Patent Publication Number: US-2007098799-A1

Title: Mineralized Hydrogels and Methods of Making and Using Hydrogels

Description:
RELATED APPLICATION  
      Pursuant to 37 C.F.R. § 1.78(a)(4), this application claims the benefit of and priority to prior filed co-pending Provisional Application Ser. No. 60/731,092, filed Oct. 28, 2005, which is expressly incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION  
      The present invention relates to mineralized hydrogels suitable for use in biomedical or other applications  
     BACKGROUND OF THE INVENTION  
      Hydrogels are water-swellable or water-swollen materials having a structure defined by a crosslinked network of hydrophilic homopolymers or copolymers. The hydrophilic homopolymers or copolymers may be water-soluble in free form, but in a hydrogel are rendered insoluble (but water-swellable) in water due to covalent, ionic, or physical crosslinking. In the case of physical crosslinking, the linkages may take the form of entanglements, crystallites, or hydrogen-bonded structures. The crosslinks in a hydrogel provide structure and physical integrity to the network.  
      Hydrogels may be classified as amorphous, semicrystalline, hydrogen-bonded structures, supermolecular structures, or hydrocolloidal aggregates. Numerous parameters affect the physical properties of a hydrogel, including porosity, pore size, the hydrogel used, molecular weight of gel polymer, and crosslinking density. The crosslinking density, for example, influences the hydrogel&#39;s macroscopic properties, including volumetric equilibrium swelling ratio (denoted “Q”), compressive modulus, and mesh size (ξ), which is the space between macromolecular chains in a cross-linked network usually characterized by the distance between adjacent cross-links.  Hydrogels in Medicine and Pharmacy , CRC Press, 1986, edited by Nicholas A. Peppas. Pore size and shape, pore density, and other factors can impact the surface properties, optical properties, and mechanical properties of a hydrogel.  
      Hydrogels have been derived from a variety of hydrophilic polymers and copolymers. Poly(vinyl alcohol) (“PVA”), Poly(ethylene glycol), poly(vinyl pyrrolidone), polyacrylamide, poly(hydroxyethyl methacrylate) (“PHEMA”), and copolymers of the foregoing, are examples of polymers from which hydrogels have been made. Hydrogels have also been formed from biopolymers such as chitosan, agarose, hyaluronic acid and gelatin, as well as (semi) interpenetrating network (“IPN”) hydrogels such as gelatin crosslinked with poly(ethylene glycol) diacrylate.  
      Hydrogels have shown promise in biomedical and pharmaceutical applications, mainly due to their high water content and rubbery or pliable nature, which can mimic natural tissue and can facilitate the release of bioactive substances at a desired physiological site. For example, hydrogels have been used and/or proposed in a variety of tissue treatment applications, including implants, tissue adhesives, and bone grafts for spinal and orthopedic treatments such as meniscus and articular cartilage replacement. One drawback to the use of conventional hydrogels in certain tissue treatment applications, and in particular bone tissue treatments, is that such hydrogels do not necessarily provide an optimal scaffolding for encouraging tissue growth and/or formation of calcified tissues. For example, conventional hydrogels do not have substantial osteoconductive characteristics, and therefore, do not suitably encourage the formation of bone tissue, either on the surface or within such hydrogel materials. Conventional hydrogels may also lack suitable mechanical properties, e.g. strength, for certain tissue treatments, in particular calcified and/or bony tissue treatments.  
      In an attempt to improve the osteoconductive characteristics and/or mechanical properties of hydrogels, calcium phosphate minerals such as hydroxyapatite have been incorporated into previously-prepared hydrogels (e.g., PHEMA or PVA), for example, by soaking the hydrogel in a concentrated calcium phosphate solution such as a simulated body fluid, by alternately immersing the hydrogel in a calcium solution and a phosphate solution, and by physically mixing previously prepared hydrogels and calcium phosphate minerals.  
      Unfortunately, each of these approaches has certain drawbacks. The first two approaches, which involve immersing pre-formed hydrogels, may not provide suitable calcium phosphate mineral distribution within the hydrogel, and the mineralization process may be difficult to control. Additionally, in-situ (e.g., in a mold) reactions may not be achievable. With the third approach, the hydrogel may not suitably bind to the mineral, and it may be difficult to prepare articles with mineral concentration gradients. Consequently, these approaches to providing combining hydrogels with calcium phosphate minerals may have significant commercial limitations.  
      Therefore, it would be beneficial to provide mineralized hydrogels and methods of making and using mineralized hydrogels that overcome one or more of the aforementioned drawbacks.  
