低强度脉冲超声对人脂肪干细胞成骨诱导影响的实验研究

[目的]探讨低强度脉冲超声在人脂肪干细胞成骨诱导中的影响。[方法]取手术患者腹部及腰背部处脂肪,采用胶原酶消化离心贴壁法分离培养脂肪间充质干细胞(adipose derived stem cells,ADSCs),流式细胞仪检测细胞表面标记。第6代细胞分为四组:(1)阴性对照组:ADSCs于完全培养基中培养,不接受LIPUS剌激;(2)LIPUS组:ADSCs于完全培养基中培养,接受LIPUS刺激;(3)成骨诱导组:ADSCs于成骨诱导培养基中培养;(4)LIPUS联合成骨诱导组:ADSCs在成骨诱导培养基中培养,接受LIPUS刺激。[结果]ADSCs形态传至10代时仍无变化。流式细胞仪分析第6代hADSCs细胞表面特异性标记结果显示,CD105:99.61%,CD29:97.54%,CD45:0.26%,CD44:97.4%,CD14:2.51%。LIPUS+诱导组的碱性磷酸酶(ALP)表达明显高于其他组,而单用LIPUS,并不能促进成人脂肪干细胞成骨分化。[结论]LIPUS可以促进成骨诱导培养基培养下的成人脂肪干细胞的成骨分化。     

人松质骨来源基质细胞克隆培养与成体干细胞特性研究

目的观察培养的人松质骨来源基质干细胞是否具有骨髓来源基质干细胞的多向分化的成体干细胞特性。方法取股骨、髂骨3mm×3mm×3mm松质骨，剪碎冲洗，用有限稀释法进行克隆培养，然后对传代细胞用化学药物或生长因子进行成骨、成软骨以及心肌细胞分化诱导培养，并检测成骨、成软骨及心肌细胞表型。结果克隆培养细胞培养2周开始表达CD29、CD59、CDll6；成骨诱导条件下2周细胞开始表达I型胶原、骨钙素，至第28天碱性磷酸酶阳性率可达95％；成软骨诱导条件下细胞表达Ⅱ胶原，甲苯胺蓝染色阳性；心肌细胞诱导条件下细胞表达肌钙蛋白，逆转录一聚合酶链反应（RT—PCR）检测有MLC-2V的mRNA表达，4周左右可见细胞连接形成合胞体样结构生长，并有自发性搏动。结论人松质骨来源基质干细胞具有多向分化能力的基质干细胞，在临床和实验研究中可以替代骨髓来源的问充质干细胞。     

体外培养的人脂肪间充质干细胞生物学特性的研究

目的:了解体外培养的人脂肪间充质干细胞的生物学特性及其体外成脂和成骨的能力.方法:体外培养人脂肪间充质干细胞并传代计数,行成骨和成脂诱导,流式细胞仪检测其细胞增殖周期与表面分子.结果:人脂肪干细胞经过体外培养均一的阳性表达CD44、CD106,而CD49d、CD34、CD45和HLA-DR表达阴性.细胞周期分析表明:G0/G1、S和G 2/M所占比例分别为79.1%、19.7%和1.3%.分离细胞在诱导体系下可以向成骨和成脂方向分化.结论:脂肪间充质干细胞具有特殊的生物学特点,体外能够向成骨和成脂方向分化.     

大鼠脂肪干细胞体外诱导成平滑肌样细胞的研究

目的:研究大鼠脂肪干细胞(ADSCs)体外单层培养条件下诱导分化为平滑肌样细胞的可行性。方法:从SD大鼠的腹股沟脂肪垫分离获取脂肪干细胞,测定其生长曲线。取第4代细胞用成脂诱导液诱导分化,并用油红O染色鉴定。取第4代细胞用成骨诱导液诱导分化,并用Von Kossa染色鉴定。取第4代细胞用含有β-巯基乙醇的成平滑肌诱导液诱导,并用免疫组化的方法检测α平滑肌肌动蛋白(α-SMA)的表达。结果:脂肪干细胞成梭形和多角形,体外生长迅速,生长曲线表明传代2 d后细胞进入对数生长期。第4代细胞成脂诱导后,油红O染色证实细胞内存在脂滴;成骨诱导后,Von Kossa染色证实有矿化结节。脂肪干细胞诱导平滑肌样细胞免疫组化结果,β-巯基乙醇诱导组和未诱导组细胞α-SMA的表达阳性率分别为(29.80±6.89)%、(2.89±1.24)%。诱导组细胞α-SMA的表达阳性率高于未诱导组,差异有统计学意义(P<0.01)。结论:脂肪干细胞经诱导后出现明显的平滑肌细胞特征,可成为平滑肌相关疾病在组织工程研究上新的种子细胞来源。     

