利用骨骼肌全器官脱细胞基质再生功能性大体积骨骼肌的实验研究

Skeletal muscle is a complex organ and its function is very important to the body.Till now, the results achieved in tissue engineered skeletal muscle are lagging behind those achieved for other tissue types such as liver and lung. Key point to explain this is the absence of an ideal scaffold that mimics the complexity of skeletal muscle extracellular matrix(ECM).Biologic scaffolds prepared from the ECM of acellular mammalian tissues have been confirmed the capability of stimulating restoration of significant muscle mass in injured sites. As the ECM of every tissue has a unique composition and structure that likely has direct effects on the host response, we hypothesize that skeletal muscle whole organ acellular matrix(SKWOACM), provides distinct advantages in muscle regeneration.In our previous work, we have manufactured the first SKWOACM in the world which presents thorough decellularization while preserving the native architecture including hierarchical and complex three-dimensional organization of parallel arrayed basal lamina with tendinous connection surrounded by capillary network. SKWOACM contains growth factors, glycosaminoglycans, and basement membrane structural proteins.In this profject, we will try to construct the in vitro tissue engineered skeletal muscle by perfusion nutrition in mechanical stimulation circumstance,test the vivo remodeling and muscule regeneration of SKWOACM in rodent partial thickness abdominal wall defect model, compared to nonhomologous ECM and other scaffolds. After heparin pretreatment, myogenic cells systemically seeded or non-seeded SKWOACMs were implanted for canine rectus defect repair with the vascular bed inside SKWOACM connected to host circulation.This project is just targeted at clinical needs and the expected results are supposed to change clinical status.The applicant has achieved much in basic research and clinical applications of acellular matrix materials, including several articles published in top journals of surgery and material science together with several patents granted. Our team represents the leading level in the field of skeletal muscle regeneration in the world.

理想的骨骼肌再生支架和平台应能精确模拟骨骼肌细胞外基质的物理和生物学特性，这方面是目前制约骨骼肌组织工程发展的关键因素。前期申请者于国际上首创新型的肌肉再生平台-骨骼肌全器官脱细胞基质（SKWOACM）并证实其细胞脱除彻底，拥有平行排列的基底膜管、血管网络、肌肉-肌腱接头等三维超微结构和生长因子、糖胺聚糖等生物活性成分，体外促进肌源性干细胞迁移、增殖和分化，是迄今最接近天然骨骼肌细胞外基质特性的支架。本课题拟验证SKWOACM能否体外构建出组织工程骨骼肌和体内实现血管化、带神经支配、具备良好收缩功能的大体积骨骼肌的诱导再生以及相关的宿主免疫反应；力争以再生医学技术解决大范围骨骼肌缺损功能性修复难题，预期结果有望改变临床现状。课题前期工作已获国际同行认可，申请者在脱细胞基质材料的基础研究和临床应用领域也有较多工作积累，已发表多篇外科学和材料学类顶尖SCI期刊论著，拥有多项相关专利。

理想的骨骼肌再生支架和平台应能精确模拟骨骼肌细胞外基质的物理和生物学特性，这方面是目前制约骨骼肌组织工程发展的关键因素。针对这一难题，本项目在前期国际上首创新型的肌肉再生平台-骨骼肌全器官脱细胞基质（SKWOACM）的基础上，开展了①建立SKWOACM取材、灌注脱细胞的方法体系；体外三维培养SKWOACM，联合灌注成肌细胞，构建组织工程骨骼肌；②对比SKWOACM与小肠粘膜下层材料修复大鼠腹壁肌部分层次缺损的治疗效果，证实SKWOACM可以实现修复区丰富的“大片肌组织”再生，新生肌肉体积占缺损区30%左右，修复疗效显著优于非组织特异性的SIS；③SKWOACM预植肝素，大鼠腹腔血液灌注抗凝模型评价SKWOACM体内抗凝时间；④对比SKWOACM与其他材料修复犬封闭的腹直肌大范围缺损的治疗效果，术后8周SKWOACM腹壁修复区重塑完成、力学性能显著优于天然腹壁组织，核素标记明确其体内重塑和最终转归。本课题研究成果已在材料学顶级期刊《Biomaterials》（IF 8.557）上发表论著1篇和方法学论著1篇，另发表中文核心期刊综述2篇，尚有SCI高分值论文1篇修回中。授权国家发明专利1项，申请PCT国际专利4项，申请国家发明专利6项。参加多次国际、国内学术会议交流，项目组负责人和成员分别获批国家863青年科学家项目1项、国家自然科学基金面上项目2项，培养博士生2名，硕士生1名。本项目实现了预期目标，为SKWOACM的临床应用奠定了基础。