人工材料作为细胞体外培养载体的观察实验研究

目的：在体外培养的条件下了解人工材料与细胞的相容性以及细胞在材料上的生长情况。方法：将冻存复苏后的传代细胞在碳纤维、头发、涤纶、几丁质四种材料联合培养，进行光镜、扫描电镜观察。结果：在Ｆ－１２培养基中，碳纤维与成纤维细胞、肌腱细胞相容性均好，有良好的吸附性；眷属材料次之；涤纶材料上两种细胞生长均少，可能与其表面结构有关；几丁质则明显抑制这两种细胞生长。同时发现两种细胞在碳纤维上坐落的数量都明显优于     

兔肌腱细胞和成纤维细胞与人工材料体外联合培养的形态学观察

在兔肌腱细胞、成纤维细胞分离培养的基础上，将冻存复苏的传代细胞与碳纤维、涤纶及几丁质三种材料体外联合培养，观察细胞与材料的相容性以及细胞在材料上的生长情况，比较这两种细胞在三种材料上的生长差异。结果发现，在体外培养条件下，碳纤维与两种细胞的相容性均好，有良好的吸附性；涤纶材料上两种细胞生长均少，可能与其表面结构有关；几丁质明显抑制这两种细胞生长。同时发现，无论肌腱细胞，还是成纤维细胞，在碳纤维上座落的数量都明显优于涤纶、几丁质。三种材料交错在一起时，胶原纤维方向是以碳纤维为轴，形成网状，交织缠绕在材料之间。随时间延长，细胞在单根碳纤维或多根碳纤维上排列均匀，包绕碳纤维，且相互衔接。研究结果，为进一步研究有生命的人工肌腱、韧带提供了依据。     

兔肌腱细胞和成纤维细胞与人工材料体外联合培养的形…

在兔肌腱细胞、成纤维细胞分离培养的基础上，将冻存复苏的传代细胞与碳纤维、涤纶及几丁质三种材料体外联合培养，观察细胞与材料的相容性以及细胞在材料上的生长情况，比较这两种细胞在三种材料上的生长差异。随着时间延长，细胞在奶碳中多根碳纤维上排列无效，包绕碳纤维，且相互衔接。     

兔自体肌腱细胞与碳纤维联合培养后体内植入实验研究

为了观察兔自体肌腱细胞与碳纤维联合培养后植入体内的改变，采用自体肌腱细胞传代后与碳纤维编织带联合体外培养，植入兔体内，分期观察了肌腱细胞的形态学改变及合成胶原的类型。结果发现：体内植入后肌腱细胞能继续增殖，并能合成Ⅰ型胶原。同时发现肌腱细胞在植入４周后脱离碳纤维支架，在编织带间增殖、合成胶原，其胶原纤维相互衔接，形成了致密的组织结构。桥接肌腱处，胶原纤维互相衔接，表明植入体与受体肌腱已愈合。扫描电镜下观察肌腱细胞在碳纤维间排列整齐、均匀，胶原纤维间相互连接形成网状，纤维条索主体与碳纤维方向一致。透射电镜下细胞核核仁清晰，细胞器丰富。为进一步研究有生命的人工材料提供了依据。     

腱细胞与碳纤维体外复合组织学变化的实验研究

目的 了解兔腱细胞与碳纤维体外复合后植入体内形成肌键的可能性与可行性。方法在体我腱细胞培养的基础上，将冻存、复苏后的传代腱细胞（Ｎ６），与碳纤维体外复合形成人工活性材料后植入兔体内修复肌腱缺损，分期观察，结果 植入４后统计分析表明：自体与同种异体组腱细胞密度与胶原纤维含量差异无显著性意义，各组间细胞密度与胶原纤维含量呈正相关关系；腱细胞在碳纤维间增殖、合成Ⅰ型胶原，植入材料表面覆有光滑的表面结构。     

