Repair of large osteoporotic rat calvarial defects with autologous adipose stem c

描述（由申请人提供）：骨质疏松症导致骨量和骨矿物质密度（BMD）降低，以及骨折愈合率和骨修复质量受损。组织工程为修复大面积骨缺损提供了新的策略，但修复大面积骨缺损，特别是骨缺损患者的骨缺损，仍然是骨科手术的一大挑战。近年来，脂肪干细胞（adipose-derived stem cells，ASCs）被报道具有向成骨细胞分化的能力。此外，ASCs可以在衰老过程中保持稳定的干细胞特性，这表明它们在骨质疏松患者的骨再生中具有潜在的应用。本研究的目的是评估增生的ASCs在体外的增殖和成骨潜能，以及自体骨诱导的ASCs在聚（乳酸-羟基乙酸共聚物）（PLGA）结构中植入增生的大鼠颅骨缺损中的骨再生能力。将80只6月龄雌性Sprague-Dawley大鼠随机分为卵巢切除组（n=40）和假手术组（n=40）。术后4个月，测量每组10只大鼠整个股骨的BMD。将来自两组的大鼠ASC与成骨培养基（骨诱导）或生长培养基（非诱导）一起单层或三维（3D，将ASC接种到PLGA网片中）培养三周。将使用免疫荧光染色、定量ELISA、生化测定和实时PCR评价ASC增殖和成骨分化。每组中剩余的30只大鼠将在双侧形成直径为5 mm的颅骨缺损。一个缺损将随机植入自体骨诱导的ASC或非诱导的基于ASC的过早结构（每组n = 15）。将PLGA补片单独植入另一个缺损中作为对照。植入后6周、12周和1年，每个亚组中将处死5只大鼠，并使用放射密度分析、BMD测量、5CT、H&E和Masson染色以及生物力学测试来评估缺损愈合。还将通过量子点（QD）标记追踪ASC，以确认ASC在骨再生过程中的直接作用。本研究将拓宽ASCs在骨组织工程中的应用范围，并为骨缺损患者的大面积骨缺损修复提供一种床旁选择。 公共卫生相关性：由于自体脂肪干细胞（adipose-derived stem cells，ASCs）独特的遗传特性，基于ASCs的组织工程技术有望成为治疗乳腺癌的有效方法。本研究的目的是评估增生的ASCs在体外的增殖和成骨潜能，以及自体骨诱导的ASCs在聚（乳酸-羟基乙酸共聚物）（PLGA）结构中植入增生的大鼠颅骨缺损中的骨再生能力。

DESCRIPTION (provided by applicant): Osteoporosis contributes to decreased bone mass and bone mineral density (BMD) as well as compromised fracture healing rates and bone repair quality. Tissue engineering offers novel strategies to repair large bone defects, but it remains a big challenge in orthopaedic surgery to repair large bone defects, especially in osteoporotic patients. Recently, adipose-derived stem cells (ASCs) were reported to be able to differentiate into the osteogenic lineage. Moreover, ASCs can maintain stable stem cell properties during the aging process, indicating their potential application in bone regeneration in osteoporotic patients. The aim of this study is to evaluate proliferation and osteogenic potential of osteoporotic ASCs in vitro, and the bone regeneration capability of autologous osteo-induced ASCs in poly(lactic-co-glycolic acid) (PLGA) constructs implanted in osteoporotic rat calvarial defects. A total of eighty 6-month-old female Sprague-Dawley rats will be randomly assigned into either the ovariectomy group (n=40) or the sham operation group (n=40). Four months after the operation, the BMD of the entire femur of ten rats in each group will be measured. The rat ASCs from both groups will be cultured in monolayer or three-dimension (3D, seeding ASCs into PLGA mesh) with osteogenic medium (osteo-induced) or growth medium (non-induced) for three weeks. ASC proliferation and osteogenic differentiation will be evaluated using immunofluorescence staining, quantitative ELISA, biochemical assays, and real-time PCR. The remaining thirty rats in each group will have 5-mm-diameter-calvarial-defects created bilaterally. One defect will be randomly implanted with autologous osteo-induced ASC- or non-induced ASC-based premature constructs (n = 15 in each group). PLGA mesh alone implanted in another defect will serve as a control. Six weeks, 12 weeks, and one year post implantation, five rats in each subgroup will be sacrificed and the defect healing will be evaluated using radiodensitometric analysis, BMD measurement, 5CT, H&E and Masson staining, and biomechanical testing. Tracking of ASCs by quantum dots (QDs) labeling will also be performed to confirm the direct role of ASCs during bone regeneration. This study may broaden ASCs' application range in bone tissue engineering and give a bench-to-bedside option for large bone defect repair in osteoporotic patients. PUBLIC HEALTH RELEVANCE: Large bone defects in osteoporotic patients are a big challenge for clinical treatment, in which autologous adipose-derived stem cells (ASCs)-based tissue engineering may be a promising approach due to ASCs unique genetic property. The aim of this study is to evaluate proliferation and osteogenic potential of osteoporotic ASCs in vitro, and the bone regeneration capability of autologous osteo-induced ASCs in poly(lactic-co-glycolic acid) (PLGA) constructs implanted in osteoporotic rat calvarial defects.