*Taken from; The Orthopaedic Surgury Online Information Centre, on February 11, 2009 Polyphosphazines in Bone Regeneration * Shayne Rybchinski University of Lethbridge February 26, 2009

Polyphosphazine Formation is a Thermal Ring-Opening Process: Prepared by a ring opening of hexachlorocyclotriphosphazene at 250°C 250°C Inert atm. 4-5 hours

Polyphosphazines have a wide range of applications due to facile addition of varying side chains through simple nucleophilic substitution: Macromolecular Substitution of Polyphosphazines: Nu :

Allograft: Risk of disease transmission Immunogenic rejection Autograft: Morbidity at sight of graph (tissue damage) C 10 (PO 4 ) 6 (OH) 2 Polyphoshpazine / Hydroxyapatite Complexes in Bone Regeneration: (Ca 4 (PO 4 ) 2 O + CaHPO 4 2H 2 O Xenograft: Risk of disease transmission Immunogenic rejection Synthetic Polymer Graft:

Low temperature formation of hydroxyapatite- poly(alkyl oxybenzoate) phosphazene composites for biomedical applications Greisha, Y. E., Benderb, J. D., Lakshmic, S., Browna, P. W., Allcock H. R., and Laurencin, C. T. ; Biomaterials 26 (2005) 1–9 Formation of hydroxyapatite - polyphosphazine polymer composites at physiologic temperature Greish, Y. E., Bender, J. D., Lakshmi, S., Brown, P. W., Allcock, H. R., and Laurencin, C. T.; Journal of Biomedical Materials Research, Part A (2006), 77A(2),

2(Ca 4 (PO 4 ) 2 O + 2CaHPO 4 2H 2 OC 10 (PO 4 ) 6 (OH) 2 + 4H2O → Tetracalcium phosphate + Dicalcium phosphate dihydrate Stoichiometric Hydroxyapatite Experimental Objective: Poly[bis(carboxylatophenoxy] phosphazene (acid- PCPP) Poly (ethyl-oxybenzoate) phosphazine-(PN-EOB) Poly (propyl-oxybenzoate) phosphazine-(PN-POB) R=

Experimental Method; Polymer formation: Poly(dichloro)phosphazine was prepared by ring opening polymerization Each oxybenzoate-substituted polyphosphazine was prepared from a mixture of Poly(dichloro)phosphazine and it’s respective sodium salt in THF Acid- PCPP was made by treating PN-POB with t-butly alkoxide in THF followed by acidic workup Structure verified with 1 H NMR (360 MHz) and 31 P NMR (145 MHz) Mw determined through gel-permeation chromotography

δH 1.29Methyl 4.21Methylene 6.71Benzoic 7.47Benzoic Structural Determination δH 0.92Methyl 1.67Methylene 4.10Methylene 6.63Benzoic 7.44Benzoic δH 6.64Benzoic 7.43Benzoic 12.62COOH Structure verified with 1 H NMR (360 MHz) and 31 P NMR (145 MHz) 1 H NMR acid-PCPP 1 H NMR PN-EOB 1 H NMR PN-POB

Experimental; Hydroxyapatite formation: HAp formation was studied as a function of time by measuring changes in [Ca 2+ ], [PO 4 - ] and pH in solution in presence of varying polymer concentration (0, 5, 10 and 15 wt %) Kinetics of HAp formation was measured by isothermal calorimetry in the presence of 5, 10 and 15 % (by weight) of each polyphosphazine at 37.5°C Scanning Electron Microscopy photographs of the Polymer- Ceramic complex taken following HAp formation in the presence or absence of polyphosphazine

pH as a function of HAp formation at 37.5°C over 24 Hr: No polymer 5 wt% acid-PCPP 15 wt% acid-PCPP

[Ca 2+ ] and [PO 4 - ] as a function of HAp formation over 24 Hr: Variation in the concentrations of calcium (a) and phosphate (b) ions in solution as a A function of time during Hap formation at 37.4°C with no polymer or 15 wt% PN-EOB and PN-POB

[Ca 2+ ] and [PO 4 - ] as a function of HAp formation over 24 Hr: Variation in [Ca 2+ ] and [PO 4 - ] as a function of time in the presence of 0 and 15 wt. acid-PCPP

X-Ray Diffraction patterns of HAp formation: XRD patterns of HAp formation as a function of time at 37.4°Cin the presence of 15 wt% PN-POB (A) and PN-EOB (B) XRD patterns indicating Hap formation in the presence of 15 wt. % acid-PCPP

Scanning Electron Micrographs of HAp formation: Scanning electron micrographs of samples containing (a) No polymer (b) 15 wt% PN-EOB, and (c) 15 wt% PN-POB prepared at 37.4°C.

Calorimetry results of hydroxyapitite formation at 37.5°C: Growth peak Nucleation peak Mixing peak Heat evolved by HAp formation at 37.5°C in the presence of varying concentration of alkyl-ester substituted polymer

Calorimetry results of hydroxyapitite formation at 37.5°C: Heat evolved by HAp formation at 37.5°C in the presence of varying concentration of carboxylic acid substituted polymer

Conclusions : Hydroxyapatite forms efficiently in the presence or absence of polymer. Although its formation may be somewhat hindered by the presence of polymer. Hap formed in the presence of polymer may not necessarily be nucleated on the polymer Authors postulated that Oxy Benzoate polyphosphazenes hinder reaction kinetics due to slow alkyl ester hydrolysis as is evidenced by reduced heat evolved. Subsequent studies in rabbit systems have indicated that polyphosphazenes may have use as a direct bone regeneration scaffolding without hydroxyapitite component