Formation and properties of composites comprised of calcium-deficient hydroxyapatites and ethyl alanate polyphosphazenes.

Composites comprised of calcium-deficient hydroxyapatite (HAp) and biodegradable polyphosphazenes were formed via cement-type reactions at physiologic temperature. The composite precursors were produced by blending particulate hydroxyapatite precursors with 10 wt% polymer using a solvent/non-solvent technique. HAp precursors having calcium-to-phosphate ratios of 1.5 (CDH) and 1.6 (CDS) were used. The polymeric constituents were poly[bis(ethyl alanato)phosphazene] (PNEA) and poly[(ethyl alanato)(1) (p-phenylphenoxy)(1) phosphazene] (PNEA(50)PhPh(50)). The effect of incorporating the phenyl phenoxy group was evaluated as a means of increasing the mechanical properties of the composites without retarding the rates of HAp formation. Reaction kinetics and mechanistic paths were characterized by pH determination, X-ray diffraction analyses, scanning electron microscopy, and infrared spectroscopy. The mechanical properties were analyzed by compression testing. These analyses indicated that the presence of the polymers slightly reduced the rate HAp formation. However, surface hydrolysis of polymer ester groups permitted the formation of calcium salt bridges that provide a mechanism for bonding with the HAp. The compressive strengths of the composites containing PNEA(50)PhPh(50) were superior to those containing PNEA, and were comparable to those of HAp produced in the absence of polymer. The current composites more closely match the structure of bone, and are thus strongly recommended to be used as bone cements where high loads are not expected.

Formation of hydroxyapatite-polyphosphazene polymer composites at physiologic temperature.

Aspects of the formation of bone analog composites at 37 degrees C are described. The composites are composed of hydroxyapatite (HAp) and the calcium salt of a biocompatible polymer and are capable of forming under in vivo conditions. Composite formation involves the formation of monolithic HAp from particulate calcium phosphate precursors while Ca ions liberated to the aqueous medium in which this reaction is occurring form crosslinks with the acidic polymer. The reactants are poly[bis(carboxylatophenoxy)phosphazene] (acid-PCPP), tetracalcium phosphate [Ca4(PO4)2O, TetCP], and anhydrous dicalcium phosphate (CaHPO4, DCPA). The effects of the proportion of polymer (5, 10, or 15 wt %) on the kinetics of HAp formation were studied. Compositional evolution of the solid calcium phosphates present was followed by X-ray diffraction and infrared spectroscopy analyses. HAp formation through a dissolution-precipitation process provided a mildly alkaline medium suitable for deprotonation of the acid-PCPP and for the formation of the calcium crosslinks, as monitored by infrared spectroscopy. Concurrence of crosslinking of the polymer and HAp formation was established, indicating true composite formation can be realized at physiologic temperature.

Composite formation from hydroxyapatite with sodium and potassium salts of polyphosphazene.

The low temperature synthesis of composites potentially suitable as bone substitutes which form in vivo, was investigated. The composites were comprised of stoichiometric hydroxyapatite (SHAp) and water-soluble poly phosphazenes. These constituents were selected because of their biocompatibility, and were mixed as powders with a phosphate buffer solution (PBS) to form the composites. The effects of poly[bis(sodium carboxylatophenoxy)phosphazene] (Na-PCPP) or poly[bis(potassium carboxylatophenoxy) phosphazene] (K-PCPP) on stoichiometric hydroxyapatite (SHAp) formation from tetracalcium phosphate and anhydrous dicalcium phosphate were assessed. The kinetics and reaction chemistries of composite formation were followed by isothermal calorimetry, X-ray diffraction, infrared spectroscopy and scanning electron microscopy. In the presence of 1% by weight of polyphosphazenes, composites comprised of SHAp and calcium cross-linked polymer salts were formed. Thus a mechanism for binding between polymer chains was established. Elevated proportions (5 and 10% by weight) of polyphosphazene, however, resulted in the inhibition of SHAp formation. This is attributed to the formation of viscous polymer solution coatings on the calcium phosphate precursors, retarding their reaction, and consequently inhibiting SHAp formation.