外源性胶原对人工活性肌腱影响的体外形态学观察

目的探讨外源性胶原在人工活性肌腱的构建中对细胞功能的影响. 方法将人发、碳纤维(carbon fiber, CF)及聚羟基乙酸(polyglycolic acid,PGA)3种材料复合外源性胶原(实验组)及不复合外源性胶原(对照组),与密度为3×106/mm3的转化人胚腱细胞进行体外联合培养,于培养后2小时、3天和5天3个时间点行倒置显微镜观察,以及培养5天行扫描电镜观察细胞数量及形态. 结果有胶原复合的实验组人工活性肌腱培养2小时后,细胞向材料周围聚集,附着于材料的部分细胞呈圆形;3天时附着的细胞增多,细胞与材料相容性好,细胞密度大.而无胶原复合的对照组3种材料上细胞附着较少.5天后实验组细胞形态由圆形→椭圆形→梭形演变,对照组细胞形态大部分仍为圆形及椭圆形,细胞数量仍少于实验组. 结论外源性胶原有助于细胞对材料的附着以及细胞自身的增殖.     

碳纤维人工腱、韧带研究进展

人工腱、韧带曾经采用各种材料,如丝织品、尼龙、涤纶等,但风行一时后均因材料萎陷而告失败。为了寻找一种合适的材料,从根本上开创一条新的径,Jinkin提出“腱、韧带诱导”的理论。纯质碳纤维由于其固有的不活泼特性和良好的生物相容性,被选作人工腱、韧带材料。最初的动物实验由Jinkin(1977)在一组绵羊跟腱置换中完成,采用编辫束碳纤维,     

人胚腱细胞培养及其生物学特征

目的：建立人腱细胞体外培养系，为肌腱愈合研究提供一种模型。方法：用贴块法培养人胚胎腱细胞，并观察了细胞生长、生化及形态特征，用免疫组化染色鉴定细胞表型。结果：人胚腱细胞在体外呈长梭形，胞质均匀透明，汇合后呈平行致密排列并表现接触抑制特征，传代后生长特征仍保持稳定，细胞免疫组化染色Ｖｉｍｅｎｔｉｎ及Ⅰ型胶原呈阳性，而Ｆｎ及Ⅲ型胶原呈阴性，人胚腱细胞最适合在含１０％胎牛血清及５％胎儿血清的ＤＭＥＭ培养基中生长。结论：本实验培养的人胚腱细胞为探索肌腱愈合机制及肌腱组织工程学研究提供了理想的实验材料     

组织工程化肌腱对T淋巴细胞亚群及其受体的影响

目的：探讨组织工程化肌腱对T淋巴细胞亚群及受体的影响。明确其组织相容性,方法：采用3月龄法国罗曼鸡75只,随机分成五组,每组15只,手术造成足第3趾屈深肌腱2．5cm缺损后,分组。A组：未手术组,B组：自体肌腱移植组;C组：新鲜同种异体肌腱移植组;D组：梯度降解材料复合腱细胞移植组;E组、;衍生肌腱材料复合腱细胞移植组,检测CD4＋,CD8＋,CD28细胞阳性率及T淋巴细胞相关抗原受体（TCR）密度变化,通过刀豆蛋白（ConA)诱导的淋巴细胞转化试验,直接淋巴细胞混合培养试验及间接淋巴细胞混合培养试验,了解机体对肌腱细胞的免疫反应水平。结果：D,E组与B组比较,早期CD4＋,CD8＋ＴＣＲ轻度增高,２周后下降,无显著性差异（Ｐ＞０．０５）,C组CD4＋,CD8＋,CD28及TCR阳性率与D,E组比较,有显著性差异（P＜0．05）,结论：肌腱细胞为弱抗原细胞,抗原脱落或可溶性抗原分泌较少,组织工程化肌腱有良好的组织相容性。     

