Liao Elly, Yaszemski Michael, Krebsbach Paul, Hollister Scott
Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA.
Tissue Eng. 2007 Mar;13(3):537-50. doi: 10.1089/ten.2006.0117.
Our approach to cartilage tissue-engineering scaffolds combines image-based design and solid free-form (SFF) fabrication to create load-bearing constructs with user-defined parameters. In this study, 3-dimensional scaffolds with cubic and ellipsoidal pore architecture were fabricated using poly(propylene fumarate) (PPF). To increase seeding efficiency and cellular retention, hydrogels were used to deliver cells into the scaffolds. The first objective of this study was to evaluate the concentrations of composite hyaluronic acid (HyA) and collagen I hydrogels best able to stimulate proteoglycan synthesis in porcine chondrocytes in vitro and in vivo. The second objective was to evaluate the differences in extracellular matrix production due to pore geometry and scaffold design. For the in vitro assessment, chondrocytes were encapsulated in collagen I hydrogels with varying concentrations of HyA. Hydrogels were cultured for 1 and 2 weeks, and then the sulfated glycosaminoglycan (sGAG) content was quantified using a dimethyl-methylene blue assay. The concentration of HyA best able to increase ECM synthesis was 5% HyA/collagen I, or 0.23 mg/mL HyA. The results from the in vitro experiment were used as culture parameters for the in vivo analysis. Composite 5% HyA/collagen I or collagen I-only hydrogels were used to seed chondrocytes into SFF-fabricated scaffolds made of PPF with designed cubic or ellipsoidal pore geometry. The scaffolds were implanted subcutaneously in immunocompromised mice for 4 weeks. Histomorphometric analyses of sections stained with Safranin O were used to quantify the amount of ECM deposited by cells in the scaffolds. Scaffolds seeded with 5% HyA/collagen hydrogels had significantly greater areas of positive Safranin O staining (approximately 60%, compared with 30% for scaffolds with collagen I hydrogels only), indicating that greater numbers of chondrocytes retained their metabolic activity in the ectopic environment. These scaffolds also had greater stain intensities (corresponding to greater amounts of sGAG in the ECM) than their counterparts seeded with collagen I hydrogels alone. Significant differences in matrix production were not found between the scaffold pore designs. Overall, these results indicate that a combination of composite HyA hydrogels and designed SFF scaffolds could provide a functional tissue-engineered construct for cartilage repair with enhanced tissue regeneration in a load-bearing scaffold.
我们用于软骨组织工程支架的方法结合了基于图像的设计和实体自由成型（SFF）制造技术，以创建具有用户定义参数的承重结构。在本研究中，使用聚富马酸丙二醇酯（PPF）制造了具有立方和椭球形孔结构的三维支架。为了提高接种效率和细胞保留率，使用水凝胶将细胞递送至支架中。本研究的首要目标是评估复合透明质酸（HyA）和I型胶原水凝胶的浓度，其最能在体外和体内刺激猪软骨细胞中的蛋白聚糖合成。第二个目标是评估由于孔几何形状和支架设计导致的细胞外基质产生的差异。对于体外评估，将软骨细胞封装在具有不同HyA浓度的I型胶原水凝胶中。水凝胶培养1周和2周，然后使用二甲基亚甲基蓝测定法定量硫酸化糖胺聚糖（sGAG）含量。最能增加细胞外基质合成的HyA浓度为5%HyA/I型胶原，即0.23mg/mL HyA。体外实验的结果用作体内分析的培养参数。使用复合5%HyA/I型胶原或仅I型胶原水凝胶将软骨细胞接种到由PPF制成的具有设计好的立方或椭球形孔几何形状的SFF制造的支架中。将支架皮下植入免疫受损小鼠体内4周。用番红O染色的切片的组织形态计量分析用于量化支架中细胞沉积的细胞外基质的量。接种5%HyA/胶原水凝胶的支架具有明显更大的番红O阳性染色面积（约60%，相比之下仅接种I型胶原水凝胶的支架为30%），表明更多的软骨细胞在异位环境中保持其代谢活性。这些支架也比单独接种I型胶原水凝胶的对应物具有更高的染色强度（对应于细胞外基质中更多的sGAG）。在支架孔设计之间未发现基质产生的显著差异。总体而言，这些结果表明，复合HyA水凝胶和设计的SFF支架的组合可为软骨修复提供功能性组织工程构建体，在承重支架中增强组织再生。