Low temperature formation of hydroxyapatite-poly(alkyl oxybenzoate)phosphazene composites for biomedical applications.

The formation of biodegradable composites which may be suitable as bone analogs is described. Polyphosphazene-hydroxyapatite (HAp) composites were produced via an acid-base reaction of tetracalcium phosphate and anhydrous dicalcium phosphate in the presence of polyphosphazenes bearing alkyl ester containing side-groups. The polyphosphazenes used were poly(ethyl oxybenzoate)phosphazene (PN-EOB) and poly(propyl oxybenzoate) phosphazene (PN-POB). The effects of temperature and the proportions of polymers, PN-EOB and PN-POB on the kinetics, reaction chemistry and phase evolution during the formation of stoichiometric HAp were studied. Kinetics, phase evolution and microstructural development were evaluated using isothermal calorimetry, X-ray diffraction and scanning electron microscopy, respectively. Analysis of solution chemistry revealed that the increases in the pH during the formation of SHAp, resulted in partial hydrolysis of the polymer surfaces, which led in turn to the formation of a calcium cross-linked polymer surface. The calcium cross-linked polymer surface appeared to facilitate the nucleation and growth of apatite deposits on the polymer. The current study illustrates the in situ formation of HAp in the presence of polyphosphazenes, where HAp is chemically bonded to the polymer.

In vitro release of colchicine using poly(phosphazenes): the development of delivery systems for musculoskeletal use.

A colchicine release system utilizing biodegradable poly(phosphazenes) was investigated in vitro for intra-articular administration. Polymer degradation and drug release studies were performed on colchicine-loaded poly(phosphazenes) containing either imidazolyl (I-PPHOS) or ethyl glycinato (EG-PPHOS) side chain substituents over a 21-day period. To study the effects of an implantable colchicine-PPHOS delivery system on local musculoskeletal tissue in vitro, osteoblast-like cells were grown on the matrices. Colchicine release was 20% for I-PPHOS and 60% for EG-PPHOS over the 21-day period. Release appeared to proceed through a combination of diffusional and degradative mechanisms. Environmental scanning electron microscopy (ESEM) studies revealed large pores in the drug-depleted devices in contrast to the control matrices without drug, which may have contributed to the release seen, especially with ethyl glycinato-containing matrices. Cell growth on matrices containing colchicine was significantly (p < 0.05) inhibited in contrast to growth on tissue culture polystyrene (TCPS) and EG-PPHOS matrices without drug. The in vitro cell kinetic data suggest that designs for in vivo studies must take into account possible toxicity of colchicine and the polymer matrix on local tissue. Biodegradable PPHOS systems are promising candidates for use as intra-articular delivery vehicles for drugs with potential for systemic toxicity.

Novel polyphosphazene/poly(lactide-co-glycolide) blends: miscibility and degradation studies.

A novel biodegradable polymer blend was developed for potential biomedical applications. A 50:50 poly(lactide-co-glycolide) (PLAGA) was blended in a 50:50 ratio with the followiing polyphosphazenes (PPHOS): poly[(25% ethyl glycinato)(75% p-methylphenoxy)phosphazene[, poly[(50% ethyl glycinato)(50% p-methylphenoxy)phosphazene], and poly[(75% ethyl glycinato)(25% p-methylphenoxy)phosphazene] to obtain Blends A, B, and C, respectively, using a mutual solvent technique. The miscibility of these blends was determined by measuring their glass transition temperature (Tg) using differential scanning calorimetry. After fabrication using a casting technique, the degradation of the matrices was examined. Differential scanning calorimetry showed one glass transition temperature for each blend which was between the Tg's of their respective parent polymers indicating miscibility of the blends. Surface analysis by scanning electron microscopy showed the matrices to have smooth uniform surfaces. Degradation studies showed near-zero order degradation kinetics for the blends with Blends A and B losing 10% of their mass after two weeks and Blend C degrading more rapidly (30% mass loss during the same period). These findings suggest that these novel biodegradable PLAGA/PPHOS blends may be useful for biomedical purposes.

