Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Belgium; Skeletal Biology and Engineering Research Center, KU Leuven, Belgium.
Prometheus, Division of Skeletal Tissue Engineering, KU Leuven, Belgium; Skeletal Biology and Engineering Research Center, KU Leuven, Belgium.
Bone. 2020 Oct;139:115520. doi: 10.1016/j.bone.2020.115520. Epub 2020 Jul 2.
The repair of deep osteochondral joint surface defects represents a significant unmet clinical need. Importantly, untreated lesions lead to a high rate of osteoarthritis. The current strategies to repair these defects include osteochondral autograft transplantation or "sandwich" strategies combining bone autografts with autologous chondrocyte implantation, with poorly documented long-term outcomes. In this study, we first investigated the capacity of juvenile osteochondral grafts (OCGs) to repair osteochondral defects in skeletally mature rats. With this regenerative model in view, we produced a new biological, bilayered and scaffold-free Tissue Engineered construct (bTEC) for the repair of a deep osteochondral defect of the rat knee.
Cylindrical OCGs were excised from the femoral intercondylar groove of the knee of skeletally immature rats (5 weeks) and transplanted into osteochondral defects created in skeletally mature rats (11 weeks). To create bTECs, micromasses (μMasses) of human periosteum-derived progenitor cells (hPDCs) and human articular chondrocytes (hACs) were produced in vitro using previously optimized chemically defined medium formulations containing growth and differentiation factors including bone morphogenetic proteins. These two μMass types were subsequently implanted as bilayered constructs into osteochondral defects in nude rats. At 4 and 16 weeks after surgery, the knees were collected and processed for subsequent 3D imaging analysis and histological evaluation. Micro-computed tomography (μCT), H&E, and Safranin O staining were used to evaluate the degree and quality of tissue repair.
The osteochondral unit of the knee joint in 5 weeks old rats exhibits an immature phenotype, displaying active subchondral bone formation through endochondral ossification and the absence of a tidemark. When transplanted into skeletally mature animals, the immature OCGs resumed their maturation process, i.e., formed new subchondral bone, established the tidemark, and maintained their Safranin O-positive hyaline cartilage at 16 weeks after transplantation. The bTECs (hPDCs + hACs) could partially recapitulate the biology as seen with the immature OCGs, including the formation of the joint surface architecture with typical zonation, ranging from non-mineralized hyaline cartilage in the superficial layers to a progressively mineralized matrix at the interface with a new subchondral bone plate.
Cell-based TE constructs mimicking immature OCGs and displaying a hierarchically organized structure comprising of different tissue forming units seem an attractive strategy to treat deep osteochondral defects of the knee.
修复深层的骨关节面缺损是一项重大的临床未满足需求。重要的是，未经治疗的病变会导致很高的骨关节炎发病率。目前修复这些缺损的策略包括骨软骨自体移植或结合骨自体移植物和自体软骨细胞移植的“三明治”策略，但长期结果记录不佳。在这项研究中，我们首先研究了幼年骨软骨移植物（OCG）修复成熟大鼠的骨关节面缺损的能力。基于这种再生模型，我们为大鼠膝关节深层骨关节缺损的修复，制作了一种新的、生物的、双层的无支架组织工程构建体（bTEC）。
从小鼠膝关节的髁间窝中切取圆柱形 OCG，并移植到成熟的大鼠（11 周龄）的骨关节缺损中。为了制作 bTEC，使用先前优化的含有生长和分化因子（包括骨形态发生蛋白）的化学定义培养基配方，体外培养人骨膜源性祖细胞（hPDC）和人关节软骨细胞（hAC）的微团（μMasses）。这两种μMass 类型随后作为双层构建体植入裸鼠的骨关节缺损中。手术后 4 周和 16 周时，收集膝关节进行后续的 3D 成像分析和组织学评估。微计算机断层扫描（μCT）、H&E 和番红 O 染色用于评估组织修复的程度和质量。
5 周龄大鼠膝关节的骨关节单元呈现出未成熟的表型，通过软骨内成骨显示出活跃的软骨下骨形成，并且没有潮线。当移植到成熟的动物体内时，未成熟的 OCG 恢复了它们的成熟过程，即在移植后 16 周形成新的软骨下骨、建立潮线并保持其番红 O 阳性透明软骨。bTEC（hPDC+hAC）可以部分再现与幼年 OCG 相似的生物学特性，包括形成具有典型分区的关节表面结构，从浅层的非矿化透明软骨到与新软骨下骨板交界的逐渐矿化基质。
模拟幼年 OCG 并显示出具有不同组织形成单元的层次结构的基于细胞的组织工程构建体似乎是治疗膝关节深层骨关节缺损的一种有吸引力的策略。
