A comparison of tenocytes and mesenchymal stem cells for use in flexor tendon tissue engineering

PURPOSE: Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts. METHODS: Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by beta-galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction. RESULTS: All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types. CONCLUSIONS: This study suggests that the four cell types may be successfully used to engineer tendons. Adipoderived mesenchymal stem cells proliferate faster in cell culture, but the cell types were similar in other respects. All could be used to successfully repopulate acellularized tendon in vivo as flexor tendon grafts.     

Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering

Articular cartilage is a load‐bearing tissue playing a crucial mechanical role in diarthrodial joints, facilitating joint articulation, and minimizing wear. The significance of biomechanical stimuli in the development of cartilage and maintenance of chondrocyte phenotype in adult tissues has been well documented. Furthermore, dysregulated loading is associated with cartilage pathology highlighting the importance of mechanical cues in cartilage homeostasis. The repair of damaged articular cartilage resulting from trauma or degenerative joint disease poses a major challenge due to a low intrinsic capacity of cartilage for self‐renewal, attributable to its avascular nature. Bone marrow‐derived mesenchymal stem cells (MSCs) are considered a promising cell type for cartilage replacement strategies due to their chondrogenic differentiation potential. Chondrogenesis of MSCs is influenced not only by biological factors but also by the environment itself, and various efforts to date have focused on harnessing biomechanics to enhance chondrogenic differentiation of MSCs. Furthermore, recapitulating mechanical cues associated with cartilage development and homeostasis in vivo, may facilitate the development of a cellular phenotype resembling native articular cartilage. The goal of this review is to summarize current literature examining the effect of mechanical cues on cartilage homeostasis, disease, and MSC chondrogenesis. The role of biological factors produced by MSCs in response to mechanical loading will also be examined. An in‐depth understanding of the impact of mechanical stimulation on the chondrogenic differentiation of MSCs in terms of endogenous bioactive factor production and signaling pathways involved, may identify therapeutic targets and facilitate the development of more robust strategies for cartilage replacement using MSCs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:52–63, 2018.     

间充质干细胞在软骨组织工程中的应用

间充质干细胞具有高度自我更新能力,能够分化为各种类型的细胞.近20年间充质细胞在软骨组织工程中得到应用,动物实验显示了良好的软骨修复潜能,成为软骨组织工程的理想的细胞来源.组织工程和干细胞技术已经确定为对软骨缺损有效的治疗方法,间充质干细胞有望在软骨组织工程中得到更广泛的应用.本文对间充质干细胞的特性和在组织工程中的应用进行了综述.     

Tendon-derived stem cells (TDSCs) promote tendon repair in a rat patellar tendon window defect model

Injured tendons heal slowly and often result in the formation of mechanically and functionally inferior fibrotic scar tissue or fibrous adhesions. This study investigated the use of tendon-derived stem cells (TDSCs) for tendon repair in a rat patellar tendon window defect model. Fibrin glue constructs with or without GFP-TDSCs were transplanted into the window defect. The patellar tendons were harvested for histology, ex vivo fluorescent imaging and biomechanical test at various time points up to week 4. Our results showed that TDSCs significantly enhanced tendon healing as indicated by the increase in collagen production as shown by hematolxylin stain-ability of the tissue, improvement of cell alignment, collagen fiber alignment and collagen birefringence typical of tendon. The labeled cells were observed at weeks 1 and 2 and became almost undetectable at week 4. Both the ultimate stress and Young's modulus were significantly higher in the TDSCs group compared to those in the fibrin glue group at week 4. In conclusion, TDSCs promoted earlier and better repair in a rat patellar tendon window defect model.     

Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering.

The use of multipotent human mesenchymal stem cells (hMSCs) for tissue engineering has been a subject of extensive research. The donor variation in growth, differentiation and in vivo bone forming ability of hMSCs is a bottleneck for standardization of therapeutic protocols. In this study, we isolated and characterized hMSCs from 19 independent donors, aged between 27 and 85 years, and investigated the extent of heterogeneity of the cells and the extent to which hMSCs can be expanded without loosing multipotency. Dexamethasone-induced ALP expression varied between 1.2- and 3.7-fold, but no correlation was found with age, gender, or source of isolation. The cells from donors with a higher percentage of ALP-positive cells in control and dexamethasone-induced groups showed more calcium deposition than cells with lower percentage of ALP positive cells. Despite the variability in osteogenic gene expression among the donors tested, ALP, Collagen type 1, osteocalcin, and S100A4 showed similar trends during the course of osteogenic differentiation. In vitro expansion studies showed that hMSCs can be effectively expanded up to four passages (approximately 10-12 population doublings from a P0 culture) while retaining their multipotency. Our in vivo studies suggest a correlation between in vitro ALP expression and in vivo bone formation. In conclusion, irrespective of age, gender, and source of isolation, cells from all donors showed osteogenic potential. The variability in ALP expression appears to be a result of sampling method and cellular heterogeneity among the donor population.     