小鼠胎肝间充质干细胞的体外成骨诱导及复合陶瓷化骨的实验研究

目的探讨小鼠胚胎肝脏间充质干细胞的体外分离培养方法，并观察其成骨分化潜能及与陶瓷化骨支架材料的复合能力。方法将小鼠胎肝组织制成单细胞悬液行原代和传代培养，流式细胞仪检测其表面标志。用化学成骨诱导体系对纯化的胎肝间充质干细胞行成骨诱导，并进行成骨功能检测。将其与经过Ⅰ型胶原表面改性的陶瓷化骨复合培养，观察细胞在其上的黏附生长情况。结果原代培养的胎肝间充质干细胞，具有集落形成能力，为梭形或多角形。易于传代，传代细胞与原代细胞大小、形态相似。流式细胞仪检测表明，传代后的细胞CD29、CD44阳性，CD34、CD45阴性，表达间充质干细胞的特征标志。成骨诱导7d后碱性磷酸酶染色可见有较多阳性细胞，Ⅰ型胶原免疫组织化学染色呈强阳性；14d后，细胞碱性磷酸酶活性定量检测明显增高；28d后，矿化结节染色呈阳性。将细胞与经胶原表面改性后的煅烧陶瓷化骨复合培养。扫描电镜见载体上有大量细胞黏附于材料表面。结论小鼠胎肝间充质干细胞易于获取及传代；体外成骨诱导后可向成骨细胞方向分化；能在陶瓷化骨支架材料上黏附生长。     

骨质疏松小鼠模型骨髓间充质干细胞特性的研究

目的 探讨骨质疏松小鼠模型骨髓间充质干细胞（mesenchymal stem cells ,MSCs）生物学特性的改变。方法 通过皮下注射地塞米松构建小鼠骨质疏松动物模型（OP组）,同时设立对照（NC组）,分离OP组及NC组骨髓MSCs进行体外培养;通过流式细胞术检测细胞表面分化抗原;运用CKK-8实验检测细胞增殖活力;通过脱氧核苷酸末端转移酶介导的dUTP缺口末端标记法（TUNEL）检测细胞凋亡水平;运用碱性磷酸酶（ALP）活性检测、油红O染色及Western blot法检测两组细胞成骨分化及脂肪分化能力。结果 经过骨组织学检测证明模型建立成功,体外培养OP组及NC组骨髓MSCs细胞形态无差异。OP-MSCs细胞表面分化抗原Scal1及CD44为阳性, CD34及CD11b为阴性,与NC-MSCs相似,但OP-MSCs增殖活力下降（P<0.05）;此外两组细胞凋亡水平类似（P>0.05）。成骨分化诱导7 d后OP-MSCs的ALP活性显著低于对照组（P<0.01）,21 d后矿化小结明显减少（P<0.001）;脂肪分化诱导8 d后OP-MSCs形成更多脂滴（P<0.05）。Western blot结果显示OP-MSCs在成骨分化中低表达转录因子Runx2（P<0.05）及Osterix (P<0.001）,但在脂肪分化早期高表达PPARγ（P<0.001）及C/EBPα（P<0.01）。结论 骨质疏松小鼠骨髓MSCs的增殖及分化潜能显著下降。     

