methylene chloride for 3 hours. The test was done with an anvil height of 7.625 and standard procedures.

Additionally, HuCMSCs were seeded on the sintered scaffolds and cultured for a period of 24 hrs and one week. Cell viability assay was carried out using Live Dead assay kit containing calcien AM and ethidium bromide dye. The constructs were imaged using confocal microscope. hUCMSCs passage number 1 were used for the live/dead assay. Cells were seeded at a density of 1 million cells per scaffold. The cells were cultured in DMEM (Low Glucose) containing 1% Pencillin/Streptomycin, 10% Fetal Bovine Serum (FBS). Live cells showed green fluorescence. Cells were viable on both the 24 hr and 1 week constructs. The cells on the week 1 constructs were properly spread and covered the bead surface and the interstitial space. The results showed that the coated beads provide a favorable non-toxic surface for cell attachment and proliferation.

Figures 11A and 11B include images of 1 mm engineered particles having the hard core and polymeric shells with live: dead cells at one week, which images are color contrasted to show the cells. The cells are human umbilical cord cells (hUCMSCs) seeded on coated glass particles in a tissue engineering scaffold.

Figure 12 includes images of 1 mm engineered particles having the hard core and polymeric shells with live:dead cells at 24 hours, which images are color contrasted to show the cells. The cells are human umbilical cord cells (hUCMSCs) seeded on coated glass particles in a tissue engineering scaffold. The spaces between the particles show the porosity (1st level porosity or interstitial space).

Figures 13A and 13B include images of 200 micron engineered particles having the hard core and polymeric shells with live: dead cells at one week, which images are color contrasted to show the cells. The cells are human umbilical cord cells (hUCMSCs) seeded on coated glass particles in a tissue engineering scaffold. The cells are shown to be in the on the particles and in spaces between the particles show the porosity (1st level porosity or interstitial space).

Figures 14A and 14B include images of 200 micron engineered particles having the hard core and polymeric shells with live:dead cells at 24 hours, which images are color contrasted to show the cells. The cells are human umbilical cord cells (hUCMSCs) seeded on coated glass particles in a tissue engineering scaffold. The cells are shown to be in the on the particles and in spaces between the particles show the porosity (1st level porosity or interstitial space). As a note, Figures 11 A, 11B, 13 A, and 13B are from snapshots taken from video files of 3D reconstruction of images generated from z stack. Figures 12, 14A, and 14B are combined images generated from z stack.

Figure 15A includes an image of 200 micron engineered particles in a sintered scaffold with interstitial spaces between the particles. Figure 15B includes an image of 1 mm engineered particles in a sintered scaffold with interstitial spaces between the particles.

Figure 16 includes a graph that illustrates the average elastic modulus of PCL coated 200 micron glass bead scaffolds compared to with 200 micron PCL microsphere scaffolds conducted in hydrated conditions. The average elastic modulus of PCL coated glass bead scaffolds (~11 MPa) is about 2.5 times higher than the average elastic modulus of PCL microsphere scaffolds (4.5 MPa). Furthermore, the range of modulus of elasticity for coated bead scaffolds is 4-30 MPa and for the microsphere scaffolds is 4-5 MPa. As such, scaffolds having the engineered particles with the hard core and polymeric shell can be tailored across a broad range of stiffness and prepared into scaffolds significantly stiffer than polymeric scaffolds.

Additionally, the scaffolds prepared from the engineered particles having the hard core and polymeric shell may be modified to be at least an order of magnitude higher stiffer than scaffolds with just polymeric microspheres. Additionally, the coating thickness and/or particle dimension (e.g., diameter) may be modulated in order to obtain scaffolds that have even greater stiffness. Moreover, the higher degree of stiffness from the engineered particles and the lower degree of stiffness of the regular polymeric microspheres allows for the formation of stiffness gradients and hard stiffness changes at interfacial transitions between the different types of microspheres.

From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. All references recited herein are incorporated herein by specific reference in their entirety: U.S. Patent Application No. 13/591,087 filed August 21, 2012; U.S. Patent Application No. 12/248,530 filed October 9, 2008 now U.S. Patent No. 8,277,832; and U.S. Provisional Patent Application No. 61/594,568 filed October 10, 2007.

Claims

1. A particle comprising:

a hard core; and

one or more polymeric shells encapsulating the core.

2. The particle of claim 1, wherein the hard core includes hydroxyapatite, tricalcium phosphate, glass, bioactive glass, calcium carbonate, titanium dioxide, or combinations thereof.

3. The particle of one of claims 1-2, wherein the one or more polymeric shells includes a biocompatible polymer.

4. The particle of one of claims 1-3, wherein the one or more polymeric shells includes polycaprolactone, poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) ( PLGA), derivatives thereof, salts thereof, or combinations thereof.

5. The particle of one of claims 1-4, wherein the particle is a nanosphere.

6. The particle of one of claims 1-4, wherein the particle is a microsphere.

7. The particle of one of claims 1-6, wherein one or more bioactive agents are encapsulated in or located on the one or more polymeric shells.

8. The particle of one of claims 1-7, wherein one or more growth factors are encapsulated in or located on the one or more polymeric shells.

