透明软骨 不含软骨下骨的单纯组织工程透明软骨移植用于骨软骨全面再生

关节软骨损伤因其创伤或退变导致骨软骨组织逐渐退化，最终导致骨关节炎。尤其是承受体重的下肢关节，一旦出现明显的关节损伤并且到达软骨下骨，由于其无血管和无神经的特性，关节软骨的自我再生能力受到严重损害。因此，创伤性骨软骨损伤的最终治疗依赖于成功的软骨移植。有研究者提出一个假设，即软骨下骨的修复可以通过自发的原位自我重建来完成，其关键在于通过适当的非自体软骨移植修复关节软骨。因此，来自于新加坡南洋理工大学化学与生物医学工程学院王东安教授团队通过构建完整的兔骨软骨缺损模型，探讨了猪源性透明软骨移植物及其脱细胞透明软骨移植物对软骨下骨缺损的修复作用。首先在体外利用大孔海藻酸钠水凝胶临时支架培养软骨细胞以制备透明软骨移植物。一旦大量软骨新组织在海藻酸钠支架内形成相互连接的网络，海藻酸钠支架即刻被柠檬酸盐缓冲液淋洗去除，只剩下呈三维海绵状的纯软骨新组织。该组织只含有活软骨细胞和由软骨细胞分泌和沉积的内源性ECMs，具有天然软骨微结构。研究者进一步通过脱细胞获得透明软骨组织特异性ECMs非细胞产物，即脱细胞的透明状软骨移植物，用于移植。实验模式图如下。

首先，研究者对LhCG和dLhCG进行组织学及生化分析。研究表明：dLhCG中DNA残留较少，同时保留了大量的II型胶原和糖胺聚糖，所以dLhCG的构建是成功且有效的。此外，dLhCG还较好的保持了软骨性ECM结构和显微结构。相反，当同样的脱细胞方法应用于天然软骨外植体时，却很难达到DNA清除目的。因此dLhCG优于dNat。

Figure 2. a) dLhCG from LhCG in comparison to decellularized native cartilage, a) histology of LhCG, dLhCG, native cartilage and decellularized native cartilage , respectively, by H&E stain, Saf O stain and IHC stain with primary antibody of collagen TypeII and counter stained by DAPI. Scale bar: 100 µm. b) Quantification of DNA residue, GAG and total collagen contents in all samples: LhCG and dLhCG in comparison to Native and dNat. * means p ≤0.05, ** means p ≤ 0.01, *** means p ≤ 0.001, and **** means p ≤ 0.0001. Positive Saf O stain of GAG appears in bright red; positive IHC stain of Col II appears as emitting green florescence; and positive DAPI stain of cell nuclei appears as emitting blue florescence.

随后研究者分别在植入手术后第50天和第100天对受损软骨部位进行表征和分析。结果发现，在植入后第50天骨软骨样新生组织完全填补了骨软骨缺损，关节面从创伤性凹陷中恢复，新生成的骨基质出现在下面的区域，新组织倾向于与周围的原生组织整合，且新基质由丰富的II型胶原和少量的I型胶原组成。再生组织的超微结构显示LhCG和dLhCG组在植入50天时软骨区及软骨下骨区均出现明显再生。植入100天后， LhCG和dLhCG组均实现了骨软骨缺损完全愈合，关节软骨和软骨下骨新生组织均在各自的缺损区域再生。

Figure 3. Histology of osteochondral repair by engraftment of cartilage implants, dLhCG and LhCG, at testing time points of day-50 and 100, respectively. a) H&E stain, b) MT stain, c) Saf O stain, and IHC stain with d) primary antibody of Type II collagen and e) Type I collagen . The negative control, namely the untreated defects that are left void of grafts, is marked as “untreated”. Two arrows are used to indicate the location and width of the experimental defects originally made. Scale bar: 500 µm. f) Polarized light microscopy of picrosirius stain, which reveals ultrastructural histology of both articular cartilage zone and subchondral bone zone of the samples. The samples include LhCG, dLhCG, the negative control “Untreated” and the positive control—intact, surrounding native Cart and Sub-B of the host—marked as “Native”. Scale bar: 500 µm. Positive Saf O stain of GAG appears in bright red; positive MT stain of collagens appears blue; positive IHC stain using DAB which precipitates in the presence of HRP conjugated Col II and Col I appears in golden yellow. Picrosirius stains relative thick matrix fibers in red or dark yellow; stains relatively delicate fibers in green or light yellow; and stains the superficial edge of articular cartilage with a bright red line.

研究者还对植入物在植入后第50天和第100天的相关指标进行定量分析，统计了软骨区和软骨下骨区的DNA测定、GAG测定、胶原测定和钙测定的定量结果。结果表明在第50天时LhCG组软骨区的GAG、胶原和钙含量皆高于dLhCG组，第100天时LhCG组中的DNA量较高；软骨下骨区第50天的测定结果两组间未见明显差异，第100天时可见LhCG组的钙含量较高。此外，对照组缺损区未能实现局部再生，而是由瘢痕等组织填充。

最后研究者对不同植入组进行了micro-CT的分析，结果表明：在植入后第50天，在LhCG和dLhCG植入样本的矢状面和冠状面均可观察到愈合进展；在植入后第100天，在dLhCG组观察到软骨下骨层的完全愈合。在未对软骨下骨进行干预的情况下，两组均实现了软骨区及软骨下骨区的有效修复。

Figure 5. Representative µCT images of coronary, sagittal and transverse sections of regenerated tissue with surrounding tissue treated by dLhCG and LhCG implants in comparison to untreated defect that were left void without any implantation of graft marked as “Untreated” at a) day-50 and b) day-100. Scale bar: 2 mm. The arrow pointed to the defect site.