Preparation And Biological Characterization Of Multifunctional Polyphosphazenes For Bone Regeneration

Polyorganphosphazenes are hybrid inorganic—organic polymers composed of a backbone containing alternative phosphorus and hitrogen atoms with two organic side groups linked to each phosphorus atom.Various properties including adjustable hydrophilicity,biocompatibility,biodegradability and machinability can be obtained due to the high flexibility in molecular design of polyphosphazene chemistry.More importantly,they are osteoactive benefiting from their phosphorus-containing chemical structure,which highlights interests in bone tissue engineering.However,the lack in mechanic properity and stability limited their application in these areas.To avoid these drawbacks,the ease of modification in polyphosphazene chemisty provide an alternative way to design materials with multifunction,and to combine with other biomaterials.In this paper,three kinds of functional polyphosphazene-based biomaterials,including fluorescent and biodegradable polyphosphazene 3D scaffold for long-term bio-imaging,photoluminescent polyphosphazene nanoparticles for in situ simvastatin delivery,injectable mineralized polyphosphazene/gelatin hydrogel for bone tissue engineering,and the correlation between the polymeric features of amino-acid substituted polyphosphaznene with their osteogenic activity,has been studied.The importance of developing photoluminescent biodegradable scaffolding materials for tissue engineering is obvious,but it meets challenges with conventional biodegradable polymers such as aliphatic polyesters.Thus,photoluminescent biodegradable polyphosphazenes(PTA)were suggested as alternatives to target for long-term in vivo tracking applications.The PTA polymers were synthesized via nucleophilic cosubstitution of linear poly(dichlorophosphazene)with a fluorescent compound(TPCA)and alanine ethyl ester.The TPCA,with high fluorescent intensity and high quantum yield(?0.5),was synthesized from citric acid and 2-aminoethanethiol.The resulted PTA polymers demonstrated adjustable degradation rates and fluorescent intensities in relating to their chemical compositions.In comparison with TPCA,the photostability of PTA polymers has been significantly improved,which made the long-term in vivo tracking feasible.PTA polymers were proven biocompatible and osteogeinc active for biomedical applications via both in vitro cell culture and in vivo implantation evaluations,and were envisioned as good choices for bone tissue regeneration as scaffolding materials with in situ bioimaging potentials.Nanomedicines have found promising applications in regulating biological behaviors of cells because of the cell endocytosis effect.To enhance osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs),which is one of the key issues in relation to bone regeneration,biodegradable simvastatin-bearing polyphosphazene prodrug was synthesized and made into nanoparticles(NPs).At the same time,photoluminescent tryptophan ethyl ester and hydrolyzable glycine ethyl ester were introduced as co-substituted side groups onto the polyphosphazene backbone.The resulting polymer,poly(simvastatin-co-ethyl tiyptophanato-co-ethyl glycinato)phosphazene(PTGP-SIM),displayed expected features of photoluminescence,degradability and sustained SIM release.Endocytosis of PTGP-SIM NPs by BMSCs and the location of internalized NPs,were visualized via the inherent photoluminescent feature of PTGP-SIM.Thus,simvastatin was released inside the cells directly along with the polymer degradation and able to play its role in promoting osteogenic differentiation efficiently at quite low local concentration.From the results,the present study suggested a very promising biomaterial to be used as flexible and functional carrier for bioactive components.Hydrogels are promising candidates for tissue engineering because of their less surgery trauma,easily forming and processing ability,and similar structure comparing with the cellular matrix.Developing complex hydrogel composed of polyphosphazene and natural polymer could effectively make up the lack in mechanic property of polyphosphazenes and to enhance the biological properties.Thus,flexible and injectable polyphosphazene hydrogel was synthesized by introducing citronellol and 2-(2-methyloxyethoxy)ethanol as co-substituted side groups onto the polyphosphazene backbone.The mechanic property and cell adhesion could be significantly improved by the addition of gelatin.However,with the increase of the gelatin contant,the integrity of the hydrogel after the injection were decreased,accompany with the change of the cross-linking density,degradation behavior and swelling ratio.The optimized addition ratio was 10 wt%.Calcium phosphate was also introduced into the hydrogel structure via in situ precipitation to further improve the osteogenic activity of hydrogel.Mineralized polyphosphazene/gelatin hydrogel was proven good substrate for the cell adhesion and proliferation via both in vitro cell culture and in vivo implantation evaluations.Amino acid ester substituted polyphosphazenes are biocompatible and biodegradable,more importantly;they are osteoactive benefiting from their phosphorus-containing chemical structure,which highlights interests in bone tissue engineering.To correlate the polymeric features with cell biological behaviors,the present study synthesized two amino acid ester substituted polyphosphazenes with different degradation rates to carry out cell studies including proliferation and osteogenic differentiation.The two polymers were poly[(ethyl alanato)0.3(ethyl glycinato)0.7phosphazene](PAGP)and poly[(ethyl phenylalanato)0.3(ethyl glycinato)0.7phosphazene](PPGP),and PAGP degraded significantly faster than PPGP.Bone mesenchymal stromal cells(BMSCs)were cultured in two manners,i.e.,non-contact manner and contact manner.In the non-contact culture manner,cells were cultured in transwell chambers containing PAGP or PPGP films,while the cells and the materials did not contact.In the contact culture manner,cells were cultured directly on PAGP or PPGP films.Thus,the possible stimulation effects from polymer surface and degradation products could be identified separately in relation to polymer degradation.To look into the effects of degradation products from polyphosphazenes on cell behaviors,solutions containing known concentrations of PAGP or PPGP degradation products were applied for cell culture using inorganic phosphate(Pi)ion as control.The results revealed that the high osteoactivity of amino acid ester substituted polyphosphazenes was ascribed to both their phosphorus-rich surface and their degradation products including phosphate,ammonium and corresponding amino acids.In comparison with PAGP,however,PPGP seemed more suitable for bone tissue engineering because its stronger synergistic stimulation effects on osteogenic differentiation came from both the cell-scaffold interaction and the degradation products,which was suggested owing to its slower degradation rate.To sum up,due to the high flexibility in molecular design of polyphosphazene chemistry,polyphosphazenes with various properties and function can be easily obtained.These functional polyphosphazenes,which owes good osteocompatibility and design freedom,can find promising applications in bone tissue engineering as scaffolds and drug delivery carriers.