Bone Tissue Engineering Using Mineralized Collagen-GAG Scaffolds

描述(由申请人提供)： 在过去的15年里，我们的实验室一直致力于合成骨移植替代品(BGS)的开发。我们的实验性骨移植替代品包括PLGA-(聚丙交酯-乙交酯共聚物)和胶原基支架，种植有自体骨髓基质细胞(BMSC)，并受到包括BMP-2在内的骨诱导剂的刺激。通过这些BGS模型，我们使用三维组织培养系统在体外和体内都产生了新的骨，导致了兔关键大小的颅骨缺损的愈合。虽然我们的研究很有希望，但临床上有用的BGS仍然难以捉摸。载体在BGS的发展中至关重要。PLGA具有良好的生物相容性和可生物降解性，已显示出广阔的应用前景。然而，PLGA在组织工程中的应用存在局限性。PLGA会导致酸性降解产物的形成，这会影响局部的PH值，可能会引发炎症。胶原蛋白是细胞外基质中天然存在的促进细胞结合的成分，抗原性低，具有良好的止血性能，因此在骨组织工程中得到了广泛的应用。我们证明，与种植在PLGA支架中的相应细胞相比，种植在胶原支架中的人骨髓间充质干细胞(MSC)具有更快、更强的矿化和骨形成能力。然而，我们在体外研究中也发现，与PLGA支架相比，细胞种植的胶原支架经历了显著的收缩。这种收缩会极大地阻碍胶原植入物在修复骨缺损方面的应用。为了限制胶原植入物的体积损失和破坏，我们建议通过在胶原-GAG纤维上掺入纳米颗粒磷酸钙(CAP)来稳定胶原支架的结构。初步实验结果表明，使用MC-GAG支架可以很好地防止hMSCs因黏附和分化而引起的收缩。在支持新骨形成方面，它也优于胶原蛋白-GAG。本研究旨在进一步探讨MC-GAG支架在骨组织工程中应用的可行性。在这个方案中，我们将比较胶原-GAG和MC-GAG支架在体外培养的兔BMSCs中支持成骨分化的能力。我们还将通过对新形成的骨进行组织学和力学分析，比较胶原-GAG和MC-GAG制成的细胞支架在修复临界大小的兔颅骨缺损中的效果。最后，我们将研究整合素及其下游信号分子，它们与胶原-GAG和MC-GAG在控制rBMSCs收缩和支持成骨分化能力方面的差异有关。我们预计，与单纯胶原相比，MC-GAG支架将(1)促进兔BMSCs的成骨分化，减少收缩；(2)与单独使用胶原相比，MC-GAG支架将促进兔颅骨缺损模型中新骨的形成。我们预计整合素信号的改变是MC-GAG支架中rBMSCs分化增强和收缩减少的原因。

DESCRIPTION (provided by applicant): Our laboratory has been focused on the development of a synthetic bone graft substitute (BGS) for the past 15 years. Our experimental bone graft substitutes have included PLGA- (Poly-lactide-co-glycolide) and collagen-based scaffolds seeded with autologous bone marrow stromal cells (BMSC), and stimulated by osteoinductive agents including BMP-2. With these BGS models, we have generated new bone both in vitro, using a 3-dimensional tissue culture system, and in vivo, resulting in the healing of critical-sized cranial defects in the rabbit. While our studies have been promising, a clinically useful BGS remains elusive. The carrier is of critical importance in the development of a BGS. PLGA has shown much promise, being both biocompatible and biodegradable. However, there are limitations to the use of PLGA in tissue engineering. PLGA induces the formation of acidic degradation products that can affect local PH, possibly inducing inflammation. Collagen based scaffolds, including collagen-GAG, have been widely used in bone tissue engineering because collagen is a naturally existing component of extracellular matrix that promotes cell binding, exhibits low antigenicity and has excellent haemostatic property. We demonstrated that human mesenchymal stem cells (MSC) seeded in collagen scaffolds exhibit accelerated and robust mineralization and bone formation in comparison to their counterparts seeded in PLGA scaffolds. However, we also discovered in our in vitro studies that cell-seeded collagen scaffolds undergo significant contraction compared to PLGA scaffolds. The contraction can greatly hamper application of collagen implants in repairing bony defects. In order to limit volume loss and destruction of collagen implants, we propose to stabilize the structure of collagen scaffolds by incorporating nanoparticulate calcium phosphate (CaP) on the collagen-GAG fibrils. Our initial test results indicate using MC-GAG scaffolds can greatly prevent contraction caused by adhesion and differentiation of hMSCs. It is also superior to collagen-GAG in supporting new bone formation. Our current proposal intends to further investigate the feasibility of MC-GAG scaffolds in bone tissue engineering. In this proposal, we will compare collagen-GAG and MC-GAG scaffolds for their ability to support osteogenic differentiation in rabbit BMSCs cultured in vitro. We will also compare the efficacy of cellular scaffolds made of collagen-GAG and MC-GAG in healing a critical-sized rabbit cranial defect by performing histological and mechanical analysis of newly formed bone. Finally, we will study integrins and their downstream signaling molecules responsible for the differences between collagen-GAG and MC-GAG in their ability to control contraction and support osteogenic differentiation in rBMSCs. We expect that MC-GAG scaffolds will (1) enhance osteogenic differentiation and reduce contraction in rabbit BMSCs compared to collagen alone, and (2) accelerate new bone formation in a rabbit cranial defect model compared to collagen alone. We anticipate that changes in integrin signaling are responsible for enhanced differentiation and reduced contraction of rBMSCs in MC-GAG scaffolds.