Synthesis and characterization of pH-sensitive poly(organophosphazene) hydrogels.

A new class of pH-sensitive hydrogels has been designed and synthesized. These are novel polyphosphazenes that bear various ratios of sodium oxybenzoate and methoxyethoxyethoxy side groups. These water-soluble macromolecules were cross-linked by 60Co gamma irradiation and the products were allowed to absorb water to form hydrogels. The hydrogels had higher equilibrium degrees of swelling in basic than in acidic buffer solutions, and polymers with a higher loading of the ionic side group showed higher swellability than those with a lower loading of this side group. The effects of ionic strength, cation charge and radiation dose on the degree of swelling were also studied. A study of the diffusion of the dye Biebrich Scarlet from the hydrogels showed complete release of the dye in 4-12 h in pH 7.4 buffer solution but significantly lower release at pH 2 even after 48 h. The release rate also varied as the side-group ratios were changed. The prehydrogel polymers were synthesized via the macromolecular substitution reactions of poly(dichlorophosphazene) with sodium methoxyethoxyethoxide and the sodium salt of propyl 4-hydroxybenzoate, followed by ester hydrolysis to yield the sodium carboxylate. The hydrogels are of interest for possible use as pH-sensitive membranes and for a number of potential biomedical applications.

A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal tissue regeneration.

Current methods for the replacement of skeletal tissue in general involve the use of autografts or allografts. There are considerable drawbacks in the use of either of these tissues. In an effort to provide an alternative to traditional graft materials, a degradable 3-dimensional (3-D) osteoblast cell-polymer matrix was designed as a construct for skeletal tissue regeneration. A degradable amino acid containing polymer, poly[(methylphenoxy)(ethyl glycinato) phosphazene], was synthesized and a 3-D matrix system was prepared using a salt leaching technique. This 3-D polyphosphazene polymer matrix system, 3-D-PHOS, was then seeded with osteoblast cells for the creation of a cell-polymer matrix material. The 3-D-PHOS matrix possessed an average pore diameter of 165 microns. Environmental scanning electron microscopy revealed a reconnecting porous network throughout the polymer with an even distribution of pores over the surface of the matrix. Osteoblast cells were found attached and grew on the 3-D-PHOS at a steady rate throughout the 21-day period studied in vitro, in contrast to osteoblast growth kinetics on similar, but 2-D polyphosphazene matrices, that showed a decline in cell growth after 7 days. Characterization of 3-D-PHOS osteoblastpolymer matrices by light microscopy revealed cells growing within the pores as well as on surface of the polymer as early as day 1. This novel porous 3-D-PHOS matrix may be suitable for use as a bioerodible scaffold for regeneration of skeletal tissue.

Poly[(amino acid ester)phosphazenes] as substrates for the controlled release of small molecules.

Three different poly[(amino acid ester)phosphazenes] have been examined in order to investigate their possible use as drug delivery vehicles. The three polymers are poly[di(ethyl glycinato)phosphazene], poly[di(ethyl alanato)phosphazene] and poly[di(benzyl alanato)phosphazene]. These macromolecules either share the same amino acid residue or the same ester group, and this facilitated comparisons of the hydrolytic decomposition and the small molecule release profiles of the polymers. The polymers were synthesized by treatment of poly(dichlorophosphazene) with an excess of the appropriate amino acid ester. Tetrahydrofuran solutions of each polymer were then thoroughly mixed with ethacrynic acid, a diuretic, or Biebrich Scarlet, an azo dye. Films cast from these solutions were immersed in aqueous media (pH 7) at 25 degrees C and at 37 degrees C for approximately 1400 h. During these experiments, the release of the small molecules was monitored by UV/visible spectroscopy. The molecular weight decline and the mass loss of the polyphosphazene films were measured.

Activity of urea amidohydrolase immobilized within poly[di(methoxyethoxyethoxy)phosphazene] hydrogels.