立体分割的肌腱段组织体外培养的组织学研究

目的：研究不同区域的肌腱组成细胞在体外培养过程中的增殖能力及行为差异，方法：将鸡Ⅱ区趾深屈肌腱立体分割成四区：腱外膜组织、外膜下腱组织、亚中心腱组织、中心腱组织。两相同区域肌腱组织按间隔0．5、1、2mm三种排列方式培养于24孔培养板中，每种排列方式培养5孔。完整肌腱段组织也以相同方式培养作为对照。每天计数每种排列方式细胞游出数目、方向及来源，培养第9d作组织学检查，结果：细胞游出最早、数目最多的组织为腱外膜组织，其它依次为亚中心腱组织，中心腱组织，外膜下膜组织，两腱外膜组织距越小细胞游出越早，而其它区域的肌腱组织则相反。组织学检查显示培养第9d的中心腱组织，两腱外膜组织间距越小细胞游出越早，而其它区域的肌腱组织则相反，组织学检查显示培养第9d的中心腱组织，其腱内膜细胞比培养前增多；而培养第9d的完整肌腱段组织的中心腱组织区域，其腱内膜细胞比培养前减少。两者差异均有显著性意义（P＜0．01）。结论：不同区域的肌腱组成细胞在体外培养过程中的增殖能力及行为存在差异。     

生物衍生材料构建组织工程肌腱体内植入的实验研究

目的 探讨用同种异体肌腱细胞与生物衍生肌腱材料体外联合培养后植入体内，构建组织工程肌腱的可行性。方法 选用四川锦猴15只，手术造成纤维鞘管内屈指深肌腱2．5cm缺损后，分三组。A组：生物衍生肌腱材料构建的组织工程肌腱移植组；B组：单纯生物衍生肌腱材料移植组；C组：自体肌腱移植组。术后1、2、3、6和12周分期观察植入物的大体形态、组织学和超微结构，BrdU标记表达。结果 A组植入体内后肌腱细胞能继续增殖，细胞形态随着时间增加而逐渐趋于正常，形成的肌腱呈白色且有光泽、致密，组织学可见胶原纤维排列较为规则，12周肌腱细胞仍成活并分泌胶原，BrdU表达为阳性；B组植入体内3周后材料逐渐变细，12周后材料被逐渐降解吸收出现中断；C组植入体内2周后桥接部有纤维连接，排列较为规则，肌腱愈合。A组分别于3、6、12周进行扫描电镜观察可见肌腱细胞排列均匀，胶原纤维相互连接形成网状，主体趋势与肌腱走行方向一致；透射电镜下可见细胞核仁清晰，细胞器丰富。随时间的增加A、C组与B组的差异明显增大。结论 同种异体肌腱细胞与生物衍生肌腱材料复合构建的组织工程肌腱，植入免疫功能正常的动物体内能够再生出肌腱样组织，植入的肌腱细胞具有生命力，新生的肌腱组织在大体、组织学方面与正常肌腱相似。     

自体组织工程化肌腱预制的初步研究

目的：应用组织工程技术研究组织工程化肌腱体内形成的可行性。方法：取成年家鸡的趾深屈肌腱，用酶消化法分离，培养肌腱细胞，将在体外扩增到一定浓度的肌腱细胞接种到聚羟基乙酸（PGA）上，形成细胞－材料复合物，体外培养5d后，将此复合物回植至自体右翼皮下，左侧以单纯PGA作为对照，培养后第3，4，6，8周取材，从大体，组织学等方面进行分析。结果；术后8周见组织工程化肌腱呈白色，有光泽，组织学见胶原组织平行排列，但仍可见未降解的PGA及少量炎性细胞，对照组则无任何组织形成，结论：自体肌腱细胞与生物材料复合后在免疫功能正常的自体动物体内能够再生出肌腱样组织，新生的肌腱样组织在大体，组织学等方面均与正常肌腱相似。     

