Bone Tissue Engineering Using Mineralized Collagen-GAG Scaffolds

使用矿化胶原蛋白-GAG 支架的骨组织工程

• 批准号: 8621974
• 负责人: Timothy A Miller
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8621974
• 关键词: 3-Dimensional; Adhesions; Affect; Afghanistan; Aging; Architecture; Autologous; Back; Binding; Biocompatible; Biological Sciences; Bone Marrow; Bone Tissue; Bone Transplantation; Caring; Cells; Cephalic; Collagen; Defect; Development; Diffusion; Dose; Excision; Exhibits; Extracellular Matrix; GAG Gene; Glycolic-Lactic Acid Polyester; Glycosaminoglycans; Goals; Harvest; Healed; Hemostatic Agents; Human; Implant; In Vitro; Infiltration; Inflammation; Injury; Integrins; Iraq; Laboratories; Limb structure; Malignant Neoplasms; Mechanics; Medical center; Mesenchymal Stem Cells; Modeling; Nutrient; Oryctolagus cuniculus; Osteogenesis; Osteomyelitis; Pain; Patients; Population; Property; Quality of Care; Recombinants; Role; Services; Signal Transduction; Signaling Molecule; Site; Solid; Stromal Cells; Structure; System; Techniques; Technology; Test Result; Testing; Tissue Engineering; Trauma; Vascularization; Veterans; Wound Healing; base; biomaterial development; bone; bone morphogenetic protein 2; calcium phosphate; combat; comparative efficacy; craniofacial; healing; improved; in vivo; mineralization; nanoparticulate; operation; osteogenic; prevent; public health relevance; reconstruction; repaired; scaffold; tissue culture

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.

描述（由申请人提供）： 过去 15 年来，我们的实验室一直专注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）和基于胶原蛋白的支架，其中接种了自体骨髓基质细胞 (BMSC)，并受到包括 BMP-2 在内的骨诱导剂的刺激。借助这些 BGS 模型，我们使用 3 维组织培养系统在体外和体内生成了新骨，从而治愈了兔子的临界尺寸颅骨缺损。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。载体对于BGS的发展至关重要。 PLGA 具有生物相容性和可生物降解性，显示出巨大的前景。然而，PLGA 在组织工程中的应用存在局限性。 PLGA 会诱导酸性降解产物的形成，影响局部 PH 值，可能引发炎症。胶原蛋白支架，包括胶原蛋白-GAG，已广泛应用于骨组织工程，因为胶原蛋白是细胞外基质中天然存在的成分，可促进细胞结合、表现出低抗原性并具有优异的止血性能。我们证明，与接种在 PLGA 支架中的对应物相比，接种在胶原蛋白支架中的人间充质干细胞 (MSC) 表现出加速且强劲的矿化和骨形成。然而，我们在体外研究中还发现，与 PLGA 支架相比，细胞接种的胶原蛋白支架会发生显着的收缩。这种收缩会极大地阻碍胶原蛋白植入物修复骨缺损的应用。为了限制胶原蛋白植入物的体积损失和破坏，我们建议通过在胶原蛋白-GAG原纤维上掺入纳米颗粒磷酸钙（CaP）来稳定胶原蛋白支架的结构。我们的初步测试结果表明，使用 MC-GAG 支架可以极大地防止 hMSC 粘附和分化引起的收缩。它在支持新骨形成方面也优于胶原蛋白-GAG。我们目前的提案旨在进一步研究 MC-GAG 支架在骨组织工程中的可行性。在本提案中，我们将比较胶原蛋白-GAG 和 MC-GAG 支架支持体外培养的兔 BMSC 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，比较由胶原蛋白-GAG 和 MC-GAG 制成的细胞支架在修复临界大小的兔颅骨缺损方面的功效。最后，我们将研究整合素及其下游信号分子，这些分子导致胶原蛋白-GAG 和 MC-GAG 在控制收缩和支持 rBMSC 成骨分化的能力方面存在差异。我们预计，与单独的胶原蛋白相比，MC-GAG 支架将（1）增强兔 BMSC 的成骨分化并减少收缩，（2）与单独的胶原蛋白相比，加速兔颅骨缺损模型中的新骨形成。我们预计整合素信号传导的变化导致 MC-GAG 支架中 rBMSC 的分化增强和收缩减少。