流式细胞仪分选纯化人脂肪干细胞体外成骨活性的实验研究

目的利用流式细胞仪分选人脂肪干细胞(Human adipose-derived stem cells,hADSCs)并检测其体外成骨活性。方法分离hADSCs,通过流式细胞仪以CD105作为表面标志进行分选,所得细胞成骨诱导培养,以CD105-细胞、未分选细胞作为对照。2周后进行碱性磷酸酶(AKP)和骨钙蛋白(OCN)定量PCR和Western-blot检测。结果流式细胞分选所得CD105+hADSCs细胞约占单核细胞的50%。定量PCR和Western-blot检测均显示分选hADSCs成骨诱导后,AKP和OCN的表达均显著高于CD105-细胞组及未分选细胞组(P<0.05)。结论诱导分选纯化后的hADSCs有较好的成骨活性,可作为骨组织工程的种子细胞。     

负载碱性成纤维细胞生长因子、骨形态发生蛋白-12基因的兔脂肪干细胞向成纤维细胞分化的实验研究

目的探讨碱性成纤维细胞生长因子(bFGF)、骨形态发生蛋白(BMP)-12基因序贯转染兔脂肪干细胞向成纤维细胞分化的可能性。方法2017年1月至2018年12月,原代培养兔脂肪干细胞(取自新西兰大耳兔颈背部皮下脂肪),细胞表面抗原CD44、CD49d、CD106检测结合成骨细胞诱导分化对培养的细胞进行鉴定,脂质体介导的方法序贯转染BMP-12和bFGF基因,蛋白质印迹法(Western blot)检测目的基因BMP-12和bFGF蛋白的表达;实时荧光定量聚合酶链反应(Real-time PCR)检测转染细胞Ⅰ型胶原和弹性蛋白RNA的表达情况。符合正态分布的计量资料以均数±标准差(±s)表示,应用单因素方差分析比较组间差异。结果兔脂肪组织中分离出来的脂肪间充质干细胞(ADSCs)CD44、CD49d表达阳性,CD106表达呈阴性。Western blot检测筛选后的ADSCs稳定表达BMP-12(0.229±0.005)和bFGF(0.208±0.019)蛋白(F=120.221,P<0.05),差异有统计学意义。双基因转染的ADSCsⅠ型胶原(2.250±0.007,F=340.103,P<0.05)和弹性蛋白(1.807±0.008,F=107.314,P<0.05)的mRNA表达最高,差异均有统计学意义。结论pcDNA3.1+绿色荧光蛋白(GFP)-BMP-12和pcDNA3.1+红色荧光蛋白(RFP)-bFGF可以促进ADSCs向成纤维细胞分化,可能在组织工程技术修复韧带损伤中具有重要作用。     

脂肪干细胞与人工骨混合后的成骨作用研究

目的探索人脂肪干细胞与不同种类的人工骨混合后的成骨效果。方法体外分离、鉴定脂肪干细胞,与3种类型（羟基磷灰石生物陶瓷、源自小牛骨的天然无机骨和重组异种骨）且不同浓度的人工骨混合培养,并设立对照组,利用茜素红染色及染色洗脱液吸光度值测定来对成骨效果进行比较。结果羟基磷灰石生物陶瓷和低浓度小牛骨与脂肪干细胞混合培养．其成骨效果要明显优于对照组（P〈O．05）：高浓度小牛骨与脂肪干细胞混合培养的成骨效果与对照组比较差异无统计学意义。重组异种骨与脂肪干细胞混合培养的成骨效果差于对照组。结论人工骨提供支架的脂肪干细胞诱导成骨的效果要优于单纯诱导培养脂肪干细胞。在一定浓度条件下．羟基磷灰石作为人工骨支架能够获得更好的成骨效果。     