滑膜间充质干细胞及其在组织工程中的应用

目的对滑膜间充质干细胞(synovium-derived mesenchymal stem cells,SMSCs)的研究进展及在组织工程中的应用进行综述。方法查阅近年SMSCs相关文献,从分离培养方法、基本特性及在组织工程中的应用三方面进行综述。结果 SMSCs具有分离方法简便、增殖能力及多分化能力较强的特点,已有研究将其应用于软骨、肌腱、韧带及骨组织工程领域。结论 SMSCs是MSCs家族的新成员,可能会成为组织工程领域新的种子细胞,还需要进一步深入研究。     

转基因骨髓间充质干细胞在软骨组织工程中的研究与应用

软骨组织工程学是用工程学和生命科学的原理和方法、依靠支架材料承载被分离的软骨细胞或其前体细胞并植入宿主体内、支架材料在宿主体内逐渐降解并释放细胞、从而形成新的有功能的软骨组织产品的技术。但目前存在的自身不足为：细胞因子的量控、种子细胞易老化、增值缓慢；细胞的定向分化、细胞与生物材料的亲和性以及动物组织基因人源化等。     

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.     

不同来源的间充质干细胞治疗骨与软骨组织疾病的研究进展

近年来,随着细胞和组织工程技术的发展,间充质干细胞广泛受到关注和研究,具有易分离获取、培养过程相对简单等优点,并且能够自我更新并分化成多种细胞类型,包括成骨细胞、软骨细胞、脂肪细胞等,是较为理想的种子细胞。在骨髓间充质干细胞大量的研究基础上,脂肪、骨骼肌、滑膜等多种不同来源的间充质干细胞也广泛应用在骨及软骨组织的体内研究和体外研究中。虽然间充质干细胞在基础性研究方面取得了飞跃进展,但在临床推广应用干细胞治疗上还面临着诸多问题,如对间充质干细胞的分化机理尚不明确,对其定向分化无法进行精确调控,且存在诸多限制骨和软骨再生的几个因素,很大程度上影响治疗的效果,故仍需进一步深入研究。     

Smad8/BMP2‐engineered mesenchymal stem cells induce accelerated recovery of the biomechanical properties of the achilles tendon

Tendon tissue regeneration is an important goal for orthopedic medicine. We hypothesized that implantation of Smad8/BMP2‐engineered MSCs in a full‐thickness defect of the Achilles tendon (AT) would induce regeneration of tissue with improved biomechanical properties. A 2 mm defect was created in the distal region of murine ATs. The injured tendons were then sutured together or given implants of genetically engineered MSCs (GE group), non‐engineered MSCs (CH3 group), or fibrin gel containing no cells (FG group). Three weeks later the mice were killed, and their healing tendons were excised and processed for histological or biomechanical analysis. A biomechanical analysis showed that tendons that received implants of genetically engineered MSCs had the highest effective stiffness (>70% greater than natural healing, p < 0.001) and elastic modulus. There were no significant differences in either ultimate load or maximum stress among the treatment groups. Histological analysis revealed a tendon‐like structure with elongated cells mainly in the GE group. ATs that had been implanted with Smad8/BMP2‐engineered stem cells displayed a better material distribution and functional recovery than control groups. While additional study is required to determine long‐term effects of GE MSCs on tendon healing, we conclude that genetically engineered MSCs may be a promising therapeutic tool for accelerating short‐term functional recovery in the treatment of tendon injuries. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1932–1939, 2012     

Studies in flexor tendon reconstruction: biomolecular modulation of tendon repair and tissue engineering

The Andrew J. Weiland Medal is presented each year by the American Society for Surgery of the Hand and the American Foundation for Surgery of the Hand for a body of work related to hand surgery research. This essay, awarded the Weiland Medal in 2011, focuses on the clinical need for flexor tendon reconstruction and on investigations into flexor tendon biology. Reconstruction of the upper extremity is limited by 2 major problems after injury or degeneration of the flexor tendons. First, adhesions formed after flexor tendon repair can cause decreased postoperative range of motion and hand function. Second, tendon losses can result from trauma and degenerative diseases, necessitating additional tendon graft material. Tendon adhesions are even more prevalent after tendon grafting; therefore these 2 problems are interrelated and lead to considerable disability. The total costs in terms of disability and inability to return to work are enormous. In this essay, published work from the past 12 years in our basic science laboratory is summarized and presented with the common theme of using molecular techniques to understand the cellular process of flexor tendon wound healing and to create substances and materials to improve tendon repair and regeneration. These are efforts to address 2 interrelated and clinically relevant problems that all hand surgeons face in their practice.     

