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密度(BMD)下降，并影响骨折愈合率和骨修复质量。组织工程学为修复大型骨缺损提供了新的策略，但在骨科手术中修复大型骨缺损仍然是一个巨大的挑战，尤其是在骨质疏松患者中。最近，脂肪来源的干细胞(ASCs)被报道能够分化为成骨细胞。此外，ASCs在衰老过程中可以保持稳定的干细胞特性，这表明其在骨质疏松患者的骨再生中具有潜在的应用前景。本研究的目的是评价骨质疏松ASCs在体外的增殖和成骨能力，以及自体骨诱导的ASCs在聚乳酸-乙醇酸(PLGA)支架植入骨质疏松大鼠颅骨缺损处的骨再生能力。6月龄雌性SD大鼠80只，随机分为卵巢切除组和假手术组，每组40只。术后4个月，测量每组10只大鼠的股骨全段骨密度。将两组大鼠的ASCs在单层或三维(3D，将ASCs种植到PLGA网状物中)中，分别加入成骨介质(成骨诱导)或生长介质(非诱导)培养三周。ASC的增殖和成骨分化将使用免疫荧光染色、定量ELISA法、生化分析和实时荧光聚合酶链式反应进行评估。每组剩下的30只大鼠将造成双侧5毫米直径的颅骨缺损。其中一个缺损区将随机植入自体骨诱导的ASC或未诱导的ASC为基础的早熟结构(每组15例)。将PLGA网片单独植入另一处缺损处作为对照。分别于植入后6周、12周、1年处死每组大鼠5只，进行放射性密度分析、骨密度测定、5CT、H&E和Masson染色及生物力学检测。还将通过量子点标记追踪ASCs，以确认ASCs在骨再生过程中的直接作用。本研究可能扩大ASCs在骨组织工程中的应用范围，并为骨质疏松患者的大块骨缺损修复提供床边选择。 公共卫生相关性：骨质疏松症患者的巨大骨缺陷是临床治疗的一大挑战，其中基于自体脂肪干细胞(ASCs)的组织工程可能是一种有前途的方法，因为ASCs具有独特的遗传特性。本研究的目的是评价骨质疏松ASCs在体外的增殖和成骨能力，以及自体骨诱导的ASCs在聚乳酸-乙醇酸(PLGA)支架植入骨质疏松大鼠颅骨缺损处的骨再生能力。

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.