应用自体脂肪干细胞修复猪关节软骨缺损的初步实验研究

目的 探索自体脂肪干细胞修复猪关节软骨缺损的可行性.方法 从猪背部脂肪组织中获取脂肪干细胞,经过体外培养扩增,以50×106/ml的浓度将脂肪干细胞接种于PLGA(polylacticacid/polyglycolicacid,PLA/PGA,PLGA)中,细胞材料复合物在体外成软骨诱导2周.于猪膝关节软骨非负重区形成2个直径8mm的环形、全层软骨缺损,实验组回植经诱导后的细胞材料复合物,对照组放置单纯支架材料.术后12周取材,缺损修复区行大体观察、组织学HE及藏红花染色、免疫组化检测.结果 术后12周实验组缺损区大部分被修复,缺损被软骨组织填充,修复区表面光滑.组织学染色显示有典型的透明软骨样结构.藏红花染色发现修复组织表达丰富的聚合蛋白多糖.对照组则未能修复关节软骨缺损,缺损区面积增大,表面覆盖薄层纤维组织.结论 猪自体脂肪干细胞可以作为组织工程种子细胞,修复猪关节软骨缺损.     

脂肪干细胞辅助自体脂肪移植隆乳术治疗小乳症的临床效果

目的 分析脂肪干细胞辅助的自体脂肪移植隆乳术治疗小乳症的临床效果。方法 选取本院2020年 7月-2023年7月收治的68例小乳症患者为研究对象,采用随机数字表法分为对照组和观察组,各34例。 对照组予以单纯自体脂肪移植隆乳术,观察组予以脂肪干细胞辅助的自体脂肪移植隆乳术,比较两组乳 房体积改善效果、三维增加值、隆起值及术后隆起增加维持率。结果 观察组乳房体积改善总有效率为 97.06%,高于对照组的76.47%（P ＜0.05）;观察组术后3个月CC、SN-N、C-N、N-MF增加值均高于对 照组（P＜0.05）;观察组术后1、3个月隆起值大于对照组（P＜0.05）;观察组术后1、3个月隆起增加维持 率高于对照组（P＜0.05）。结论 脂肪干细胞辅助的自体脂肪移植隆乳术在小乳症中应用价值较高,能更 好地改善乳房形体,且术后维持效果较好,值得临床应用。     

两种细胞辅助脂肪移植隆乳的临床效果观察

目的观察两种细胞辅助脂肪移植技术(Cell-assisted lipotransfer,CAL)隆乳的临床疗效。方法 CAL技术隆胸30例,根据术中提取的基质血管成分细胞(Stromal vascular fraction,SVF)来源分为两组,来源于脂肪组织的称为固相组(S组,n=24),来源于吸脂液的为液相组(L组,n=6)。术后均随访12个月。流式细胞仪检测两种来源SVF中脂肪干细胞(Adipose-derived stem cells,ADSCs)比例,MRI检测术前及术后6、12个月的乳房体积,计算移植脂肪组织的吸收率。结果两组新鲜分离的SVF细胞群中,ADSCs平均比例分别为S组40.73%、L组3.75%。MRI测量及统计结果显示,S组术后6、12个月时乳房体积均明显大于术前(P0.05)。12个月时脂肪吸收率为(51.83±15.28)%,与6个月时相比无明显差异(P>0.05);L组12个月时脂肪吸收率为(75.47±12.20)%,与6个月时相比无明显差异(P>0.05);MRI影像见囊肿2例,均来自S组。结论脂肪组织相比吸脂液,其SVF细胞群中ADSCs含量明显更高;CAL辅助脂肪移植技术隆乳术安全有效,术后6个月乳房体积即可保持基本稳定。     

CD204阴性人脂肪来源细胞体外成软骨潜能的初步研究

目的比较CD204+和CD204-两种人脂肪来源细胞的干细胞特性和体外诱导成软骨的能力。方法分离培养人脂肪来源细胞并进行流式细胞分选,分别获得CD204+和CD204-两种细胞。将这两种细胞培养扩增至第2代,观察其形态学特点;成脂、成骨定向诱导分化,油红O染色、茜素红染色定性;同时将两种细胞进行pellet成软骨诱导分化,比较体外诱导成软骨的能力,并以HE、Safranin O和Ⅱ型胶原免疫组化染色等进行鉴定。结果经流式细胞分选,脂肪来源细胞中CD204表达阳性率约为10%。分选后的CD204+和CD204-细胞均呈成纤维细胞样贴壁生长,但CD204-细胞具有更强的增殖能力。成脂诱导10 d后,两组细胞油红O染色均可见胞浆内有大量红染脂滴,但CD204+细胞具有更强的成脂潜能。成骨诱导2周后,两组细胞茜素红染色均可见钙结节红染,而CD204-细胞具有更强的成骨潜能。同时,细胞pellet成软骨诱导4周后,大体观可见CD204-细胞组pellet形成白色透明样软骨成分,而CD204+细胞组pellet外观微黄。HE和Ⅱ型胶原免疫组化染色均显示CD204-组pellet可见成熟软骨陷窝形成,Safranin O染色显示CD204-组软骨特异性细胞外基质沉积;而CD204+细胞组pellet则呈纤维化改变。结论脂肪组织来源的CD204-细胞具有干细胞的生物学特点,且有良好的成软骨潜能,可以成为软骨组织工程良好的种子细胞来源。     

