新型软骨脱细胞基质海绵的制备及生物相容性评价

PREPARATION AND BIOCOMPATIBILITY EVALUATION OF NOVEL CARTILAGE ACELLULAR MATRIX SPONGE

目的探讨利用猪耳软骨脱细胞基质制备海绵状多孔支架材料的方法,及其用作组织工程关节软骨支架材料的可行性。方法取新鲜猪耳软骨低温粉碎后,筛取粒径90μm以下软骨微粒经低温脱细胞处理,冻干,用1.5%乙酸分散制备质量体积比为2%的胶状悬液,低温冻干成型。行理化性能检测测定其孔径、孔隙率及吸水率,并行组织学及扫描电镜观察。24只SD大鼠脊柱两侧皮下分别植入制备的软骨脱细胞基质海绵(实验组)和ColⅠ海绵(对照组);于术后1、2、4、8周取材行组织学观察,分析软骨脱细胞基质海绵在体内的吸收降解情况。取1周龄新西兰大白兔股骨骨髓,培养获取第3代BMSCs,分别采用浓度为50%(A组)及100%(B组)的软骨脱细胞基质海绵浸提液及DMEM培养液(C组)进行培养,于培养后2、4、6dMTT法测定细胞增殖。结果制备的软骨脱细胞基质海绵呈白色多孔状,组织学观察示材料中无软骨细胞碎片残留,主要由胶原构成。扫描电镜显示材料呈多孔蜂巢状,孔洞相互连接,孔径均匀。吸水率为2029%±253%,孔径为(90.66±21.26)μm,孔隙率为90.10%±2.42%。支架材料植入SD大鼠皮下后可见组织长入材料,血管生成,炎性反应较对照组轻,材料可按一定速率降解吸收。MTT法检测结果示,浸提液培养2、4d,3组间吸光度(A)值差异无统计学意义(P>0.05);6d,3组间比较差异均有统计学意义(P<0.05)。结论将软骨脱细胞基质进行改性处理制备的海绵支架材料脱细胞彻底,保留了软骨ECM主要成分,无毒,具备合适的孔径和孔隙率,孔隙分布均匀,生物相容性好,可作为软骨组织工程的支架材料。

Objective To explore the method of preparing spongy and porous scaffold materials with swine articular cartilage acellular matrix and to investigate its applicability for tissue engineered articular cartilage scaffold. Methods Fresh swine articular cartilage was freeze-dried and freeze-ground into microparticles. The microparticles with diameter of less than 90 μm were sieved and treated sequentially with TNE, pepsin and hypotonic solution for decellularization at cryogenic temperatures. Colloidal suspension with a mass/volume ratio of 2% was prepared by dissolving the microparticles into 1.5% HAc, and then was 1yophilized for molding and cross-linked by UV radiation to prepare the decellularized cartilage matrix sponge. Physicochemical property detection was performed to identify aperture, porosity and water absorption rate. Histology and scanning electron microscope observations were conducted. The prepared acellular cartilage matrix sponge was implanted into the bilateral area of spine in 24 SD rats subcutaneously （experimental group）, and the implantation of Col Ⅰ sponge served as control group. The rats were killed 1, 2, 4, and 8 weeks after operation to receive histology observation, and the absorption and degeneration conditions of the sponge in vivo were analyzed. BMSCs obtained from femoral marrow of 1-week-old New Zealand white rabbits were cultured. The cells at passage 3 were cultured with acellular cartilage matrix sponge lixivium at 50% （group A）, acellular cartilage matrix sponge lixivium at 100% （group B）, and DMEM culture medium （group C）, respectively. Cell proliferation was detected by MTT method 2, 4, and 6 days after culture. Results The prepared acellular cartilage matrix sponge was white and porous. Histology observation suggested that the sponge scaffold consisted primarily of collagen without chondrocyte fragments. Scanning electron microscope demonstrated that the scaffold had porous and honeycomb-shaped structure, the pores were interconnected and even in size. The water absorption rate was 20.29% ± 25.30%, the aperture was （90.66 ± 21.26） μm, and the porosity of the scaffold was 90.10% ± 2.42%. The tissue grew into the scaffold after the subcutaneous implantation of scaffold into the SD rats, angiogenesis was observed, inflammatory reaction was mild compared with the control group, and the scaffold was degraded and absorbed at a certain rate. MTT detection suggested that there were no significant differences among three groups in terms of absorbance （A） value 2 and 4 days after culturing with the lixivium （P 〉 0.05）, but significant differences were evident among three groups 6 days after culturing with the lixivium （P 〈 0.05）. Conclusion With modified treatment and processing, the cartilage acellular matrix sponge scaffold reserves the main components of cartilage extracellular matrix after thorough decellularization, has appropriate aperture and porosity, and provides even distribution of pores and good biocompatibility without cytotoxicity. It can be used as an ideal scaffold for cartilage tissue engineering.