     SUMMARY OF THE INVENTION  
      The present invention provides mineralized hydrogels and methods of making and using mineralized hydrogels, in which calcium phosphate minerals are dispersed within a hydrogel polymer. In one embodiment, a calcium phosphate dispersion may first be formed by combining a first mixture including a calcium derivative, a second mixture including a phosphate derivative, and a hydrogel precursor to form a calcium phosphate dispersion containing the hydrogel precursor. The hydrogel precursor present in the calcium phosphate dispersion is then crosslinked to form a mineralized hydrogel, in which the calcium phosphate minerals may be substantially uniformly dispersed within the mineralized hydrogel. Optional treatments may be employed to modify the calcium phosphate dispersion and/or minerals into a desired form such as a calcium-deficient apatite, which mimic biological bone and/or other calcified tissues. 
    
    
     BRIEF DESCRIPTION OF FIGURES  
      The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with a general description of the invention given above, and the detailed description given below, serve to explain the invention.  
       FIG. 1  is a flow-chart illustrating a method of forming mineralized hydrogels according to an embodiment of the present invention.  
       FIGS. 2A-2B  illustrate an instrument for delivering mineralized hydrogels according to an embodiment of the present invention.  
       FIGS. 3A-3B  are Fourier Transform Infrared Spectroscopy (FTIR) spectra of samples formed according to Example 1.  
       FIGS. 4A-4B  are Scanning Electron Microscope (SEM) images of samples formed according to Example 1.  
       FIGS. 5A-5C  are images of samples formed according to Example 4.  
       FIGS. 6A-6B  are images of samples formed according to Example 5. 
    
    
     DETAILED DESCRIPTION  
       FIG. 1  is a flow-chart summarizing a method of forming a mineralized hydrogel according to one embodiment of the present invention. In  10  and  12 , a first mixture including a calcium derivative and a second mixture including a phosphate derivative are separately prepared and combined in  14  to form a calcium phosphate dispersion. A hydrogel precursor may be added to either or both of the first and second mixtures prior to being combined. Alternatively, the hydrogel precursor may be added during or after the first and second mixtures are combined to form the calcium phosphate dispersion. In either case, in  16 , the hydrogel precursor contained in the calcium phosphate dispersion is subsequently crosslinked to form a mineralized hydrogel including calcium phosphate minerals. In  18 , optional pH treatments may also be carried out to modify the calcium phosphate mineral. Each of these steps is described in detail below.  
      Preparation of First and Second Mixtures  
      In one embodiment, in  10 , the first mixture includes a calcium derivative, an optional hydrogel precursor and an aqueous carrier (e.g., deionized water or miscible solutions of water and organic solvents). Suitable calcium derivatives include CaCl 2 , Ca(OH) 2  and Ca(NO 3 ) 2 . The calcium derivative may be combined with the aqueous carrier to form an aqueous calcium dispersion. The optional hydrogel precursor may be combined with the aqueous dispersion via conventional mixing methods.  
      In another embodiment, in  12 , the second mixture includes a phosphate derivative, an optional hydrogel precursor and an aqueous carrier. Examples of suitable phosphate derivatives include K 2 HPO 4 , Na 2 HPO 4 , H 3 PO 4  and (NH 4 ) 2 HPO 4 . The phosphate derivative may be combined with the aqueous carrier to form an aqueous phosphate dispersion. The optional hydrogel precursor may then be combined with the aqueous phosphate dispersion using conventional methods.  
      As used herein, the term “hydrogel precursor” refers to hydrogels and hydrogel polymer source materials (e.g. macromers, monomers, oligomers, homopolymers, copolymers, and/or (semi)-interpenetrating polymer networks (IPNs), which form hydrogels) that may be processed into a hydrogel. Suitable hydrogel precursors may be derived from a variety of polymer materials including poly(vinyl alcohol) (“PVA”), poly(glycols) such as poly(ethylene glycol) dimethacrylate (“PEGDMA”), poly(ethylene glycol) diacrylate (“PEGDA”), poly(hydroxyethyl methacrylate) (“PHEMA”), poly(vinyl pyrrolidone), poly(acrylamide), poly(acrylic acid), hydrolyzed poly(acrylonitrile), poly(ethyleneimine), ethoxylated poly(ethyleneimine) and poly(allylamine), polypeptides, as well as monomers, oligomers, macromers, copolymers and/or other derivatives of the foregoing. Biopolymers may also be used in certain embodiments. Suitable biopolymers include anionic biopolymers such as hyaluronic acid, cationic biopolymers such as chitosan, amphipathic polymers such as collagen, gelatin and fibrin, and neutral biopolymers such as dextran and agarose. Interpenetrating polymer networks (e.g. combinations of water soluble polymers and water insoluble polymers), polymers modified or grafted with (poly)peptides or proteins, and polymers having backbones modified with calcium or phosphate derivatives may also be suitable for use in certain embodiments. Additional polymers which may be suitable for use in certain embodiments are reported in U.S. Pat. No. 6,224,893 to Langer et al., the contents of which are hereby incorporated by reference in their entirety. Suitable hydrogel blends are reported in U.S. application Ser. No. 11/358,383 entitled “Blend Hydrogels and Methods of Making,” incorporated herein by reference in its entirety.  
