Department of Pathology, Microbiology, and Immunology, University of California, Davis, One Shields Ave., Davis, CA, 95616, USA.
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, 4206 VM3A, Davis, CA, 95616, USA.
Ann Biomed Eng. 2022 Jan;50(1):56-77. doi: 10.1007/s10439-021-02897-7. Epub 2021 Dec 27.
This study in dogs explored the feasibility of using cartilage fragments removed and discarded during routine palliative surgery for osteochondritis dissecans (OCD) as a source of primary chondrocytes for scaffold-free cartilage tissue-engineering. Primary chondrocytes were obtained from three OCD donors and one age-matched healthy articular cartilage (HAC) donor. After monolayer expansion of primary cells, a three-dimensional spherical suspension culture was implemented. Following this stage, cells were seeded at a high density into custom-made agarose molds that allowed for size and shape-specific constructs to be generated via a method of cellular self-assembling in a scaffold-free environment. Fifty-eight neocartilage constructs were tissue-engineered using this methodology. Neocartilage constructs and native cartilage from shoulder joint were subjected to histological, mechanical, and biochemical testing. OCD and HAC chondrocytes-sourced constructs had uniformly flat morphology and histology consistent with cartilage tissue. Constructs sourced from OCD chondrocytes were 1.5-times (32%) stiffer in compression and 1.3 times (23%) stronger in tension than constructs sourced from HAC chondrocytes and only 8.7-times (81%) less stiff in tension than native tissue. Constructs from both cell sources consistently had lower collagen content than native tissue (22.9%/dry weight [DW] for OCD and 4.1%/DW for HAC vs. 51.1%/DW native tissue). To improve the collagen content and mechanical properties of neocartilage, biological and mechanical stimuli, and thyroid hormone (tri-iodothyronine) were applied to the chondrocytes during the self-assembling stage in two separate studies. A 2.6-fold (62%) increase in compressive stiffness was detected with supplementation of biological stimuli alone and 5-fold (81%) increase with combined biological and mechanical stimuli at 20% strain. Application of thyroid hormone improved collagen content (1.7-times, 33%), tensile strength (1.8-times, 43%), and stiffness (1.3-times, 21%) of constructs, relative to untreated controls. Collectively, these data suggest that OCD chondrocytes can serve as a reliable cell source for cartilage tissue-engineering and that canine chondrocytes respond favorably to biological and mechanical stimuli that have been shown effective in chondrocytes from other animal species, including humans.
本研究在狗中探索了使用在常规姑息性手术中切除和丢弃的软骨碎片作为用于无支架软骨组织工程的原代软骨细胞的来源的可行性。从三个骨软骨病 (OCD) 供体和一个年龄匹配的健康关节软骨 (HAC) 供体中获得原代软骨细胞。在原代细胞单层扩增后，进行三维球形悬浮培养。在这个阶段之后，细胞以高浓度接种到定制的琼脂糖模具中，通过在无支架环境中细胞自组装的方法允许生成具有特定大小和形状的构建体。使用这种方法组织工程了 58 个新软骨构建体。新软骨构建体和肩关节的天然软骨进行了组织学、力学和生物化学测试。OCD 和 HAC 软骨细胞来源的构建体具有均匀平坦的形态和组织学，与软骨组织一致。源自 OCD 软骨细胞的构建体在压缩时硬度提高 1.5 倍（32%），在拉伸时强度提高 1.3 倍（23%），而与天然组织相比仅在拉伸时降低了 8.7 倍（81%）。源自两种细胞源的构建体的胶原蛋白含量始终低于天然组织（OCD 为 22.9%/干重 [DW]，HAC 为 4.1%/DW，而天然组织为 51.1%/DW）。为了提高新软骨的胶原蛋白含量和力学性能，在两个单独的研究中，在自组装阶段向软骨细胞施加生物和力学刺激以及甲状腺激素（三碘甲状腺原氨酸）。单独补充生物刺激可检测到压缩刚度提高 2.6 倍（62%），在 20%应变下联合生物和力学刺激可提高 5 倍（81%）。应用甲状腺激素可提高构建体的胶原蛋白含量（提高 1.7 倍，33%）、拉伸强度（提高 1.8 倍，43%）和刚度（提高 1.3 倍，21%），与未处理的对照相比。总的来说，这些数据表明，OCD 软骨细胞可以作为软骨组织工程的可靠细胞来源，并且犬软骨细胞对已证明对包括人类在内的其他动物物种的软骨细胞有效的生物和力学刺激反应良好。