Osteogenic Induction of Adipose-derived Stromal Cells: Not a Requirement for Bone Formation In Vivo

Osteogenic induction was regarded as an indispensable step for adipose-derived stromal cells (ADSCs) to have osteogenic ability. Non-induced ADSCs can also produce bone in vivo and heal skeletal defects. The present study aimed to compare the bone-forming ability of osteogenically induced ADSCs and non-induced ADSCs in vivo. Tissue-engineered constructs were prepared from osteogenically induced or non-induced ADSCs and porous hydroxyapatite/beta-tricalcium phosphate scaffolds. A scaffold without cells and an empty defect group were used as control. All were implanted in rat critical calvarial defects. After implantation for 6 and 12 weeks, bone formation was analyzed using histomorphometry and microcomputed tomography; there were no significant differences in the formation of new bone between osteogenically induced ADSCs and non-induced ADSCs ( P  > 0.05). In conclusion, osteogenic induction of ADSCs is not an indispensable step for bone formation in vivo. Non-induced ADSCs can also be used as seeding cells to construct bone tissue.     

Osteogenesis of Adipose-Derived Stem Cells

Current treatment options for skeletal repair, including immobilization, rigid fixation, alloplastic materials and bone grafts, have significant limitations. Bone tissue engineering offers a promising method for the repair of bone deficieny caused by fractures, bone loss and tumors. The use of adipose derived stem cells (ASCs) has received attention because of the self-renewal ability, high proliferative capacity and potential of osteogenic differentiation in vitro and in vivo studies of bone regeneration. Although cell therapies using ASCs are widely promising in various clinical fields, no large human clinical trials exist for bone tissue engineering. The aim of this review is to introduce how they are harvested, examine the characterization of ASCs, to review the mechanisms of osteogenic differentiation, to analyze the effect of mechanical and chemical stimuli on ASC osteodifferentiation, to summarize the current knowledge about usage of ASC in vivo studies and clinical trials, and finally to conclude with a general summary of the field and comments on its future direction.     

人脂肪来源干细胞与胶原支架共培养的实验研究

目的观察评价人脂肪来源干细胞(Human adipose derived stem cells,hADSCs)在胶原支架中的生长情况,为进一步体内组织修复研究提供依据。方法取人抽脂术后脂肪,经胶原酶解、过滤、离心获得hADSCs,代传代扩增后,接种到胶原支架上。细胞-材料复合物分别体外培养1周、2周,裸鼠体内培养2周、4周后,HE染色观察细胞在支架上的生长情况,免疫组化HLA-Ⅰ检测经裸鼠体内培养后的复合物上的细胞的种属来源。结果原代培养的hADSCs呈"梭形"或"成纤维细胞"样,并以克隆团形式生长。免疫荧光Vimentin染色阳性。第3代hADSCs经流式细胞鉴定,CD29、CD44、CD105表达阳性,CD45、CD34表达阴性。胶原支架复合hADSCs经过体外、体内培养,hADSCs均能长入胶原支架的空隙内,且体内培养比体外培养有更多的细胞长入支架。体外培养1周,已有细胞粘附生长在胶原支架的边缘,体外培养2周后,更多的细胞渗透到材料内部。体内培养2周后,大量细胞占据材料的边缘,有部分细胞能渗透到材料内部甚至材料全层。体内培养4周后,大量细胞渗透支架全层。HLA-Ⅰ抗体检测发现,支架材料内部细胞阳性表达,说明胶原支架内部的细胞来源于hADSCs。结论胶原支架与hADSCs具有较好的相容性,可作为hADSCs的载体材料,用于组织工程缺损修复的研究。     

Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects

We compared bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) of adult rabbits under identical conditions in terms of their culture characteristics, proliferation capacity, osteogenic differentiation potentials induced by adenovirus-containing bone morphogenetic protein 4 (Ad-BMP4) in vitro, and capacity to repair calvarial defects in the rabbit model by autologous transplantation ex vivo. According to the results of growth curve, cell cycle, and telomerase activity analysis, ADSCs possess a higher proliferation potential. Both of the Ad-BMP4 transduced MSCs expressed BMP4 mRNA and protein and underwent osteogenic differentiation. Up-regulated mRNA expression of all osteogenic genes was observed in differentiated BMSCs and ADSCs, but with different patterns confirmed by real-time RT-PCR. Deposition of calcified extracellular matrix was significantly greater in differentiated ADSCs compared with differentiated BMSCs. X-ray and histological examination indicated significant bone regeneration in the calvarial defects transplanted with Ad-BMP4 transduced autologous MSCs compared to the control groups. There was no significant difference in new bone formation in Ad-BMP4 transduced MSCs based on quantitative digital analysis of histological sections. The use of ADSCs often resulted in the growth of fat tissue structures in the control groups, and the fat tissue structures were not seen with BMSC cells. Our data demonstrate that BMP4 can be potently osteoinductive in vivo, resulting in bone repair. ADSCs may be an attractive alternative to BMSCs for bone tissue engineering under appropriate stimuli. But the easy adipogenic differentiation needs to be considered when choosing adipose tissue for specific clinical application.     

Stem Cell Therapy for the Treatment of Hip Osteonecrosis: A 30-Year Review of Progress

Avascular necrosis of the femoral head is caused by a multitude of etiologic factors and is associated with collapse with a risk of hip arthroplasty in younger populations. A focus on early disease management with the use of stem cells was proposed as early as 1985 by the senior author (PH). We undertook a systematic review of the medical literature to examine the progress in cell therapy during the last 30 years for the treatment of early stage osteonecrosis.     

大鼠骨髓基质细胞的体外培养

目的：观察大鼠骨髓基质细胞在体外培养的条件,为骨组织工程学研究奠定一定实验基础。方法：取幼年SD大鼠,处死后分离骨髓,原代培养骨髓基质细胞,传代后分别观察其生长特性,绘制生长曲线,测定分裂指数和贴壁率。结果：大鼠骨髓基质细胞在第7代以前生长形状相对稳定,生长曲线相似;第4d分裂指数最高,为16％;传代后12h贴壁率最高,为90％。结论：本实验方法中,骨髓基质细胞在体外培养条件下,早期生长性状稳定,增殖速度快,适应性强,可作为骨组织工程学研究的种子细胞。     

脂肪干细胞在骨组织工程中的研究进展

脂肪干细胞上一类存在于脂肪组织中,能够自我更新、具有多向分化潜能的成体干细胞,在一定的条件下可以分化成许多具有特定功能的细胞系,具有一般干细胞的特点,可作为组织工程的种子细胞。本文就脂肪干细胞的生物学特性及其在骨组织工程中的研究进展进行综述。     

脂肪干细胞通过旁分泌作用改善异种脱细胞真皮基质体内植入效果的机制研究

目的 探讨脂肪干细胞(ADSCs)在异种脱细胞真皮基质(HADM)微环境中能否有效发挥其旁分泌功能,从而改善HADM体内植入的效果。方法 将ADSCs分别接种于HADM及培养皿上。分别收集两组细胞的总RNA,通过q RT-PCR检测ADSCs旁分泌基因的表达。将ADSCs预处理前后的HADM分别植入大鼠体内,4周后取材行组织学观察。结果 在HADM上培养的ADSCs与在普通培养皿上培养的ADSCs相比,其促血管形成因子(VEGF、HGF、bFGF)、炎症调节因子(TGF-β、i NOS、TSG-6、COX-2)及干性因子(SOX-2)均有所上调。经ADSC体外预培养后的HADM与未经处理的HADM相比,体内植入后表现出更好的血管化能力且炎症反应也更低。结论 HADM能为ADSCs发挥旁分泌作用提供合适的微环境,且HADM经过ADSCs体外预培养,其血管化程度更高,而炎症反应则得以缓解及局限化。     

骨髓基质干细胞的单克隆化培养和多向分化的实验研究

目的通过对骨髓基质干细胞（marrow stromal cells，MSCs）的单克隆化培养、鉴定和扩增，获得从一个单细胞扩增出的大量均质MSCs，并研究其多向分化能力。方法无菌条件下获取大鼠骨髓细胞，在培养的过程中挑出相应的单细胞克隆。将挑出的单细胞克隆与饲养细胞混合培养，快速进行扩增和细胞表面标志的鉴定，并进一步行增殖和细胞分化实验。结果在含有特定饲养层细胞的培养条件下，单克隆来源的MSCs能在抑制分化的状态下大量扩增，并保持细胞的均质性；而且在这种培养条件下，MSCs的增殖能力明显增强，并能成功被诱导向软骨、神经样细胞分化。结论通过骨髓细胞的单克隆培养，获得了均质和多向分化潜能的MSCs，为进一步更精确的研究MSCs的生物学特性提供了基础。