      If a hydrogel precursor is added to both the first and second mixtures, the hydrogel precursor used in each mixture may be the same hydrogel precursor. Alternatively, the hydrogel precursors may be different than, but capable of crosslinking with, one another. In one embodiment, about 10 w/w % hydrogel precursor may be added to each mixture.  
      In certain embodiments of the present invention, the hydrogel precursor is poly(vinyl alcohol), or a derivative thereof. Poly(vinyl alcohol) may be produced by free-radical polymerization of vinyl acetate to form poly(vinyl acetate), followed by hydrolysis to yield PVA. The hydrolysis reaction does not go to completion, which leaves pendent acetate groups at some points along the polymer chain. The extent of the hydrolysis reaction determines the degree of hydrolysis of the PVA. Commercially available PVA can have a degree of hydrolysis over 98% in some cases. PEGDA and PEGDMA are also suitable for use in particular embodiments.  
      In certain embodiments, ions such as F − , Cl − , Na + , K + , Mg 2+ , Sr 2+ , Ba 2+ , HPO 4   2 , CO 3   2− , and/or SO 4   2−  (or salts thereof) may be added to the first and/or second mixture to form calcium deficient apatites when the first and second mixtures are combined as reported in greater detail below.  
      Optionally, a radiation sensitive material such as a photoinitiator may be added to the first and/or second mixture (or subsequently to the calcium phosphate dispersion) to facilitate crosslinking of the hydrogel precursor as reported below. IRGACURE® brand photoinitiators (available from Ciba Specialty Chemicals) is an example of a group of suitable radiation sensitive materials. Other optional additives include biocompatible preservatives, surfactants, colorants and/or other additives conventionally added to polymer mixtures.  
      The properties and/or physical characteristics of the first and second mixtures may vary depending on the type and amount of carrier and the type and concentration of hydrogel precursor added to the carrier. In certain embodiments, the mixtures will have characteristics similar to a traditional solution. In other embodiments, for example embodiments utilizing PVA hydrogel precursors, the mixture may take on characteristics (e.g. water swellable) similar to a hydrogel without any additional crosslinking steps.  
      Combining First and Second Mixtures  
      In  14 , the first and second mixtures may be combined under conditions suitable to form a calcium phosphate dispersion including calcium phosphate minerals (generally nano- to micro-sized particles formed from precipitation) into which the hydrogel precursor is dispersed, dissolved or are otherwise contained. As used herein, the phrase “calcium phosphate dispersion” broadly encompasses dispersions, slurries, solutions, and any other mixture formed by combining the calcium and phosphate derivatives reported herein. The conditions under which the first and second mixtures are combined may affect the resulting reaction between the calcium and phosphate derivatives.  
      The order in which the first and second mixtures are combined may be varied. In one embodiment, the second mixture is added to the first mixture at a controlled rate and under continuous agitation to form the desired calcium phosphate dispersion. In another embodiment, the first mixture is added to the second mixture at a controlled rate and under continuous agitation to form the desired calcium phosphate dispersion.  
      Reaction temperature may also be varied. In one embodiment, the mixtures may be combined at room temperature, i.e., at a temperature of about 25° C. In another embodiment, the mixtures may be combined at physiological temperature, i.e., at a temperature of about 37° C. In another embodiment, the mixtures may be combined at a temperature between about 25° C. and about 37° C.  
      Additional conditions that may be varied include the atmosphere (e.g., air, inert or CO 2 ), the concentration of the calcium and or phosphate derivative in each mixture, the calcium:phosphate ion ratio, and the pH of the first and second mixtures. These conditions may affect the calcium phosphate mineral or minerals formed when the first and second mixtures are combined. The types of calcium phosphate minerals that may be formed include amorphous calcium phosphates, dicalcium phosphate dihydrate, tricalcium phosphate, octacalcium phosphate, calcium deficient apatites and hydroxyapatite.  
      Amorphous calcium phosphate minerals formed according to embodiments of the present invention do not show traditional crystalline peaks from X-ray diffraction (XRD) characterization and may not have a fixed chemical structure, but instead are defined by their substantially non-crystalline and/or nano-crystalline structure.  
      Dicalcium phosphate dehydrate formed according to embodiments of the present invention has the following formula:
 
CaHPO 4 .2H 2 O
 
      Calcium-deficient apatites formed according to embodiments of the present invention have the following formula:
 
(C a , M) 10 (PO 4 , CO 3 , X) 6 (OH, F, Cl) 2 
 
 wherein M includes minor ions and/or trace elements such as Na + , K + , Mg 2+ , Sr 2+ , Ba 2+ , and X is HPO 4   2−  or So 4   2− . The presence of minor ions in calcium deficient apatites may be achieved by adding the ion (such as in the form of a salt) to the first and/or second mixture. 