脂肪干细胞诱导分化成淋巴管内皮样细胞的初步研究

目的 探讨利用血管内皮细胞生长因子C(VEGF-C) (156s)等生长因子诱导脂肪干细胞成为淋巴管内皮样细胞的方法. 方法 取健康成人脂肪组织,胰酶消化法获得脂肪组织来源的间质干细胞(adipose-derived stem cell),通过流式细胞仪检测其表面标记,并进行成脂肪、成骨诱导等体外分化能力鉴定.取生长状态良好的P3代细胞进行诱导实验:设置含VEGF-C156 s、bFGF等生长因子的诱导液组为实验组,并设空白对照组(常规L-DMEM培养基).观察诱导前后两组细胞的形态变化,并于10 d后进行LYVE-1免疫荧光的鉴定. 结果 成功分离纯化脂肪干细胞,流式细胞结果显示,CD13、CD29、CD44、CD105高表达,CD31、CD34、CD45、HLA-DR极低表达,具有间充质干细胞特性;体外成脂和成骨能力鉴定也取得成功;在体外成功利用含VEGF-C156 s、bFGF等生长因子的诱导液将脂肪干细胞诱导为淋巴管内皮样细胞,实验组LYVE-1免疫荧光显色呈强阳性,而对照组则未见到阳性染色;荧光强度定量结果差异有统计学意义(P＜0.01). 结论 利用含有VEGF-C156 s的诱导液可以将脂肪干细胞诱导分化为淋巴管内皮样细胞.     

髌下脂肪垫对全膝关节置换术后患者膝关节功能恢复影响的meta分析

目的通过meta分析评价髌下脂肪垫对全膝关节置换患者术后功能恢复的影响。 方法检索Cochrane Library、Embase、PubMed英文数据库和万方数据库、维普数据库以及中国知网数据库有关于髌下脂肪垫对全膝关节置换术后影响的临床随机对照试验（RCTs）和回顾性队列研究实验（RCSs）,使用Revman5.3软件进行meta分析。 结果最终共纳入8篇文献,其中英文文献4篇,中文文献4篇,共纳入全膝关节置换术病例1 474例,其中髌下脂肪垫切除术696例,髌下脂肪垫保留组778例。与髌下脂肪垫切除相比,髌下脂肪垫保留组可降低术后髌韧挛缩（MD=-2.34,95% CI：-3.38~-1.31,P<0.05）,麦克马斯特骨关节炎指数（WOMAC）评分（MD=2.34,95% CI：2.04~2.65,P<0.05）;提高膝关节功能评分（MD=-0.77,95% CI：-1.02~-0.52,P<0.05）,降低术后膝关节前侧疼痛发生率（RR=12.58,95% CI：3.24~48.75,P<0.05）。两组患者在术后膝关节活动度（MD=-7.53,95% CI：-20.27~5.21,P>0.05）,髌腱长度与髌骨最大长轴比值（Insall-Savatict）评分法（MD=-0.04,95% CI：-0.09~0.01,P>0.05）;术后膝关节返修率：（RD=0.01,95% CI：-0.01~0.02,P>0.05）方面无明显差异。 结论与髌下脂肪垫去除组相比,全膝关节置换术后髌下脂肪垫保留可以降低髌韧带痉挛程度,提高膝关节功能评分,同时可降低膝关节前侧疼痛。     

Patellar tendon length after knee arthroplasty with and without preservation of the infrapatellar fat pad

Our aim was to assess whether there was any significant difference in change in patellar tendon length after knee arthroplasty, when the infrapatellar fat pad was either preserved or excised. Three-year radiographic follow-up was studied on 73 primary knee arthroplasty patients. The infrapatellar fat pad was completely preserved in 38 cases and completely excised in 35. At 3 years there was a significant patellar tendon shortening of 4.2% (P = .0004) in the fat pad excision group and no significant change in the fat pad preservation group (P = .82). The difference between the 2 groups was significant (P = .004). Our results show that patella tendon length does not always shorten after knee arthroplasty and that preservation of the infrapatellar fat pad may be a factor in preventing such shortening.     

