J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, FL, United States of America.
J Neural Eng. 2021 Jun 22;18(4). doi: 10.1088/1741-2552/ac07bf.
. Chondroitinase ABC (ChABC) has emerged as a promising therapeutic agent for central nervous system regeneration. Despite multiple beneficial outcomes for regeneration, translation of this enzyme is challenged by poor pharmacokinetics, localization, and stability.. This study explored the function andapplication of engineered ChABC fused to galectin-3 (Gal3). Two previously developed ChABC-Gal3 oligomers (monomeric and trimeric) were evaluated for functionality and kinetics, then applied to ancellular outgrowth model using dorsal root ganglia (DRGs). The fusions were combined with two formulations of hyaluronan (HA)-based scaffolds to determine the extent of active enzyme release compared to wild type (WT) ChABC.. Monomeric and trimeric ChABC-Gal3 maintained digestive capabilities with kinetic properties that were substrate-dependent for chondroitin sulfates A, B, and C. The fusions had longer half-lives at 37 °C on the order of seven fold for monomer and twelve fold for trimer compared to WT. Both fusions were also effective at restoring DRG outgrowth. To create a combination approach, two triple-component hydrogels containing modified HA were formulated to match the mechanical properties of native spinal cord tissue and to support astrocyte viability (>80%) and adhesion. The hydrogels included collagen-I and laminin mixed with either 5 mg mlof glycidyl methacrylate HA or 3 mg mlHystem. When combined with scaffolds, ChABC-Gal3 release time was lengthened compared to WT. Both fusions had measurable enzymatic activity for at least 10 d when incorporated in gels, compared to WT that lost activity after 1 d. These longer term release products from gels maintained adequate function to promote DRG outgrowth.. Results of this study demonstrated cohesive benefits of two stabilized ChABC-Gal3 oligomers in combination with HA-based scaffolds for neural applications. Significant improvements to ChABC stability and release were achieved, meriting future studies of ChABC-Gal3/hydrogel combinations to target neural regeneration.
. 软骨素酶 ABC（ChABC）已成为中枢神经系统再生的一种很有前途的治疗药物。尽管这种酶在再生方面有多种有益的结果，但由于其药代动力学、定位和稳定性较差，其翻译仍具有挑战性。本研究探讨了与半乳糖凝集素-3（Gal3）融合的工程化 ChABC 的功能和应用。评估了两种先前开发的 ChABC-Gal3 低聚物（单体和三聚体）的功能和动力学特性，然后将其应用于使用背根神经节（DRG）的无细胞外生模型。融合物与两种透明质酸（HA）基支架制剂结合，以确定与野生型（WT）ChABC 相比，活性酶的释放程度。单体和三聚体 ChABC-Gal3 保持了消化能力，动力学特性对软骨素硫酸盐 A、B 和 C 具有底物依赖性。与 WT 相比，融合物在 37°C 下的半衰期更长，单体为 7 倍，三聚体为 12 倍。两种融合物也能有效地恢复 DRG 外生。为了创建一种组合方法，两种含有改性 HA 的三组分水凝胶被配方制成与天然脊髓组织的机械性能相匹配，并支持星形胶质细胞的活力（>80%）和粘附。水凝胶包含胶原蛋白-I 和层粘连蛋白，与 5mg ml 的甲基丙烯酸缩水甘油酯 HA 或 3mg mlHystem 混合。与支架结合时，与 WT 相比，ChABC-Gal3 的释放时间延长。与 WT 相比，在凝胶中掺入时，两种融合物的酶活性至少可维持 10 天，而 WT 在 1 天后失去活性。凝胶中这些长期释放的产物保持了足够的功能，以促进 DRG 外生。本研究的结果表明，两种稳定的 ChABC-Gal3 低聚物与基于 HA 的支架相结合，在神经应用方面具有协同作用。ChABC 稳定性和释放得到了显著改善，为未来研究 ChABC-Gal3/水凝胶组合以靶向神经再生奠定了基础。
