Venkatesan Jagadeesh K, Frisch Janina, Rey-Rico Ana, Schmitt Gertrud, Madry Henning, Cucchiarini Magali
Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany.
Department of Orthopaedic Surgery, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421, Homburg/Saar, Germany.
J Exp Orthop. 2017 Dec;4(1):22. doi: 10.1186/s40634-017-0097-1. Epub 2017 Jun 21.
Evaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions.
Fresh human bone marrow aspirates were transduced with rAAV-FLAG-hsox9 (40 μl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of rAAV vector; reporter rAAV-lacZ, rAAV-RFP, and rAAV-luc gene transfer).
Prolonged, significant sox9 overexpression via rAAV was achieved in the aspirates for at least 28 days when applying the rAAV-FLAG-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments.
The current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by rAAV sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces.
评估基于基因的方法在机械刺激条件下靶向人类骨髓抽吸物，旨在重现自然关节环境，这可能有助于开发针对关节软骨损伤的改进治疗方法。在本研究中，我们研究了重组腺相关病毒（rAAV）介导的sox9基因转移在既定流体动力学条件下增强人类骨髓抽吸物软骨形成分化过程的潜力，并与更常用的静态培养条件进行比较。
用rAAV-FLAG-hsox9（40μl）转导新鲜人类骨髓抽吸物，并在动态流动旋转生物反应器的机械诱导条件下，于软骨形成培养基中维持长达28天，相对于静态培养条件，该反应器允许组织生长和基质沉积。然后对样本进行处理，以检查与对照处理（无rAAV载体；报告基因rAAV-lacZ、rAAV-RFP和rAAV-luc基因转移）相比，sox9过表达对细胞活性（基质合成、增殖）和软骨形成分化潜能的潜在影响。
当应用rAAV-FLAG-hsox9构建体时，抽吸物中通过rAAV实现了至少28天的sox9长时间显著过表达，导致相对于对照处理，基质生物合成水平更高且持续时间更长，软骨形成活性增强，尤其是在对样本进行机械刺激时。然而，sox9的施用并未影响抽吸物中的增殖指标。值得注意的是，无论与对照处理相比应用何种培养条件，sox9基因转移也持久地延迟了样本中的肥大和成骨分化。
目前的观察结果表明，在机械刺激下用rAAV sox9对人类骨髓抽吸物进行基因改造，作为一种有前景的策略，可用于未来通过植入受自然机械力作用的损伤组织来改善软骨修复的治疗。