Urea amidohydrolase (urease) was immobilized within poly[di(methoxyethoxyethoxy)phosphazene] (MEEP) hydrogels. This was accomplished by mixing an aqueous solution (pH 7) of the soluble polymer with the enzyme. Films of the conjugate were cast and the solvent removed to yield an MEEP/enzyme composite. The conjugate films were dried in a vacuum and were then cross-linked by exposure to 0.2 or 0.5 Mrad of 60Co gamma-radiation to give an MEEP network with the enzyme entrapped within its matrix. The cross-linked films were sectioned into strips and were washed with pH 7 buffer to remove enzyme adhering to the surface. The films were then allowed to swell to form a hydrogel in pH 7 buffer to which was added a 1.0 M aqueous urea solution. The increase in pH from the conversion of urea to ammonia was monitored over a 24 h period. The immobilized enzyme could be recycled at least five times without significant loss of activity. Several control experiments were also performed by monitoring the pH of buffer solutions that contained hydrogels devoid of entrapped urease, and by monitoring the pH of solutions of the free, non-irradiated and free, irradiated urease after the addition of the urea solution. The polymer-free, irradiated urease lost little to no activity compared with its non-irradiated counterpart. The MEEP gel-immobilized enzyme retained approximately 80% of the activity of the non-irradiated, polymer-free urease.

Use of polyphosphazenes for skeletal tissue regeneration.

The hydrolytically unstable polyphosphazenes, poly [(imidazolyl) (methylphenoxy) phosphazenes] and poly [ethyl glycinato) (methylphenoxy) phosphazenes], were studied as potential polymeric supports for cells in tissue regeneration. For bone repair, their specific function would be to support osteoblast growth, forming a bone-polymer matrix. MC3T3-E1 cells (an osteogenic cell line) were seeded onto polymer matrices and cell adhesion and growth as well as polymer degradation were examined. Both imidazolyl- and ethyl glycinato-substituted polyphosphazenes supported the growth of MC3T3-E1 cells. An increase in the content of the imidazolyl side group resulted in a reduction in cell attachment and growth on the polymer surface and an increase in the rate of degradation of the polymer. In contrast, substitution with the ethyl glycinato group favored increased cell adhesion and growth and also an increase in the rate of degradation of the polymers. Thus, the polyphosphazenes represent a system whereby cell growth and degradation can be modulated by varying the nature of the hydrolytically unstable side chain. This in vitro evaluation suggests that the polyphosphazenes may be suitable candidate biomaterials for the construction of a cell-polymer matrix for tissue regeneration.

Design of synthetic polymeric structures for cell transplantation and tissue engineering.

Two approaches for cell transplantation and new tissue constructions are discussed. In one case, a novel synthetic polyphosphazene has been synthesized that can be gelled by simply adding ions to it at room temperature under aqueous conditions. This polymer has been shown to be compatible for several different cell types. Microcapsular membranes based on the complex of this polymer with poly (L-lysine) allow the inward diffusion of nutrients to nourish the encapsulated cells, but are impermeable to antibodies. In a second approach, biodegradable polyesters have been designed as scaffolds for liver cells and cartilage cells to aid in organ regeneration. Design of the polymer scaffold including the characterization of the surface chemistries for cell attachment, as well as in-vitro and in-vivo data on cell behavior are presented.

Rational design and synthesis of new polymeric material.

Polymers constitute one of the four main areas of materials science. Modern research in polymers focuses on the ways in which tailored macromolecules organize themselves in the solid state and on materials design and synthesis at the interface between polymer science and the fields of ceramics, metals, and electroactive or electrooptical materials.

Antibacterial activity and mutagenicity studies of water-soluble phosphazene high polymers.