9. The particle of claim 8, wherein the growth factors include insulin- like growth factor-I (IGF-I), a transforming growth factor, TGF-betal, TGF-beta3, a bone morphogenetic protein, BMP -2, BMP-7, or combinations thereof.

10. The particle of one of claims 1-9, wherein the particle has only one core.

11. The particle of one of claims 1-10, wherein the particle has only one polymeric shell. 12. The particle of one of claims 1-11, wherein the core has a shape and the one or more polymeric shells have the shape of the core or more spherical shape thereof.

13. The particle of one of claims 1-12, wherein an outer-most shell is substantially devoid of an active agent.

14. The particle of one of claims 1-13, wherein the hard core of the particle has a dimension in a range of about 5 μιη to 1200 μιη and the one or more polymeric shells has a thickness from about about 0.025 μιη to 125 μιη. 15. The particle of one of claims 1-14, wherein a ratio of the coating thickness to core diameter is between about 0.005 and 1.5.

16. A method of manufacturing a particle of one of claims 1-15, the method comprising:

providing a plurality of the hard core particles;

providing one or more polymer compositions for use in forming the one or more shells; and

coating each of the hard core particulates with the one or more polymeric shells. 17. The method of claim 16, wherein the coating is performed with a circulating fluidized bed (CFB) protocol.

18. The method of one of claims 16-17, comprising:

providing a bioactive agent to be included in the one or more polymeric shells; and

incorporating the bioactive agent into the one or more polymeric shells.

19. The method of one of claims 16-18, wherein a bioactive agent is incorporated into one or more polymer compositions before being applied to the hard core particulates. 20. The method of one of claims 16-19, comprising drying the particles.

21. A particulate composition comprising:

a plurality of the particles of claims 1-15. 22. A particulate composition of claim 21, comprising a liquid containing the plurality of particles.

23. A particulate composition of claim 21, wherein the composition is a free- flowing powder.

24. A particulate composition of one of the claims 21-22, wherein the composition is a suspension of particles in the liquid.

25. A particulate composition of claim 21, wherein the composition is a paste.

26. A particulate composition of one of the claims 21-25, wherein two or more particles are melded together such that the polymeric shells thereof are bound together.

27. A particulate composition of one of the claims 21-25, wherein the composition is configured as an implant.

28. A biocompatible implant comprising:

a plurality of the particles of claims 1-15 melded together to form a structure having a plurality of pores between the particles.

29. A biocompatible implant as in claim 28, wherein the implant has a shape configured as a shape of a defect in a hard tissue of a subject.

30. A biocompatible implant as in one of the claims 28-29, wherein the implant has a complex shape that is unattainable by a mold.

31. A biocompatible implant as in one of the claims 28-30, wherein the implant has one or more apertures, pores, holes, internal chambers or combinations thereof.

32. A biocompatible implant as in one of the claims 28-31, wherein the implant has one or more internal solid members unattainable by a mold.

33. A biocompatible implant as in one of the claims 28-32, wherein the implant has one or more internal solid members connected to one or more external solid members, with space or gaps around the one or more internal solid members. 34. A biocompatible implant as in one of the claims 28-33, wherein the melding of adjacent particles is from a solvent meld from a liquid solvent, vapor solvent or C0₂.

35. A biocompatible implant as in one of the claims 28-33, wherein the meld is from a laser meld or general heat meld.

36. A biocompatible implant as in one of the claims 28-35, comprising one or more bioactive agents each in an active form. 37. A biocompatible implant as in one of the claims 28-36, the implant being configured for as a bone implant.

38. A biocompatible implant as in one of the claims 28-37, the implant being configured as a tooth implant.

39. A biocompatible implant as in one of the claims 28-38, the implant being configured to have a portion for hard tissue regeneration and a portion for soft tissue regeneration.

40. A biocompatible implant as in one of the claims 28-39, comprising a first gradient of the plurality of particles and a second gradient of different particles. 41. A biocompatible implant as in one of the claims 28-40, comprising a hardness in the mega-Pascal (MPa) range.

42. A biocompatible implant as in one of the claims 28-40, comprising a hardness in the giga-Pascal (GPa) range.

43. A biocompatible implant as in one of the claims 28-42, comprising a hardness of at least two orders of magnitude higher than an implant having only polymeric particles of the polymer shell. 44. A biocompatible implant as in one of the claims 28-43, comprising a first level of porosity from interstitial spacing between the particles and a second level of porosity formed by selective layer sintering with a laser.

45. A biocompatible implant as in one of the claims 28-44, comprising a first level of porosity of about 40% from interstitial spacing between the particles and a second level of porosity of macroscopic pores formed by selective layer sintering with a laser and removing particles to form the macroscopic pores.

46. A biocompatible implant as in one of the claims 28-45, comprising a compressive modulus of about 3.7 MPa at 37 °C in hydrated conditions and a higher compressive modulus in dry conditions.

47. A biocompatible implant as in one of the claims 28-46, comprising a substantially homogeneous distribution of the particles throughout the implant.

