摘要
目的制备骨软骨一体化多相支架并进行相关力学性能检测与生物学性能评估,为骨软骨缺损修复提供一种新技术与方法。方法以猪源脱细胞软骨细胞外基质、纳米级羟基磷灰石和海藻酸钠为原材料,按不同成分比例混合,利用冷冻干燥技术和物理化学法交联,制备以脱细胞软骨细胞外基质为软骨层,海藻酸钠和脱细胞软骨细胞外基质为中间层,纳米级羟基磷灰石、海藻酸钠和脱细胞软骨细胞外基质为骨层的骨软骨一体化多相支架。通过大体观察、扫描电镜观察、Micro-CT观察、支架孔隙率和孔径测定、支架吸水能力测定、力学检测(压缩模量、界面间黏附强度)、生物相容性检测[MTT法检测鼠L929成纤维细胞在支架上的增殖情况以及绿色荧光蛋白(green fluorescent protein,GFP)标记的大鼠BMSC_s在支架上的生长情况],对骨软骨一体化多相支架进行力学与生物学性能评价。结果大体观察及Micro-CT观察均显示支架各层间结合紧密,未出现明显不连续和相互分离。扫描电镜观察示,骨层结构相对致密,中间层与软骨层结构则比较疏松,各层内的孔隙结构相互连通且均具有立体多维性。支架软骨层、中间层、骨层孔隙率分别为93.55%±2.90%、93.55%±4.10%、50.28%±3.20%,骨层显著低于软骨层和中间层(P<0.05),软骨层与中间层比较差异无统计学意义(P>0.05);孔径分别为(239.66±35.28)、(153.24±19.78)、(82.72±16.94)μm,各层间比较差异均有统计学意义(P<0.05)。支架软骨层、中间层、骨层亲水性分别为(15.14±3.15)、(13.65±2.98)、(5.32±1.87)m L/g,骨层显著低于软骨层和中间层(P<0.05),软骨层与中间层比较差异无统计学意义(P>0.05);压缩模量分别为(51.36±13.25)、(47.93±12.74)、(155.18±19.62)k Pa,骨层显著高于软骨层和中间层(P<0.05),软骨层与中间层比较差异无统计学意义(P>0.05)。骨软骨一体化多相支架每两层间结合紧密,其中软骨层与中间层的界面黏附强度为(18.21±5.16)k Pa,骨层与中间层的界面黏附强度则为(16.73±6.38)k Pa,比较差异无统计学意义(t=0.637,P=0.537)。MTT法检测显示鼠L929成纤维细胞在支架上生长良好,GFP标记的大鼠BMSC_s在支架上生长分布均匀,显示支架材料无细胞毒性且具有良好的生物相容性。结论骨软骨一体化多相支架各层间的性能各有侧重,实现了对正常骨软骨组织成分上与结构上的双重仿生,为进一步动物体内实验奠定了基础,有望成为骨软骨缺损修复与再生治疗的一种新策略。
ObjectiveThe tissue engineered osteochondral integration of multi-layered scaffold was prepared and the related mechanical properties and biological properties were evaluated to provide a new technique and method for the repair and regeneration of osteochondral defect.MethodsAccording to blend of different components and proportion of acellular cartilage extracellular matrix of pig, nano-hydroxyapatite, and alginate, the osteochondral integration of multi-layered scaffold was prepared by using freeze-drying and physical and chemical cross-linking technology. The cartilage layer was consisted of acellular cartilage extracellular matrix; the middle layer was consisted of acellular cartilage extracellular matrix and alginate; and the bone layer was consisted of nano-hydroxyapatite, alginate, and acellular cartilage extracellular matrix. The biological and mechanics characteristic of the osteochondral integration of multi-layered scaffold were evaluated by morphology observation, scanning electron microscope observation, Micro-CT observation, porosity and pore size determination, water absorption capacity determination, mechanical testing （compression modulus and layer adhesive strength）, biocompatibility testing [L929 cell proliferation on scaffold assessed by MTT assay, and growth of green fluorescent protein （GFP）-labeled Sprague Dawley rats’ bone marrow mesenchumal stem cells （BMSCs） on scaffolds].ResultsGross observation and Micro-CT observation showed that the scaffolds were closely integrated with each other without obvious discontinuities and separation. Scanning electron microscope showed that the structure of the bone layer was relatively dense, while the structure of the middle layer and the cartilage layer was relatively loose. The pore structures in the layers were connected to each other and all had the multi-dimensional characteristics. The porosity of cartilage layer, middle layer, and bone layer of the scaffolds were 93.55%±2.90%, 93.55%±4.10%, and 50.28%±3.20%, respectively; the porosity of the bone layer was significantly lower than that of cartilage layer and middle layer （P〈0.05）, but no significant difference was found between cartilage layer and middle layer （P〉0.05）. The pore size of the three layers were （239.66±35.28）, （153.24±19.78）, and （82.72±16.94） μm, respectively, showing significant differences between layers （P〈0.05）. The hydrophilic of the three layers were （15.14±3.15）, （13.65±2.98）, and （5.32±1.87） mL/g, respectively; the hydrophilic of the bone layer was significantly lower than that of cartilage layer and middle layer （P〈0.05）, but no significant difference was found between cartilage layer and middle layer （P〉0.05）. The compression modulus of the three layers were （51.36±13.25）, （47.93±12.74）, and （155.18±19.62） kPa, respectively; and compression modulus of the bone layer was significantly higher than that of cartilage layer and middle layer （P〈0.05）, but no significant difference was found between cartilage layer and middle layer （P〉0.05）. The osteochondral integration of multi-layered scaffold was tightly bonded with each layer. The layer adhesive strength between the cartilage layer and the middle layer was （18.21±5.16） kPa, and the layer adhesive strength between the middle layer and the bone layer was （16.73±6.38） kPa, showing no significant difference （t=0.637, P=0.537）. MTT assay showed that L929 cells grew well on the scaffolds, indicating no scaffold cytotoxicity. GFP-labeled rat BMSCs grew evenly on the scaffolds, indicating scaffold has excellent biocompatibility.ConclusionThe advantages of three layers which have different performance of the tissue engineered osteochondral integration of multi-layered scaffold is achieved double biomimetics of structure and composition, lays a foundation for further research of animal in vivo experiment, meanwhile, as an advanced and potential strategy for osteochondral defect repair.
作者
李建伟
张学亮
郭全义
张景春
曹艳杰
张新国
黄建军
王琪
刘小刚
郝春香
LI Jianwei1, ZHANG Xueliang1,2, GUOQuanyi2, ZHANG Jingchun1, CAO Yanjie1, ZHANG Xinguo1, HUANG Jianjun1, WANG Qi1, LIU Xiaogang1, HAO Chunxiang2(1. Shanxi Institute of Traditional Chinese Medicine, Taiyuan Shanxi, 030012, P.R.China 2. Institute of Orthopaedics, Beijing Key Laboratory of Regenerative Medicine in Orthopaedics, Key Laboratory of Musculoskeletal Trauma ＆ War Injuries, Chinese PLA General Hospital, Beijing, 100853, P.R.Chin)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2018年第4期434-440,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家重点研发计划(2017YFC1104102)
国家自然科学基金资助项目(81772319)~~
关键词
骨软骨
组织工程
多相支架
细胞外基质
力学性能
生物相容性
Osteochondral
tissue engineering
multi-layered scaffold
extracellular matrix
mechanical property
biocompatibflity
作者简介
通信作者：郝春香，Email：haochunxiang@163．com