Eight water-soluble phosphazene high polymers, [NPR2]n (R, organic, water-solubilizing side-group; n, approx: 15,000) and the small-molecule counterparts of the polymers were examined for antibacterial activity against six different strains of bacteria (Escherichia coli, Salmonella typhimurium (TA 100), Salmonella pullorum, Streptococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa). Antibacterial testing was carried out by measuring zones of inhibition and changes in solution turbidity over time. In addition, the antibacterial activity of the surfaces of cross-linked poly[di(methoxyethoxyethoxy)phosphazene] (MEEP) hydrogels were investigated. A number of the high polymers, as well as the MEEP hydrogels, impeded bacterial growth. Only E. coli was unaffected by the phosphazenes. A possible explanation for the antibacterial character of the polymers is presented. The same compounds were monitored for potential mutagenic activity using the Salmonella typhimurium tester strains TA 100 and TA 98. None of the high polymers or their small-molecule analogues showed mutagenic activity in either strain of Salmonella at the concentrations tested. The use of these materials as coatings for artificial implants is discussed.

A novel synthetic method for hybridoma cell encapsulation.

We report here what we believe is the first example of the encapsulation of hybridoma cells within a synthetic polymer by a simple gelation with dissolved cations in water, and at room temperature. Two lines of hybridoma cells were encapsulated within calcium cross-linked polyphosphazene gel microbeads without affecting their viability or their capability to produce antibodies. Interaction of these gel beads with the positively-charged polyelectrolyte, poly(L-lysine), of 102-kD molecular weight, produced a semipermeable membrane that was capable of retaining the cell-secreted antibodies inside the beads. Cell density increased 3.5-fold within 13 days concomitant with a 6.4-fold increase in antibody production. These synthetic membranes have the potential to aid in protein recovery schemes.

6-Oxo-2,2,4,4,6-pentaphenoxycyclotri-lambda 5-phosphazane-1,3-diene.

C30H26N3O6P3, Mr = 617.48, monoclinic, P2(1)/n, a = 13.200(4), b = 16.714(3), c = 13.438(9) A, beta = 96.38(3) degrees, V = 2946.3 A3, Z = 4, Dx = 1.392 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.23 mm-1, F(000) = 1280, T = 293(1) K, R = 0.037 for 4089 observed data with I greater than 3 sigma (I). The P3N3 ring has a boat conformation with a P atom and an N atom 0.396(1) and 0.118(2) A, respectively, out of the plane of the remaining atoms of the ring. One of the P atoms involves a double bond P = O [1.470(1) A] and a P-N single bond [1.683(1) A]. The P-O single bonds and P-N ring bonds range between 1.564(1) and 1.5941) A [mean 1.576(1) A] and 1.546(1) and 1.607(1) A [mean 1.574(1) A], respectively.

Amphiphilic polyphosphazenes as membrane materials: influence of side group on radiation cross-linking.

The amphiphilic mixed-substituent polyphosphazenes, [NP(OCH2CF3)x (NHCH3)y)]n and [NP(OC6H5)x (NHCH3)y]n, have been prepared by the sequential replacement of chlorine in [NPCI2]n by trifluorethoxide or phenoxide and methylamine. Thin films of these species were cross-linked by exposure to gamma radiation and the semipermeability of the resultant membranes was monitored. The radiation-induced cross-linking and membrane-forming properties of these polymers were compared with those of the single substituent polymers, [NP(OCH2CF3)2]n, [NP(OC6H5)2]n, and [NP(NHCH3)2]n. The radiation-cross-linking and appeared to involve free radical reactions at the methyl groups of the methylamino substituents. The possible utility of these materials in biomedical research is discussed.

Hydrophilic polyphosphazenes as hydrogels: radiation cross-linking and hydrogel characteristics of poly[bis(methoxyethoxyethoxy)phosphazene].

The water-soluble polyphosphazene, [NP(OCH2CH2OCH2CH2OCH3)2]n, cross-links when exposed to gamma rays to form hydrophilic, water-swellable membranes and hydrogels. The degree of cross-linking increases with irradiation dose in a manner that can be utilized to change the properties. gamma irradiation studies of the small molecule model compound, [NP(OCH2CH2OCH2CH2OCH3)2]4, were also carried out to probe the relationship between irradiation dose and cross-link density.