School of Medicine (Keele campus), Institute for Science & Technology in Medicine, University of Keele, Staffordshire, ST5 5BG, UK.
Eur Cell Mater. 2010 Feb 26;19:86-95. doi: 10.22203/ecm.v019a09.
The injectable and hydrophilic nature of hydrogels makes them suitable candidates for cartilage tissue engineering. To date, a wide range of hydrogels have been proposed for articular cartilage regeneration but few studies have quantitatively compared chondrocyte behaviour and extracellular matrix (ECM) synthesis within the hydrogels. Herein we have examined the nature of ECM synthesis by chondrocytes seeded into four hydrogels formed by either temperature change, self-assembly or chemical cross-linking. Bovine articular cartilage chondrocytes were cultured for 14 days in Extracel, Pluronic F127 blended with Type II collagen, Puramatrix and Matrixhyal. The discriminatory and sensitive technique of fluorophore-assisted carbohydrate electrophoresis (FACE) was used to determine the fine detail of the glycosaminoglycans (GAG); hyaluronan and chondroitin sulphate. FACE analysis for chondroitin sulphate and hyaluronan profiles in Puramatrix closely matched that of native cartilage. For each hydrogel, DNA content, viability and morphology were assessed. Total collagen and total sulphated GAG production were measured and normalised to DNA content. Significant differences were found in total collagen synthesis. By day 14, Extracel and Puramatrix had significantly more total collagen than Matrixhyal (1.77+/-0.26 microg and 1.97+/-0.26 microg vs. 0.60+/-0.26 microg; p<0.05). sGAG synthesis occurred in all hydrogels but a significantly higher amount of sGAG was retained within Extracel at days 7 and 14 (p<0.05). In summary, we have shown that the biochemical and biophysical characteristics of each hydrogel directly or indirectly influenced ECM formation. A detailed understanding of the ECM in the development of engineered constructs is an important step in monitoring the success of cartilage regeneration strategies.
水凝胶的可注射性和亲水性使其成为软骨组织工程的候选材料。迄今为止,已经提出了广泛的水凝胶用于关节软骨再生,但很少有研究定量比较水凝胶内软骨细胞的行为和细胞外基质(ECM)合成。在此,我们研究了通过温度变化、自组装或化学交联形成的四种水凝胶中接种的软骨细胞的 ECM 合成的性质。牛关节软骨细胞在 Extracel、与 II 型胶原混合的 Pluronic F127、Puramatrix 和 Matrixhyal 中培养 14 天。使用荧光辅助碳水化合物电泳(FACE)的鉴别和敏感技术来确定糖胺聚糖(GAG)的详细信息;透明质酸和硫酸软骨素。Puramatrix 中硫酸软骨素和透明质酸图谱的 FACE 分析与天然软骨非常匹配。对于每种水凝胶,评估 DNA 含量、活力和形态。测量总胶原蛋白和总硫酸化 GAG 产生,并标准化到 DNA 含量。在总胶原蛋白合成方面发现了显著差异。到第 14 天,Extracel 和 Puramatrix 的总胶原蛋白含量明显高于 Matrixhyal(1.77+/-0.26μg 和 1.97+/-0.26μg 与 0.60+/-0.26μg;p<0.05)。所有水凝胶中均发生 sGAG 合成,但 Extracel 在第 7 天和第 14 天保留了明显更多的 sGAG(p<0.05)。总之,我们表明,每种水凝胶的生化和生物物理特性直接或间接影响 ECM 的形成。在监测软骨再生策略的成功方面,详细了解工程化构建体中的 ECM 是重要的一步。