Leni and Peter W. May Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
Eur Cell Mater. 2020 May 12;39:211-226. doi: 10.22203/eCM.v039a14.
Back pain is a leading cause of global disability associated with intervertebral disc (IVD) pathologies. Discectomy alleviates disabling pain caused by IVD herniation without repairing annulus fibrosus (AF) defects, which can cause accelerated degeneration and recurrent pain. Biological therapies show promise for IVD repair but developing high-modulus biomaterials capable of providing biomechanical stabilisation and delivering biologics remains an unmet challenge. The present study identified critical factors and developed an optimal formulation to enhance the delivery of AF cells and transforming growth beta-3 (TGFβ-3) in genipin-crosslinked fibrin (FibGen) hydrogels. Part 1 showed that AF cells encapsulated in TGFβ-3-supplemented high-modulus FibGen synthesised little extracellular matrix (ECM) but could release TGFβ-3 at physiologically relevant levels. Part 2 showed that AF cells underwent apoptosis when encapsulated in FibGen, even after reducing fibrin concentration from 70 to 5 mg/mL. Mechanistic experiments, modifying genipin concentration and integrin binding site presence demonstrated that genipin crosslinking caused AF cell apoptosis by inhibiting cell-biomaterial binding. Adding integrin binding sites with fibronectin partially rescued apoptosis, indicating genipin also caused acute cytotoxicity. Part 3 showed that FibGen formulations with 1 mg/mL genipin had enhanced ECM synthesis when supplemented with fibronectin and TGFβ-3. In conclusion, FibGen could be used for delivering biologically active compounds and AF cells, provided that formulations supplied additional sites for cell-biomaterial binding and genipin concentrations were low. Results also highlighted a need for developing strategies that protect cells against acute crosslinker cytotoxicity to overcome challenges of engineering high-modulus cell carriers for musculoskeletal tissues that experience high mechanical demands.
背痛是全球残疾的主要原因，与椎间盘（IVD）病变有关。椎间盘切除术可缓解因椎间盘突出引起的致残性疼痛，而无需修复纤维环（AF）缺陷，否则会导致加速退化和复发疼痛。生物疗法为 IVD 修复带来了希望，但开发能够提供生物力学稳定性并输送生物制剂的高模量生物材料仍然是一个未满足的挑战。本研究确定了关键因素并开发了最佳配方，以增强 AF 细胞和转化生长β-3（TGFβ-3）在京尼平交联纤维蛋白（FibGen）水凝胶中的递送。第 1 部分表明，在补充 TGFβ-3 的高模量 FibGen 中包封的 AF 细胞合成的细胞外基质（ECM）很少，但可以在生理相关水平释放 TGFβ-3。第 2 部分表明，即使将纤维蛋白浓度从 70 降至 5mg/mL，AF 细胞在包封于 FibGen 中时仍会发生细胞凋亡。通过改变京尼平浓度和整合素结合位点存在的机制实验表明，京尼平交联通过抑制细胞-生物材料结合导致 AF 细胞凋亡。用纤维连接蛋白添加整合素结合位点部分挽救了细胞凋亡，表明京尼平也引起了急性细胞毒性。第 3 部分表明，当用纤维连接蛋白和 TGFβ-3 补充时，1mg/mL 京尼平的 FibGen 制剂具有增强的 ECM 合成。总之，只要配方提供了更多的细胞-生物材料结合位点并且京尼平浓度较低，FibGen 就可以用于输送生物活性化合物和 AF 细胞。结果还强调了需要开发策略来保护细胞免受急性交联剂细胞毒性的影响，以克服为经历高机械需求的肌肉骨骼组织工程高模量细胞载体所面临的挑战。