 
      Hydroxyapatite formed according to embodiments of the present invention has the following formula:
 
Ca 10 (PO 4 ) 6 (OH) 2  
 
      In one embodiment, the concentration of the calcium ions in the first mixture and the phosphate ions in the second mixture may be between about 0.1 mM and about 5 M, more particularly between about 0.5 M and about 2 M. In another embodiment, the first mixture includes about a 1M concentration of calcium ions and the second mixture includes about a 0.67 M concentration of phosphate groups.  
      The resulting ratio of Ca ions to phosphate ions in the first and second mixtures may range from about 1:1 to about 2:1. Ratios between about 1:1 and about 1.5:1 may encourage the formation of amorphous calcium phosphate minerals. Ratios between about 1.5:1 and 2:1, more particularly between about 1.5:1 and about 1.67:1, may encourage the formation of calcium-deficient minerals.  
      When the first and second mixtures are combined in a manner that encourages the formation of amorphous calcium phosphate, but a more crystalline form of calcium phosphate (e.g. calcium-deficient apatite) is desired, the calcium phosphate dispersion may be further treated in a pH environment suitable for transforming the amorphous calcium phosphate to a crystalline form. In one embodiment, the pH of the calcium phosphate dispersion may be adjusted to between about 6.5 and 7.5 (i.e. approximately neutral pH), more particularly between about 7.2 and about 7.4 to encourage transformation from amorphous calcium phosphate to a crystalline form. In another embodiment, the pH may be adjusted to a pH of greater than 7.5 (i.e. a basic dispersion), more particularly between about 10 and 11 to transform amorphous calcium phosphate to a crystalline form at a different reaction rate and/or kinetics. For example, the calcium phosphate dispersion may be combined with a basic solution such as an ammonia solution or a sodium hydroxide solution, or a buffer solution such as a phosphate buffered saline, or a tris-buffer. Additional description of the conversion of amorphous calcium phosphate to crystalline calcium phosphate may be found in LeGeros R. Z., “Calcium phosphates in oral biology and medicine,” Monograph in Oral Science, Vol. 15, pp. 1-201, and Chow et al., “Octacalcium Phosphate,” Monograph in Oral Science, Vol. 18, pp. 94-112 and 130-148. In alternative embodiments, similar pH treatments may also be employed after the formation of the mineralized hydrogel.  
      In one embodiment, the calcium phosphate dispersion is formed and/or treated to encourage the formation of calcium-deficient apatite. One benefit of calcium-deficient apatite as opposed to other calcium phosphate minerals is that calcium deficient apatite is known to more closely mimic biological apatite in the body, and may therefore provide improved osteoconductive properties compared to other calcium phosphate minerals.  
      The properties and/or physical characteristics of the mixture will also affect the characteristics of the calcium phosphate dispersion. In certain embodiments, the calcium phosphate dispersion will have characteristics similar to a traditional dispersion. In other embodiments, for example embodiments utilizing PVA hydrogel precursors, the calcium phosphate dispersion may take on characteristics (e.g. water swellable) similar to a hydrogel without additional crosslinking steps.  
      Prior to crosslinking the hydrogel precursor as reported below, the calcium phosphate may be subjected to continuous mixing to provide a substantially uniform dispersion. Alternatively, the dispersion could be placed in a centrifuge device, and subjected to a sufficient force to create a calcium phosphate mineral gradient in the dispersion.  
      Crosslink Hydrogel Precursor  
      After forming the desired calcium phosphate dispersion, in  16 , the hydrogel precursor in the dispersion is optionally crosslinked to form a mineralized hydrogel, in which the calcium phosphate minerals are dispersed and/or distributed within the mineralized hydrogel. A variety of approaches may be used to crosslink the hydrogel precursor, including photoinitiation, irradiation, physical crosslinking (e.g., freeze thaw method), and chemical crosslinking.  
      In one embodiment of the present invention, crosslinking is achieved by exposing the calcium phosphate dispersion to ultraviolet or visible blue light (collectively referred to herein as “UV/Vis radiation”). In this embodiment, a photoinitiator such as an IRGACURE® brand initiator may be combined with the calcium phosphate dispersion, or with one of the mixtures used to form the calcium phosphate dispersion. Suitable UV/Vis radiation sources according to one embodiment may operate at a wavelength ranging between about 200 to 700 nm, more particularly between about 320 nm and about 700 nm, and even more particularly between about 350 and about 520 nm. Suitable energy levels for the UV/Vis radiation source may range between about 10 μW/cm 2  and about 20 W/cm 2 . In a particular embodiment, a wavelength of approximately 365 nm and an energy level of 300 μW/cm 2  may be suitable. Crosslinking by electron-beam or gamma irradiation, such as by using a Co 60  source, may also be employed according to embodiments of the present invention.  
