两种交联剂对β-磷酸三钙明胶复合骨支架理化及生物学性能影响的比较

Influence of two different crosslinkers on physiochemical properties and bioactivity of a composite bone scaffold composed of beta-tricalcium phosphate and gelatin

背景:不同交联方法会改变支架的理化性能与生物学性能。目的:分别以京尼平、戊二醛作为交联剂制备β-磷酸三钙/明胶复合骨支架,比较支架的理化及生物学性能差异。方法:分别以京尼平(交联72 h)、戊二醛(交联24 h)作为交联剂,通过相分离/冷冻干燥技术制备β-磷酸三钙/明胶复合骨支架,检测两组支架的孔隙率、交联度、抗压强度、体外溶胀度及降解率。(1)体外细胞毒性实验:分别以浓度为25%、50%、100%的两组支架浸提液培养兔骨膜成骨细胞24,48,72 h,检测细胞增殖率,评定细胞毒性分级;(2)体内修复实验:在18只兔颅骨两侧制作直径8 mm的骨缺损模型,两侧分别分别植入京尼平与戊二醛交联的β-磷酸三钙/明胶复合骨支架,植入后4,8,12周进行缺损处大体、X射线及组织学观察。结果与结论:(1)两组支架孔隙率、抗压强度与最大压缩力比较差异无显著性意义;京尼平交联组支架交联度高于戊二醛交联组(P<0.05),体外溶胀度及降解率低于戊二醛交联组(P<0.05);(2)京尼平交联支架浸提液仅100%浓度组培养24 h时细胞生长抑制不超过总数的50%,毒性为2级,其余为1或0级;戊二醛交联支架浸提液100%浓度组24 h细胞生长抑制超过总数50%,毒性为3级,25%、50%浓度组培养24 h及100%浓度组培养48 h的细胞毒性为2级,其余为1级;(3)X射线及组织学观察显示,随时间植入时间的延长,两组新骨组织从周围长入,支架材料呈向心性降解,京尼平交联组植入8,12周的新骨形成率高于戊二醛交联组(P<0.05);(4)结果表明,采用京尼平、戊二醛交联制备的β-磷酸三钙/明胶复合骨支架理化性能接近,京尼平交联需要的时间较长,但制备的支架生物活性更优。关键词:

BACKGROUND:The physiochemical properties and bioactivity of composite scaffolds can be altered by different crosslinkers.OBJECTIVE:To compare the physiochemical properties and bioactivity of composite scaffolds composed ofβ-tricalcium phosphate and gelatin,which are crosslinked by 1%genipin or gluteraldehyde,respectively.METHODS:Porous scaffolds composed ofβ-tricalcium phosphate and gelatin were made by phase separation/freeze-drying technique.Crosslinking time was 72 hours when genipin acted as a crosslinker and 24 hours when glutaraldehyde as a crosslinker.Physiochemical properties including porosity,degree of cross-linking,in vitro swelling ratio,degradation rate and compressive strength were detected.Bioactivities analyses were performed through co-culturing rabbit periosteal osteoblasts with 25%,50%and 100%scaffold extracts for 24,48,72 hours.The proliferation rate and cytotoxicity gradation were evaluated.In addition,bilateral 8-mm skull defects were made in 18 rabbits and repaired with scaffolds crosslinked by genipin or gluteraldehyde,respectively.Gross observation,X-ray analysis and histological observation were performed at 4,8 and 12 postoperative weeks.RESULTS AND CONCLUSION:(1)The porosity,compressive strength and maximum compressive force showed no statistical difference between the two crosslinker groups.Compared with the gluteraldehyde group,higher degree of crosslinking and lower swelling ratio and degradation rate were observed in the genipin group(P<0.05).(2)In the genipin group,less than 50%growth inhibition was observed when co-cultured with 100%scaffold extract for 24 hours.Thus,the cytotoxicity was graded as 2,and the remains were graded as 1 or 0.In the gluteraldehyde group,excessive 50%growth inhibition was observed when co-cultured with 100%scaffold extract for 24 hours,and the cytotoxicity was graded as 3.For 25%and 50%subgroups(culture for 24 hours)and 100%subgroup(culture for 48 hours),the cytotoxicity was graded as 2,and the remains were graded as 1.(3)X-ray and histological observation showed the in-growth of new bone tissues from the periphery of the defect and the scaffold degraded centripetally.New bone formation was better in the genipin group than the gluteraldehyde group at 8 and 12 postoperative weeks(P<0.05).To conclude,both genipin and gluteraldehyde can be used as crosslinkers to prepare the composite bone scaffold composed ofβ-tricalcium phosphate and gelatin.Two scaffolds have similar physicochemical properties;however,the former has a superior bioactivity except for a longer time for crosslinking with genipin.