| Bone tissue engineering brings new hope for many patients suffering bone diseasesas it will provide a new way to treat trauma, fracture or defect. The design of boneregeneration material is a key factor for the success of bone tissue regeneration. Thematerial should meet with several basic demands such as certain mechanical strength,controllable biodegradation and reasonable surface properties. However, there are fewmaterials suitable for bone tissue engineering. The aim of this study is to design andsynthetic a novel bone tissue engineering material, which can integrate the advantagesof certain mechanical property, controllable degradation, hydrophilicity and feasiblesurface functional modification. Based on a novel PEG derivate-poly （amino terminatedpolyethylene glycol-co-pyromellitic dianhydride） imide （PAPI） copolymerization withD,L-lactide （D,L-LA）, a series of PAPI-PDLLA copolymer with adjustable propertieswere synthesized. The structure of the copolymers was characterized by nuclearmagnetic resonance spectrometer （NMR）, Fourier transform infraredspectrometer（FTIR）, Ultraviolet-visible spectrometer （UV）, Gel permeationchromatography with multi-angle laser light scattering （GPC-MALLS）, Differentialscanning calorimeter （DSC）, Scanning electron microscopy （SEM）, Atomic forcemicroscope （AFM）, and X-ray photoelectron spectrometer（XPS）. Then, thehydrophilicity/hydrophobicity and biodegradation of PAPI-PDLLA copolymers weresystemically examined. And the mechanical property and mechanical property duringdegradation of the copolymer were evaluated by tensile testing and compression testing.Furthermore, amino groups and hydroxyl groups were grafted on the surface ofcopolymers. Finally, the cell biocompatibility of the copolymers was evaluated. Themain works and conclusions of this study were listed as follows:1． A novel PEG derivate PAPI was synthesized by polycondensation ofamino-terminated polyethylene glycol （ATPEG） and PMDA. The reaction conditionwas optimized. An extensive effort was expended in investigating the effects of themolar ratios of monomers, reactive time and temperature on the molecular weight. Theimide process was monitored during the reaction. The introduced imide rings in thepolymer provided reactive sites to graft functional groups. The possibility of PAPIreaction with BDA and EtA was evaluated. The structure of PAPI was characterized. ①FTIR,1H NMR,13C NMR, GPC-MALLS and UV demonstrated that thepolymerization of ATPEG and PMDA was successful. When the molar ratio of ATPEGand PMDA is1.05, the molecular weight of the polymer reached higher and all theamides in the polymer turned imides almost completely after reaction at the set gradienttemperature and time. Thermal gravity （TG） analysis suggested that the thermal stabilityof PAPI improved compared with PEG.②FTIR verified that the imide rings of PAPI were successfully reacted with BDAand EtA without catalysis under room temperature, that means imide rings in PAPI canprovide reactive site for grafting functional groups.③A gel was generated quickly when BDA added into PAPI solution. The gelshowed multi-pore and certain mechanical strength, having promising to apply in drugdelivery and tissue engineering.2. PAPI-PDLLA copolymers were prepared by the melt ring-openingpolymerization of D,L-lactide under the co-initiated system of PAPI and Sn （Oct）2.Effects have been done to understanding the reaction mechanism, the dosage, thetemperature and the reaction time towards the molecular weight. The structure andthermal property of copolymers were analyzed.①FTIR,1H NMR,13C NMR and GPC indicated that PAPI-PDLLA copolymerswere obtained successfully under the co-initiator of PAPI-NH2and Sn （Oct）2. Theoptimization condition of the ring-opening copolymerization was that the reaction wasperformed under vacuum condition at150℃for36h.②A series of PAPI-PDLLA copolymer with different molecular weight wereprepared by varying the ratio of PAPI/D, L-lactide. The molecular weight of the graftedPDLLA in the copolymer was calculated by1HNMR. The PAPI-PDLLA copolymerswith different components and molecular weight have different physicochemicalproperties, which can be adjusted for the best application in bone tissue regeneration.③The data of DSC shown that the PAPI-PDLLA copolymers exhibited only a glasstransition temperature （Tg）, and Tg decreased with the increased of feed ratio ofPAPI/D,L-lactide. The TG curve presented that the copolymers have two decomposedtemperature: The first turning point indicated PDLLA segment decomposed, then, PAPIdecomposed. Therefore, according the TG records, the weight ratio of the two segmentsin the copolymers can be calculated.3. The hydrophilicity/hydrophobility and biodegradation property of PAPI-PDLLAcopolymers were evaluated. Both of the static contact angle and water absorption were used to estimate the hydrophilic/hydrophobic ability of the PAPI-PDLLA copolymers.While in vitro biodegradation of PAPI-PDLLA copolymers was investigated by weightloss ratio, molecular weight changing, pH and the surface morphology variation duringdegradation.