      In another embodiment, physical crosslinking may be achieved by conventional freeze-thaw techniques, for example, as described in Peppas, et al.,  Adv. Polymer Sci.  153, 37 (2000).  
      Examples of suitable chemical crosslinking agents for addition to the calcium phosphate dispersion (or the first or second mixture) include monoaldehydes such as formaldehyde, acetaldehyde, or glutaraldehyde in the presence of a solvent such as methanol. Other suitable crosslinking agents include diisocyanate compounds, which can result in urethane linkages, or epoxy compounds. Crosslinking achieved using enzymes such as a calcium independent microbial transglutaminase, which catalyzes transamidation reactions to form N-ε-(γ-glutamyl)lysine crosslinks in proteins, may also be suitable according to embodiments of the present invention.  
      A combination of crosslinking techniques may also be utilized in the invention. For example, a freeze-thaw cycle could be used to provide physical crosslinking, followed by irradiation or photoinitiation to provide more complete crosslinking. As other examples, chemical crosslinking could be followed by irradiation or photoinitiation, or a freeze-thaw step could be performed after crosslinking by any of chemical, irradiation or photoinitiation.  
      The type, concentration and chemical properties (e.g. molecular weight) of the hydrogel precursor added to the first and/or second mixtures, as well as the employed crosslinking technique, may affect the properties and/or characteristics of the formed hydrogels. For example, swelling ratio, water content, mesh and/or pore size of the hydrogel may be varied by controlling the type, molecular weight and concentration of the hydrogel material. Also, the resulting hydrogel may be formed to be biodegradable or stable in vitro and/or in vivo by varying the chemistry of the hydrogel. For example, hydrogels formed from PEGDA are generally biostable, but hydrogels formed from copolymers of Poly(lactic acid) and PEGDA may be formed with controllable degradation properties (See, e.g., Anseth, et al.,  Journal of Controlled Release,  78 (2002), 199-209).  
      Additionally, the type of hydrogel precursor used may result in enhanced properties such as adhesion. For example, PEGDA, interpenetrating networks of PEGDA, and gelatins (including gelatin crosslinked by Ca-independent enzymes) may have enhanced or improved hydrogel adhesion properties. The addition of non-hydrogel phases or fillers (e.g. solid fillers) into the hydrogel, either prior to or after crosslinking, may also modify the properties of the hydrogel. The employed method of crosslinking the hydrogel precursor to form a mineralized hydrogel may also affect hydrogel properties. In certain embodiments, additional bioactive agents may be incorporated into the hydrogel prior to or after the hydrogel is formed. Examples of suitable bioactive agents include, without limitation, proteins such as bone morphogenic proteins (“BMPs”), growth factors, pharmaceuticals such as analgesics or antibiotics, enzymes and/or genes.  
      As previously noted, certain embodiments of the present invention may form a mineralized hydrogel material without additional crosslinking steps. For example, certain PVA polymers may form a hydrogel when the first and second mixtures are prepared. In such cases, a final crosslinking step is optional, but may further improve certain characteristics of the mineralized hydrogel.  
      In the foregoing manner, the form of the mineralized hydrogel may be varied depending on the specific application and the desired result. In certain applications for example, it may be desirable to disperse and/or distribute the calcium phosphate derivate in a substantially uniform and/or homogenous manner throughout the mineralized hydrogel. In these applications, a bulk mineralized hydrogel may be formed as reported herein by combining the first and second mixtures and crosslinking. Alternatively, the first and second mixtures may be combined in a desired mold to form an implant.  
      In other embodiments, a surface region having a uniform distribution of the calcium phosphate minerals, or a concentration gradient of the calcium phosphate minerals may be desirable. This could be accomplished by applying multiple layers of separately formed mineralized hydrogels having varying forms or concentrations of calcium phosphate minerals. Still further, it may be desirable to adhere layers of mineralized hydrogels with layers of conventional hydrogels.  
      The mineralized hydrogels of the present invention may be used in a wide range of medical applications, including orthopedic and spinal uses as implants and/or implant coatings, bone grafts and or bone cements, adhesive/fixation materials, orthobiologics (e.g. tissue engineering scaffolds and constructs), mineralized fillers, drug, growth factor and gene delivery, and in dental applications. In one embodiment, the mineralized hydrogels could be used to provide artificial articular cartilage as described, e.g., by Noguchi, et al.,  J. Appl. Biomat.  2, 101 (1991), or as artificial meniscus or articular bearing components. The hydrogels could also be employed to repair or replace the temporomandibular joint, proximal interphalangeal joint, metacarpophalangeal joint, metatarsalphalanx joint, or in hip capsule joint repair.  
