The Study Of Preparation And Biocompatibility Of Bioactive PDLLA Modified By Bone Repair Factors

Bone fractures or defects resulted from various reasons such as natural disasters,traffic accidents, occupational injury, sport, bone tumor incision, congenital bonediseases or metabolic osteoporosis have become one of the most popular diseases. It isestimated that the world-wide medical cost for bone fractures or defects has reachedabout$17billion per year, and the clinical treatment of bone fractures or defects inChina is about30million person-time per year. Therefore, it is highly important andurgent to design and prepare a bone repair/replacement biomaterial with clinicaleffectiveness. In this study, a series of polylactic acid (PLA) based bioactive materialswere design and prepared by using adhesive molecules RGDS/collagen, stressshielding-prevention factor MGF-Ct24E and anti-coagulation factorglycerophosphorylcholine (GPC) as model bioactive factors and PLA as an initialpolymer. The platforms for covalent incorporation of these bioactive factors have beenestablished, and the effects of the concentration of various bioactive factors in thepolymers on osteoblasts were evaluated and an optimal concentration was proposed,aiming to provide a theoretical and technological support for design and preparation ofclinically effective bone implants integrated with multiple bioactive functions and toprepare a specific biomaterial which combines the merits to promote specific celladhesion and prevent stress shielding and blood coagulation. The main works andconclusions are listed as follows.1. Preparation of the reactive PLAs①Preparation of type I and Ⅱreactive PLAvia grafting method1) Preparation of type I reactive PLA from maleic anhydride-grafted PLA (MPLA):Maleic anhydride was grafted onto poly(D,L-lactic acid)(PDLLA)(100kDa,polydispersity index (PDI)<=1.3) by using free radical reactions. FTIR and13C NMRwere used to qualitatively characterize MPLA. The results indicated that MAH has beensuccessful grafted onto PDLLA. When the Mw of PDLLA is100kDa, three MPLApolymers with a MAH content of1.53%,2.45%and3.04%were prepared.2) Preparation of type Ⅱ reactive PLA from butanediamine-grafted MPLA: TheMPLAs with MAH content of1.53%,2.45%and3.04%were used to react withbutanediamine (BDA) through the reaction of anhydride with-NH2, giving the desiredtype Ⅱ reactive PLA (DPLA). FTIR and13C NMR were used to qualitatively characterize DPLA.Ninhydrin test results showed that the content of BDA in DPLAswere1.37%,2.20%and2.59%, respectively.②Preparation of type Ⅲ reactive PLAvia copolymerization methodIn order to increase the reactive sites and thus introduce more bioactive factors, atype Ⅲ reactive PLA was designed and prepared through the copolymerization ofPDLLA oligomer with methacrylic anhydride (MAA) and itaconic anhydride (ITA).Firstly, PDLLA oligomer (=5000) was synthesized, and then reacted with MAA toproduce PDLLA-MAA. Secondly, PDLLA-MAA further reacted with ITA via meltingfree radical copolymerization using benzoyl peroxide (BPO) as an initiator, resulting inITA-modified PLA (PITLA).Finally, PITLA reacted with aliphatic diamine to prepare the type Ⅲ reactive PLA--DPITLA. FTIR,1HNMR showed that DPITLA was prepared successfully. Ninhydrintest results showed that the content of HMD in DPITLA were4.23%,5.79%.2. Preparation of a series of functionalized PLAs①Incorporation of adhesive molecules RGDS/collagenCell adhesive molecules RGDS or collagen was incorporated into DPLA via thereaction between–NH2in RGDS or collagen and–COOH in DPLA withdicyclohexylcarbodiimide (DCC) as a condensation agent, producing the desiredbioactive PLA with enhanced cell adhesive activity (RGDS-PLA or COL-PLA). Thecontent of RGDS or collagen in the polymers could be controlled by regulating theirconcentrations in the raw materials. The results of amino acid analysis (AAA)demonstrated that the conversion of RGDS for low RGDS concentration had a higherconversion40%-60%while for high RGDS concentration the conversion was10%-30%.The conversion of collagen followed the similar trend although it was obviously lowerthan that of RGDS.②Incorporation of MGF-Ct24EMGF-Ct24E is a kind of peptide consisting of24amino acids. It was incorporatedinto DPLA by using the similar technology for RGDS peptide. The obtained polymerMGF-PLA was qualitatively characterized by means of FTIR and1H NMR. The resultsindicated that MGF-Ct24E has been successfully incorporated into DPLA. The AAAresults indicated that the content of MGF-Ct24E in MGF-PLA is0.31umol/g、0.55umol/g and0.83umol/g.③Incorporation of glycerophosphorylcholineGlycerophosphorylcholine (GPC) was incorporated into MPLA through the direct reaction of–OH in GPC with the anhydride groups in MPLA. The results of FTIR and1H NMR showed that GPC has been successfully introduced into DPLA. XPS detectionshowed that the atom percentage of GPC-PLA.3. Structures and performance study on functionalized PLAs①Chemical structures: Various active factors, especially various branchedstructures were introduced by the means of grafting in side chains to provide differentchemical functional groups on the surface of polylactic acid, which provided diverseparent metals for subsequent cell compatible experiments.②Topological structures: Various polylactic acid surfaces of distinct topologicalstructure were obtained based on: first, different active factors possess differentmolecular structures and weights, second different active factors form differentrotational structure when dissolve in different solvents.③Hydrophilicity and hydrophobicity: The results of hydrophilicity andhydrophobicity show that the hydrophilicity of functional polylactic acid increased afterthe introduction of different active factors, and the more molecular weight with higherhydrophilicity; with increased active factor concentration, the hydrophilicity offunctionalized polylactic acid improved.4. Cytocompatibility evaluation of functionalized PLAsThe cell morphology, adhesion and spreading, proliferation, differentiation andmineralization of osteoblasts on various bioactive polymers and the blood compatibilitywere were mainly investigated. The results revealed that:①Low concentration of bioactive factor might improve the cytocompatibility.②Compared to low concentration of bioactive factor, high concentration ofbioactive factors was good for proliferation but not good for cell adhesion.③With the increasing content of MGF-Ct24E in MGF-PLA, the cell proliferationwas enhanced. Incorporation of MGF-Ct24E significantly improved the differentiationand mineralization of osteoblasts, however, they were delayed compared to RGDS. Thecells show same performance after the introduction of MGF-Ct24E or stress loading,which provides basis for the preparation of anti-stress shieding bone repair materials.④Incorporation of glycerophosphorylcholine to MPLA decreased the adsorptionof fibronectin and blood platelet on GPC-PLA. Glycerophosphorylcholine has similarchemical structure to cell membrane so that it is good for the natural conformation ofproteins, and thus promote the adhesion and growth of osteoblasts. This study providesopportunity for the application of polylactic acid for bone wound repair.