48. A biocompatible implant as in one of the claims 28-47, wherein the biocompatible implant is configured as a tissue engineering scaffold for growing cells.

49. A biocompatible implant configured as a tissue engineering scaffold comprising:

a plurality of biocompatible particles linked together so as to form a three- dimensional matrix having a plurality of pores defined by and disposed between the particles, said plurality of particles having a surface area sufficient for growing cells within the plurality of pores, said plurality of biocompatible particles comprising:

a first set of particles having a first characteristic, the first set of particles having a first predetermined spatial distribution with respect to a first end of the three-dimensional matrix, the first set of particles having a hard core and one or more polymeric shells; and

a second set of particles having a second characteristic that is different from the first characteristic, the second set of particles having a second predetermined spatial distribution with respect to a second end of the three- dimensional matrix and that is different from the first predetermined spatial distribution with respect to the three-dimensional matrix.

50. A biocompatible implant as in claim 49, wherein the first predetermined spatial distribution is distinct from and adjacent to the second predetermined spatial distribution.

51. A biocompatible implant as in one of the claims 49-50, wherein the first predetermined spatial distribution forms a first concentration gradient of the first set of particles and the second predetermined spatial distribution forms a second concentration gradient of the second set of particles.

52. A biocompatible implant as in one of the claims 49-51, wherein the three dimensional matrix comprises:

a first portion having a majority of particles of the first set; and

a second different portion having a majority of particles of the second set.

53. A biocompatible implant as in one of the claims 49-52, wherein the three- dimensional matrix comprises:

a first portion having a majority of particles of the first set; a second portion having a majority of particles of the second set; and

a third portion disposed between the first portion and the second portion, wherein the first predetermined spatial distribution in the third portion forms a first concentration gradient of the first set of particles and the second predetermined spatial distribution in the third portion forms a second concentration gradient of the second set of particles.

54. A biocompatible implant as in one of the claims 49-53, the first and second characteristics are independently selected from the group consisting of the following: composition; polymer; particle size; core size; shell thickness; shell layer thickness; particle size distribution; type of bioactive agent; type of bioactive agent combination; bioactive agent concentration; amount of bioactive agent; rate of bioactive agent release; mechanical strength; flexibility; rigidity; color; radiotranslucency; or radiopaqueness.

55. A biocompatible implant as in one of the claims 49-54, said scaffold further comprising live cells and a medium sufficient for growing the cells disposed in the pores.

56. A biocompatible implant as in one of the claims 49-55, said scaffold further comprising a plurality of live cells attached to the plurality of particles.

57. A biocompatible implant as in one of the claims 49-56, said scaffold being further characterized by the following:

a first cell type associated with the first set of particles; and

a second cell type associated with the second set of particles.

58. A biocompatible implant as in one of the claims 49-57, a first end of the scaffold having a majority of the first set of particles and an opposite send end of the scaffold having a majority of the second set of particles.

60. A method of manufacturing a biocompatible implant, the method comprising:

providing the particles of one of claims 1-15; and

melding the particles together so as to form the biocompatible implant.

61. A method as in claim 60, comprising melding the particles so as to form interstitial spaces between adjacent melded particles. 62. A method of manufacture as in one of the claims 60-61, comprising sintering the particles so as to form pores of sufficient size for cellular migration and culturing.

63. A method of manufacture as in one of the claims 60-62, comprising shaping the implant to correspond with a hard tissue defect of a subject.

64. A method of manufacture as in one of the claims 60-63, comprising:

obtaining an image of a hard tissue defect of a subject;

computing, with a computing system, the shape of the implant to match the hard tissue defect using the image; and

shaping the implant to correspond with the hard tissue defect.

65. A method of manufacture as in one of the claims 60-64, wherein the sintering comprises solvent liquid sintering.

66. A method of manufacture as in one of the claims 60-64, the sintering comprising solvent vapor sintering.

67. A method of manufacture as in one of the claims 60-66, the sintering comprising liquid or vapor solvent sintering with ethanol, ethanol-acetone, dense-phase carbon dioxide, chloroform, methylene chloride, or combinations thereof.

68. A method of manufacture as in one of the claims 60-67, comprising sintering the particles so as to retain activity of the bioactive agent.

69. A method of manufacture as in one of the claims 60-68, comprising distributing the particles in a substantially homogenous distribution.

70. A method of manufacture as in one of the claims 60-68, comprising distributing the particles in a concentration gradient with respect to a set of different second particles. 71. A method of manufacture as in one of the claims 60-70, comprising laser sintering.

72. A method of manufacture as in one of the claims 60-71, comprising selective layer sintering with a laser.

73. A method of manufacture as in one of the claims 60-72, comprising general heating for sintering.

74. A method of manufacture as in one of the claims 60-73, comprising removing particles that are not melded from the implant.

75. A method of manufacture as in one of the claims 60-74, comprising sintering the particles without heat. 76. A method of correcting a defect in a hard tissue of a subject, the method comprising:

providing an implant having a plurality of particles sintered together, the particles including a hard core and one or more polymeric shells, the implant having a plurality of pores between adjacent sintered particles; and

implanting the implant into the defect in the hard tissue of the subject.