      In another embodiment, the mineralized hydrogels of the present invention may be used to replace or rehabilitate the nucleus pulposus of an intervertebral disc. Degenerative disc disease in the lumbar spine is marked by a dehydration of the intervertebral disc and loss of biomechanical function of the spinal unit. A recent approach has been to replace only the central portion of the disc, called the nucleus pulposus. The approach entails a less invasive posterior surgery, and can be done rather rapidly. Bao and Higham developed a PVA hydrogel suitable for nucleus pulposus replacement, as reported in U.S. Pat. No. 5,047,055. The hydrogel material, containing about 70% water, acts similarly to the native nucleus, in that it absorbs and releases water depending on the applied load.  
      The mineralized hydrogels of the invention may also be employed in a spinal disc prosthesis used to replace a part of or all of a natural human spinal disc. By way of example, a spinal disc prosthesis may comprise a flexible nucleus, a flexible braided fiber annulus, and end-plates. The hydrogel may be employed in the flexible nucleus, for example. A spinal disc prosthesis is described in U.S. Pat. No. 6,733,533 to Lozier, for instance.  
      According to one embodiment, the mineralized hydrogels of the present invention may be formed into an implantable prosthetic device of the types reported herein. Such an implant may be formed by initially placing the first mixture in an implant mold. The second mixture may then be rapidly mixed and/or injected into the first mixture. The resulting calcium phosphate dispersion may then be crosslinked according to one of the methods reported herein to form a mineralized hydrogel implant. Alternate processing methods include solution casting, injection molding, or compression molding. In general, these methods may be used prior to or after crosslinking.  
      According to another embodiment of the present invention, a mineralized hydrogel may be delivered to a physiological site prior to, coincident with, or immediately after combining the first and second mixtures.  FIGS. 2A-2B  are schematic illustrations of an instrument  20  capable of combining the first and second mixtures and delivering the mixtures to a physiological site. The instrument  20  includes a syringe  22 , a plunger  24 , and a cannula  26 . The syringe  22  includes a divider  28  for separating the first and second mixtures into respective reservoirs  30 ,  32 . The plunger  24  includes a button  34 , a shaft  36  having a channel  38 , which slides relative to the divider  28  to deliver the first and second mixtures into the cannula  26 . In this embodiment, the first and second mixtures first initially combine in the cannula  26 .  
      In an alternate embodiment, separate plungers  24  for the first and second mixtures could be utilized such that the first and second mixtures could be delivered separately. In a further embodiment, the syringe  22  and/or cannula  26  could have a dual lumen construction such that the first and second mixtures do not contact until after exiting the cannula  26 . The water-swellable material can then be crosslinked and/or hydrated after delivery and formation to provide a hydrogel.  
      The following examples are provided to illustrate the invention and are not intended to limit the same.  
     EXAMPLE 1A  
      At room temperature, equal volumes of CaCl 2 . 2 H 2 O (1M) and Na 2 HPO 4  (0.67M) were reacted. A calcium phosphate powder was obtained by filtering and drying the precipitate. The calcium phosphate powder was then analyzed under FTIR and SEM, which indicated that dicalcium phosphate (DCPD) formed. FTIR spectra were acquired using a Bruker Vertex 70 spectrometer (Bruker Optics Inc., Billerica, Mass., USA), and SEM images were acquired using a LEO™ 1550 Variable Pressure field emission SEM (Carl Zeiss SMT Inc., Thornwood, N.Y., USA).  FIG. 3A  is the FTIR spectra of the calcium phosphate material.  FIG. 4A  is the SEM image of the calcium phosphate material.  
     EXAMPLE 1B  
      At room temperature, equal volumes of CaCl 2 .2H 2 O (IM) and Na 2 HPO 4  (0.67M) were reacted. The resulting calcium phosphate dispersion was adjusted to a pH between 10 and 12 using 1M NaOH. A calcium phosphate powder was obtained by filtering and drying the precipitate. The calcium phosphate powder was analyzed under FTIR and SEM, which indicated that calcium-deficient apatite formed.  FIG. 3B  is the FTIR spectra of the calcium phosphate material.  FIG. 4B  is the SEM image of the calcium phosphate material. Thus, by controlling the pH of the calcium phosphate material, calcium-deficient apatite was formed.  
     EXAMPLE 2  
      A first mixture containing 10 w/w % PEGDMA macromers (synthesized as reported in Lin-Gibson et al.  Biomacromolecules,  2004, 5, 1280-1287) having a molecular weight between about 2000 and 5000 g/mol, a 1M solution of CaCl 2 .2H 2 O, and 0.05-1 w/w % (relative to concentration of PEGDMA) IRGACURE® 2959 brand photoinitiator (Ciba Specialty Chemicals) was prepared. A second mixture containing 10w/w % PEGDMA macromers, a 0.67 M solution of Na 2 HPO 4 , and 0.05-1 w/w % (relative to concentration of PEGDMA) IRGACURE® 2959 brand photoinitiator was also prepared.  