A giant extrasynovial osteochondroma in the infrapatellar fat pad: end stage Hoffa’s disease

The infrapatellar fat pad of Hoffa is commonly injured but rarely discussed in the orthopaedic literature. Hoffa’s disease is the extension of various traumatic events due to impingement and inflammation of the infrapatellar fat pad and known as a vague reason for anterior knee pain. Inflammation is foreground during acute phase of the disease while impingement due to fibrosis and scar tissue of infrapatellar fat pad plays a major role in the chronic phase. The osteochondroma of the infrapatellar fat pad secondary to the Hoffa’s disease can be more problematic. Although, the fibrocartilaginous transformation and osteochondral metaplasia of infrapatellar fat pad was pointed out frequently in the literature, the published papers seem far from clarifying the relation between chronic impingement and formation of osteochondroma. We present a case of a giant ossifying chondroma in the infrapatellar fat pad that resulted from chronic Hoffa’s disease. Complete open resection was performed successfully after arthroscopic examination. The infrapatellar fat pad contains the entire progenitor cells for the development of an osteochondroma and chronic impingement may have promoter affect on this issue, thus, an osteochondroma may occur at the end-stage Hoffa’s disease.     

Distribution of substance-P nerves inside the infrapatellar fat pad and the adjacent synovial tissue: a neurohistological approach to anterior knee pain syndrome

Introduction The purpose of the study was to determine the distribution and number of nerves inside the infrapatellar fat pad and the adjacent synovium, in particular with regards to nociceptive substance-P nerves.Materials and methods The infrapatellar fat pad of the knee was resected from 21 patients (4 male, 17 female, mean age 69 years) during the course of standard total knee arthroplasty operations performed in our clinic. The fat pad was dissected into five standardized segments, fixed in formalin and embedded in paraffin. Immunohistochemical techniques using antibodies against S-100 protein and substance-P (SP) were employed to determine and specify the nerves.Results Studying all the detectable nerves present in 50 observation fields (200-fold magnification), we found an average of 106 S-100 versus 25 SP nerves (24%) in the synovium and 27 S-100- versus 7 SP nerves (26%) in the interior of the fat pad. The total nerve count was significantly ( P <0.001) higher in the synovium than in the fat pad for both marker types. The number of S-100 nerves was significantly ( P <0.05) higher in the central and lateral segments of the fat pad, while SP nerves were equally distributed throughout all segments of the fat-pad. SP nerves were significantly more frequently associated with blood vessels inside the fat pad (43%, P <0.05) than in the synovial tissue (28%).Conclusion The occurrence and distribution of SP nerves inside the infrapatellar fat pad suggest a nociceptive function and a neurohistological role in anterior knee pain syndrome. The data support the hypothesis that a neurogenous infection of the infrapatellar fat pad could contribute to anterior knee pain syndrome.     

Influence of an infrapatellar fat pad edema on patellofemoral biomechanics and knee kinematics: a possible relation to the anterior knee pain syndrome

Introduction  An edema of the infrapatellar fat pad following knee arthroscopy or in case of chronic anterior knee pain syndrome is suspected to increase the patellofemoral pressure by a modification of the patellofemoral glide mechanism. The study was performed to evaluate this hypothesis. Materials and methods  Isokinetic knee extension from 120° of flexion to full extension was simulated on 10 human knee cadaver specimens (six males, four females, average age at death 42 years) using a knee kinemator. Joint kinematics was evaluated by ultrasound sensors (CMS 100^TM, Zebris, Isny, Germany), and retro-patellar contact pressure was measured using a thin-film resistive ink pressure system (K-Scan^TM 4000, Tekscan, Boston). Infrapatellar tissue pressure was analyzed using a closed sensor cell which was implanted inside the fat pad (GISMA, Buggingen, Germany). An inflatable fluid cell was implanted by ultrasound control in the center of the infrapatellar fat pad and filled subsequently with water to simulate a fat pad edema. All parameters were recorded and analyzed from 0 to 5 ml volume of the fluid cell. Results  Simulating a fat pad edema resulted in a significant (P < 0.01) increase of the infrapatellar fat pad pressure (247 mbar at 0 ml to 615 mbar at 5 ml volume). In knee extension and flexion the patella flexion (sagittal plane) was decreased while we did not find any other significant influence of the edema on knee kinematics. During the analysis of the patellofemoral biomechanics, a simulated fat pad edema resulted in a significant (P < 0.05) decrease of the patellofemoral force between 120° of knee flexion and full extension. The contact area was reduced significantly near extension (0°–30°) by an average of 10% while the contact pressure was reduced at the entire range of motion up to 20%. Conclusion  An edema of the infrapatellar fat pad does not cause an increase of the patellofemoral pressure or a significant alteration of the patellofemoral glide mechanism. Anterior knee pain in case of a fat pad edema may be related to a significant increase of the tissue pressure and possible histochemical reactions. The study was funded by the German Speaking Association of Artroscopy (AGA).     