①Water absorption of PAPI-PDLLA copolymers was more than PDLLA controlsand the static water contact angle was smaller than PDLLA controls. The reason is thehydrophilic PAPI segment into PAPI-PDLLA could bind more water molecular.Therefore, the hydrophilicity of PAPI-PDLLA copolymers is better than PDLLA.Meanwhile, the hydrophilicity of PAPI-PDLLA copolymers is elevating by theincreasing the feed ratio of PAPI segment in copolymers.②Hydrolytic degradation of PAPI-PDLLA copolymers was lasted for12weeks.The data illustrated that the degradation of PAPI-PDLLA copolymers during the first5weeks was more rapidly than PDLLA controls. However, the weight loss, molecularweight and pH values PDLLA controls degraded significantly after degradation4-6week due to the degraded acid substances accumulation leading to acid catalyzedauto-accelerating degradation. During the whole degradation process, the PAPI-PDLLAcopolymers exhibited more controllable degradation and the degradation speed ofPAPI-PDLLA can be fitted by Mnmolecular model. The reason is the hydrophilicsegment PAPI in copolymers can accelerate the acidic material diffusion during thePDLLA segment degradation without resulting in acid auto-catalyzed degradation. Thesurface morphology of PAPI-PDLLA copolymers also showed more uniformdegradation than PDLLA.4. The mechanical property of the copolymers was tested by tensile andcompression experiments. The results illustrated the materials exhibited certain tensilestrength and compression strength and the strength raised with the feed ratio ofPAPI/D,L-lactide decrease. The tensile modulus of copolymers was adjusted in therange of48.8to280MPa, and the compression modulus in the range of108to780MPa,which is much match with the cancellous bone. During the degradation, the mechanicalproperty of the materials is loss and the speed is rapid with the feed ratio of PAPIincreasing. After degradation for4weeks, the mechanical property of PAPD4/15istotally loss, but PAPI4/25is only loss about its25%.5. The surface of PAPI-PDLLA member was grafted functional groups-aminogroups and hydroxyl groups by wetting chemistry. The grafted amino groups andhydroxyl groups were characterized qualitatively and quantitatively by XPS, colorimetric method, and AFM The surface amino group density of PAPI-PDLLAcopolymer reached3.41×10-6mol/cm2for PAPD4/15-BDA material by colorimetricmethod. The surface morphology of the PAPI-PDLLA copolymer became rough afterreaction with BDA and EtA.6. The cytocompatibility of PAPI precursor and PAPI-PDLLA copolymers wasstudied by empolying primary SD rat osteoblasts as the model cells and PDLLA ascontrol. The osteoblasts morphology, attachment and spreading, proliferation,differentiation and mineralization ability were detected to evaluate the cytocompatibilityof PAPI-PDLLA copolymers. The results are following:①Osteoblasts cultured in the PAPI soaking culture medium shown no apparentdifferences in cell morphologies and cell proliferation compared to black controls. Andosteoblasts cultured in PMA dissolved medium also exhibited normal morphology anddid not affect cell proliferation. The amounts of PMA were based on the maximumamounts that could be released from0.2g of completely degraded polymer. All theresults indicated that the PAPI and PAPI-PDLLA are no cytotoxicity.②Compared to PDLLA, a certain amount of PAPI introduced into PAPI-PDLLAcopolymers could promote osteoblasts adhesion and spreading, but a large of PAPI inthe PAPI-PDLLA such as PAPD4/15was unfavorable for cell adhesion and spreadingbecause the hydrophilicity of the material is too strong leading to detriment of celladhesion. The functional surface of the copolymers is benefit for cell adhesion andspreading. The cell adhesion on amino group functional surface had significantimprovement contrast to other samples.③A certain amount of PAPI introduced into PAPI-PDLLA copolymers （i.e.PAPD4/20） and their functional surface are favor to osteoblasts proliferation.④PAPI-PDLLA copolymers member and the amino and hydroxyl groupsfunctional surfaces stimulated osteoblasts differentiation and mineralization, whichwere reflected by the production of ALP, mineralization and the expression of collagen.And the order of priority of the copolymers for differentiation and mineralization isPAPD-NH2, PAPD-OH, PAPD4/20, and PDLLA. |
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