      40 μL of the first mixture was injected into a cylindrical mold having a height of 3 mm and a thickness of 6 mm. 40 μL of the second mixture was then injected into the mold. A calcium phosphate dispersion formed rapidly. The calcium phosphate dispersion was then exposed to a long wavelength UV source (365 nm, 300 μW/cm 2 ) for 10-30 minutes to crosslink the hydrogel to form a mineralized hydrogel.  
     EXAMPLE 3  
      A first mixture was prepared by adding 10 w/w % poly(vinyl alcohol) (“PVA”) polymer having a molecular weight of about 140,000 g/mol and a 1M dispersion of CaCl 2 .2H 2 O to an aqueous dispersion of 25w/w% dimethyl sulfoxide (“DMSO”). A second mixture was prepared by adding 10 w/w % of the same PVA polymer and a 0.67 of dispersion of Na 2 HPO 4  to an aqueous dispersion of 25 w/w % DMSO.  
      The first and second mixtures were separately loaded into a DUO-PAK™ brand twist-lock cartridge having a 3/16 in. tip (available from McMaster-Carr). The cartridge was connected to a dispensing gun, and the first and second mixtures were injected from the dispensing gun, through a static mixer (available from McMaster-Carr), and into a mold. The mold can be subjected to a conventional freeze-thaw cycle or other conventional crosslinking technique to crosslink the hydrogel to form the mineralized hydrogel.  
     EXAMPLE 4  
      0.05 g IRGACURE® 2959 brand photoinitiator (2-Hydroxy-1-[ 4 -(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone, Ciba Specialty Chemicals) was dissolved in 5 mL poly(ethylene glycol) diacrylate (PEGDA, Aldrich Inc., Mn ˜575, water soluble). The resulting PEGDA solution was mixed with 5 mL 1M CaCl 2  to form a first mixture. A 0.67M solution of Na 2 HPO 4  was also prepared to form a second mixture. The second mixture was added dropwise to the first mixture under moderate stirring on a magnetic stirring plate (speed 3-4 in a 0-10 scale) to form a dispersion. Stirring of the dispersion was continued for another 15-30 min. The dispersion was then transferred to several UHMWPE cylinder molds having a diameter of 0.25 inch and a thickness of 0.25 inch. The molds were then positioned on a piece of glass and covered with a Mylar film. Photopolymerization of the dispersion was carried out for 3 min in a UV box (LOCTITE®ZETA® 7411-S UV Flood System, with a lamp power of 400 W and optimized wavelength between 315 and 400 nm) to form a mineralized hydrogel. After being removed from the molds, several gel specimens were soaked in a 1M NaOH solution. Control gel samples were prepared in the same matter except that no Ca and phosphate derivatives were included.  
       FIGS. 5A-5C  show images of several samples formed as reported above.  FIG. 5A  shows two mineralized samples and two control samples. One mineralized sample and one control sample were soaked in NaOH. All four samples swelled when contacted with water, with the NaOH-soaked samples swelling to a greater degree.  
       FIGS. 5B-5C  show microscopic views of a control sample ( 5 A) and a mineralized sample ( 5 B) obtained by light microscopy using a Zeiss Stemi 2000-C microscope (Carl Zeiss SMT Inc., Thornwood, N.Y., USA ). Notably, a substantially uniform dispersion of a calcium phosphate mineral is visible in the mineralized sample.  
      Alternatively, a mineralized hydrogel is prepared according to Example 4 except that the calcium phosphate dispersion is centrifuged prior to being added to the mold. The resulting mineralized hydrogel contains a gradient of calcium phosphate minerals.  
     EXAMPLE 5  
      Poly(ethylene glycol) dimethacrylate (PEGDMA, Mn ˜4000, water soluble) macromers were synthesized as reported in the literature (Lin-Gibson et al.  Biomacromolecules,  2004, 5, 1280-1287). A first mixture was prepared by dissolving 0.28 g PEGDMA and 0.0003 g (˜0.1 wt % relative to PEGDMA macromers) IRGACURE® 2959 brand photoinitiator in 1.0 mL 1M CaCl 2  solution. A second mixture was provided by preparing a 1.0 mL 0.67M solution of Na 2 HPO 4 . The second mixture was added dropwise to the first mixture under moderate stirring on a magnetic stirring plate (speed 3-4 in a 0-10 scale) to form a calcium phosphate dispersion. The calcium phosphate dispersion was then stirred for another 15-30 min. A 10 wt % photoinitiated PEGDMA solution was prepared by dissolving 0.2 g PEGDMA in 1.8 g deionized water, along with 0.0002 g (0.1 wt % relative to PEGDMA macromers) Irgacure 2959 brand photoinitiator (2-Hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone, Ciba Specialty Chemicals). No calcium or phosphate materials were added to the 10 wt % photoinitiated PEGDMA solution. Several UHMWPE cylinder molds having a diameter of 0.25 inch and a thickness of 0.25 inch were positioned on a piece of glass and partially filled with the 10 wt % photoinitiated PEGDMA solution. The molds were then covered with a Mylar film. Photopolymerization of the partially filled molds was carried out for 4 min in a UV box (LOCTITE®ZETA® 7411-S UV Flood System, with a lamp power of 400 W and optimized wavelength between 315 and 400 nm) to form a PEGDMA hydrogel. The calcium phosphate dispersion was then added over the crosslinked PEGDMA hydrogel to fill up the molds. A Mylar film was again applied to cover the filled molds. Photopolymerization of the calcium phosphate dispersion was again carried out under the same conditions to form a composite hydrogel having bottom layer of PEGDMA hydrogel and the top layer of mineralized PEGDMA hydrogel.  