衍生肌腱支架材料的细胞相容性研究

目的 探讨衍生肌腱支架材料（tendon derivation biomaterials，TDBM）与肌腱细胞的细胞相容性及肌腱细胞在此三维支架上培养的生物学行为，为肌腱组织工程新型支架材料的应用提供依据。方法将肌腱细胞与TDBM体外复合培养，设置单纯肌腱细胞培养为对照组，进行形态学观察，并检测细胞增殖、细胞周期、细胞DNA倍体水平及肌腱细胞凋亡率。通过^3H-脯氨酸（^3H-Proline）掺入试验了解材料对肌腱细胞胶原合成的影响。结果 肌腱细胞在TDBM上呈梭形生长，TDBM组细胞第2天进入对数增长期，倍增时间为3d，而单纯肌腱细胞培养对照组倍增时间为3．75d。TDBM组与单纯肌腱细胞培养对照组比较，^3H-Proline掺入值差异无统计学意义（P〉0．05），说明细胞功能未受影响。与支架材料复合培养的肌腱细胞DNA指数为0．96，增殖指数较对照组高10．1％，提示肌腱细胞在三维胶原支架上生长速度快，增殖能力强。结论TDBM具有良好的细胞相容性，可作为肌腱细胞的有效载体应用于组织工程化肌腱的构建。     

梯度降解三维支架材料与肌腱细胞复合培养的实验研究

目的　探讨梯度降解肌腱支架材料 (GDBM)与肌腱细胞的生物相容性及肌腱细胞在三维支架上培养的生物学行为。　方法　将肌腱细胞与梯度降解肌腱支架材料体外复合培养 ,单纯培养为对照组。在 8h、2 4h、72h、7d、2个月进行倒置相差显微镜及扫描电镜形态学观察。 1、2、3、4、5、6、7、8d检测细胞增殖 (MTT法 )。流式细胞仪检测细胞周期、细胞DNA指数及肌腱细胞凋亡。通过3 H Proline掺入实验探讨支架材料对肌腱细胞胶原合成的影响。　结果　肌腱细胞在支架材料上呈梭形复层生长 ,GDBM组肌腱细胞第 2天进入对数增长期 ,倍增时间为 3 .2 5d ,而单纯培养对照组倍增时间为 3 75d。GDBM组与单纯培养对照组比较3 H Proline掺入值差异无显著性 (P >0 .0 5 ) ,说明细胞功能未受影响。与支架材料复合培养的肌腱细胞DNA指数为 0 96,增殖指数比对照组高 2 1% ,提示肌腱细胞在三维支架上生长速度快 ,增殖能力强。　结论　梯度降解肌腱支架材料具有良好的生物相容性 ,可作为肌腱细胞的有效载体应用于组织工程化肌腱的构建。     

Extracellular matrix expression of human tenocytes in three‐dimensional air–liquid and PLGA cultures compared with tendon tissue: Implications for tendon tissue engineering

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1170–1177, 2010     

Mechanism of mast cell adhesion to human tenocytes in vitro

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell‐fibroblast co‐culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte‐conditioned media; the tenocyte‐derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte‐derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte‐derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:9–16, 2015.

     

Characterization of a purified exosome product and its effects on canine flexor tenocyte biology

Flexor tendon injuries and tendinopathy are very common but remain challenging in clinical treatment. Exosomes-based cell-free therapy appears to be a promising strategy for tendon healing, while limited studies have evaluated its impacts on tenocyte biology. The objective of this study was to characterize a novel purified exosome product (PEP) derived from plasma, as well as to explore its cellular effects on canine tenocyte biology. The transmission electron microscope revealed that exosomes of PEP present cup-shaped structures with the diameters ranged from 80 to 141 nm, and the NanoSight report presented that their size mainly concentrated around 100 nm. The enzyme-linked immunosorbent assay kits analysis showed that PEP was positive for CD63 and AChE expression, and the cellular uptake of exosomes internalized into tenocyte cytoplasm was observed. The cell growth assays displayed that tenocyte proliferation ability was enhanced by PEP solution in a dose-dependent manner. Tenogenic phenotype was preserved as is evident by that tendon-related genes expression (SCX, COL1A, COL3A1, TNMD, DCN, and MKX) were expressed insistently in a high level, while tenocytes were treated with 5% PEP solution. Furthermore, migration capability was maintained and total collagen deposition was increased. More interesting, dexamethasone-induced cellular apoptosis was attenuated during the incubation of tenocytes with a 5% PEP solution. These findings will provide the basic understandings about the PEP, and support the potential use of this biological strategy for tendon healing.     

Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.