Departments of Orthopaedic Surgery and Cell Biology, Duke University Medical Center, Durham, NC 27710.
Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):E798-806. doi: 10.1073/pnas.1321744111. Epub 2014 Feb 18.
The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.
在肌肉骨骼工程学中，开发具有促进快速组织修复或再生的基质组成和生物力学特性的组织构建体仍然是一个持久的挑战。目前的方法需要在体外进行广泛的细胞操作，使用外源性生长因子来驱动组织特异性分化、基质积累和机械性能，从而限制了它们的潜在临床应用。诱导和维持干细胞在体内分化的能力可以绕过这些步骤，提高组织再生工程方法的成功率。本研究的目的是利用基于慢病毒的方法生成一种自包含的生物活性支架，能够介导干细胞分化和形成软骨细胞外基质 (ECM)。我们首先表明，聚-L-赖氨酸可以将慢病毒固定在聚（ε-己内酯）薄膜上，并促进人间充质干细胞 (hMSC) 的转导。然后我们证明了使用 3D 编织聚（ε-己内酯）支架进行支架介导的转化生长因子β3 (TGF-β3) 基因传递可以诱导 hMSCs 产生强健的软骨 ECM。通过支架介导的基因传递诱导的软骨生成与涉及 TGF-β3 培养基补充的传统分化方案一样有效，如基因表达、生化和生物力学分析所示。通过将固定在生物力学功能支架上的慢病毒载体，我们开发了一种系统，可通过 hMSCs 实现持续的转基因表达和 ECM 形成。这种方法为生物活性植入物的开发开辟了新途径，通过实现原位组织工程的长期目标，避免了体外组织生成的需要。