Multipotent stromal cells derived from the infrapatellar fat pad of the knee

Tissue engineering approaches for promoting the repair of skeletal tissues have focused on cell-based therapies involving multipotent stromal cells. Recent studies have identified such cells in several tissues in the adult human, including skin, muscle, bone marrow, and subcutaneous fat. This study examined the hypothesis that the infrapatellar fat pad of the adult knee contains progenitor cells that have the ability to differentiate into chondrocytes, osteoblasts, or adipocytes under appropriate culture conditions. Cells isolated from the fat pad stroma had a profile of cell-surface molecules similar but not identical to that of bone marrow-derived mesenchymal stem cells. Using defined culture conditions, fat pad-derived stromal cells were induced to differentiate cells with phenotypic characteristics of: (1) chondrocytes, synthesizing cartilage matrix molecules; (2) adipocytes, producing lipid vacuoles and leptin; or (3) osteoblasts, forming mineralized tissue. The culture conditions also modulated the expression of characteristic gene markers for each lineage. This study supports the hypothesis that multipotent stromal cells are present in many connective tissues in the adult human. Given its location and accessibility, the fat pad may prove to be a potential source of progenitor cells for musculoskeletal tissue engineering.     

髌下脂肪垫来源间充质干细胞软骨分化的研究进展

间充质干细胞(MSCs)为重点的组织工程学研究已成为软骨损伤修复与再生的研究热点,髌下脂肪垫(IPFP)是脂肪间充质干细胞(ASCs)的新型优良组织来源,其取材方便,供区损伤小。且IPFP-ASCs体外增殖快,成软骨分化能力较强。低氧,转化生长因子β1、β3,骨形态发生蛋白7等可以促进其分化。体内试验表明其可以有效改善软骨损伤患者症状,提高关节功能。虽然目前仍存在生物学和技术上的困难需克服,但IPFP-ASCs有望成为修复包括骨关节炎在内的关节软骨损伤的良好策略。     

类风湿膝关节置换术中髌下脂肪垫切除与否对髌骨高度的影响

目的:比较类风湿关节炎膝关节置换术中切除或保留髌下脂肪垫对术后髌骨高度的影响。方法:回顾性分析2013年10月至2017年10月行全膝关节置换术的类风湿关节炎患者48例,分为切除及保留髌下脂肪垫组。切除组23例,男9例,女14例;年龄48～69(55.83±5.65)岁;术中采取常规切除髌下脂肪垫。保留组25例,男6例,女19例;年龄49～70(55.52±6.28)岁;术中采取完整保留髌下脂肪垫。观察两组患者术后并发症情况,术后1年采用视觉模拟疼痛评分(visual analogue scale,VAS)评价疼痛缓解程度,采用美国特种外科医院(Hospital for Specical Surgery,HSS)评分进行膝关节疗效评价,并采用Insall-Salvati比值法(I-S比)比较两组术后髌骨高度改变情况。结果:所有患者获得随访,时间12～39(23.85±8.82)个月。两组患者术后伤口愈合良好,未发生感染并发症,术后随访无假体松动、翻修发生。两组术后1年VAS评分较术前明显降低,但术后1年两组间VAS评分比较差异无统计学意义(0.05)。术后1年两组HSS评分较术前改善(P0.05),但术后1年两组之间HSS评分比较差异无统计学意义(P0.05)。术后1年切除组I-S比(1.03±0.04)小于髌下脂肪垫保留组(1.06±0.06),差异有统计学意义(P0.05)。结论:类风湿关节置换术中切除或保留髌下脂肪垫在术后疼痛缓解及功能恢复方面都能获得良好疗效,但术中保留髌下脂肪垫有利于髌骨高度的恢复。