      A first control sample was prepared in the manner described above except that the molds were filled completely with the calcium phosphate dispersion and crosslinked to form a mineralized hydrogel. A second control sample was formed in the same manner except that the molds were filled completely with the 10 wt % photoinitiated PEGDMA solution and crosslinked to form a PEGDMA hydrogel.  
      FIGS.  6 A-B shows images of several samples formed as reported above. Samples 1 and 2 are images of the first and second control samples, respectively. Specimens 3 and 4 are two-layer composite hydrogels with varying thicknesses of mineralized PEGDMA hydrogel layers and PEGDMA hydrogel layers.  
       FIG. 6B  show a close-up image of a two-layer sample with a top layer of mineralized PEGDMA hydrogel and a bottom layer of PEGDMA hydrogel.  
      Alternately, a layered hydrogel can be prepared as described above in Example 5, except that the calcium phosphate dispersion is added to the mold and photopolymerized first, followed by the addition and photopolymerization of the PEGDMA solution such that the bottom layer is mineralized PEGDMA hydrogel and the top layer is PEGDMA hydrogel.  
     EXAMPLE 6  
      Calcium phosphate minerals in the absence of a hydrogel are prepared as in Example 1, using the concentrations of calcium and phosphate and pH control to yield amorphous calcium phosphate as demonstrated by FTIR, XRD and SEM. The amorphous calcium phosphate can then be converted to calcium-deficient apatite using appropriate pH adjustments, as described above. Alternately, calcium phosphate minerals in the absence of a hydrogel can be prepared as in Example 1, but using the concentrations of Ca and phosphate and pH control to yield calcium-deficient apatite as demonstrated by FTIR, XRD and SEM, as described above.  
     EXAMPLE 7  
      Prepare and combine first and second mixtures according to Example 2. Prior to crosslinking, combine the calcium phosphate dispersion with a phosphate buffered saline solution (pH about 7.4) for up to about 24 hours and periodically test dried samples under FTIR, XRD and SEM until the desired conversion of amorphous calcium phosphate to calcium-deficient apatite and/or other crystalline calcium phosphates is achieved. Also, identify the microstructure of the inside of a sample using light microscopy or similar methods. Further, test the mechanical properties of samples using conventional mechanical tests procedures according to ASTM standards.  
     EXAMPLE 8  
      Prepare the first and second mixtures according to Example 2. Separately load each mixture into a DUO-PAK™ cartridge with a 3/16 in. tip, which is connected to a dispensing gun, as described in Example 3. Dispense the first and second mixture from the dispensing gun, through a static mixture and into a mold or Petri dish. Crosslink via physical or chemical crosslinking and characterize the hydrogel using light microscopy, XRD, FTIR and SEM.  
     EXAMPLE 9  
      Prepare a layered hydrogel as described in Example 5, except that a third mixture is prepared by dissolving 0.28 g PEGDMA and 0.0003 g (˜1 wt % relative to PEGDMA macromers) IRGACURE® 2959 brand photoinitiator in 1.0 mL 0.1M CaCl 2  solution. A 1.0 mL 0.067M solution of Na 2 HPO 4  is also prepared to form a fourth mixture. The fourth mixture is added dropwise to the third mixture under moderate stirring on a magnetic stirring plate (speed 3-4 in a 0-10 scale) to form an additional calcium phosphate dispersion. This additional calcium phosphate dispersion is filled into a mold over the previously-formed two-layer hydrogel described in Example 5. The mold is then covered with a Mylar film and photopolymerized under the conditions described in Example 5 to form a three-layer gel with the top two layers providing a calcium phosphate concentration gradient. Alternatively the ordering of the layers can be modified such that the bottom two layers form a calcium phosphate gradient and the top layer is a PEGDMA gel.  
      While the present invention has been illustrated by the description of one or more embodiments thereof, and while the embodiments have been described in considerable detail, they are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and method and illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the scope of the general inventive concept.