人脐带间充质干细胞与蚕丝蛋白支架构建组织工程脂肪的研究

目的：将体外以人脐带间充质干细胞（hUCMSCs）与蚕丝蛋白支架初步构建的组织工程脂肪移植到大鼠体内,观察其演变过程。方法：hUCMSCs与蚕丝蛋白支架复合培养10天后,进行成脂诱导;6周后将其移植到Wi st ar大鼠后肢肌肉内,同时,以同体积支架材料作为对照;分别于移植后4周和8周取材,行油红O染色、HE染色以及扫描电镜观察。结果：hUCMSCs与蚕丝蛋白支架复合培养及成脂诱导6周后,见大量成脂样细胞生成,并与支架牢固粘附。移植4周,移植物体积略小,质稍硬,表面有透明薄膜形成,膜中分布新生血管网;油红O染色见支架内新生脂肪组织及细胞呈橙红色;HE染色显示支架网眼内有新生脂肪组织,并可见少量炎性细胞浸润;扫描电镜见支架网眼内有球形、表面光滑的脂肪细胞。移植8周,移植物体积进一步缩小,质变软,表面薄膜内血管网丰富;油红O染色见支架中着橙红色组织较前明显增多,部分呈片状融合;HE染色显示新生脂肪明显增多,仍有少量炎性细胞浸润;扫描电镜显示脂肪细胞较前增生明显。对照组同样可见炎性细胞浸润,未见新生脂肪组织生成,支架材料8周时较4周时降解更加明显。结论：随着时间推移,蚕丝蛋白支架网眼内脂肪细胞逐渐增多,支架材料在体内呈现逐步降解趋势,说明体内环境有利于组织工程化脂肪的进一步形成。同时,也提示支架材料在组织相容性方面尚存不足。     

Tendon stem progenitor cells: Understanding the biology to inform therapeutic strategies for tendon repair

Tendon and ligament injuries are a leading cause of healthcare visits with significant impact in terms of economic cost and reduced quality of life. To date, reparative strategies remain largely restricted to conservative treatment or surgical repair. However, these therapies fail to restore native tendon structure and function; thus, the tissue may re‐rupture or degenerate with time. To improve tendon healing, one promising strategy may be harnessing the innate potential of resident tendon stem/progenitor cells (TSPCs) to guide tenogenic regeneration. In this review, we outline recent advances in the identification and characterization of putative TSPC populations, and discuss biochemical, biomechanical, and biomaterial methods employed for their culture and differentiation. Finally, we identify limitations in our current understanding of TSPC biology, key challenges for their use, and potential therapeutic strategies to inform cell‐based tendon repair. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1270–1280, 2019.     

脂肪组织来源干细胞与脂肪瘤间充质干细胞特性的比较研究

目的 比较体外培养的脂肪来源干细胞(adipose-derived stem cells,ASCs)与脂肪瘤间充质干细胞(lipoma-derived mesenchymal stem cells,LMSCs)的生物特性,以探讨ASCs移植的安全性.方法 对正常脂肪组织和脂肪瘤组织进行切片染色,分离培养ASCs和LMSCs,观察细胞形态;MTS比色法检测细胞活性并绘制细胞生长曲线;流式细胞仪测定细胞周期及表面分子表达;QRT-PCR检测高迁移率族蛋白2(HMGA2)表达水平;免疫组织化学染色法鉴定端粒酶逆转录酶(hTERT)的表达.结果 正常脂肪组织和脂肪瘤组织切片差异明显;ASCs细胞形态一致性好,而LMSCs具有不均质性;MTS活性测定ASCs增殖活性要远低于LMSCs细胞(P=0.000);流式细胞仪检测结果显示ASCs与LMSCs在干细胞标志CD29、CD44、CD105上表达类似,而在肿瘤干细胞标志CD133表达上,ASCs(5.35%)要远低于LMSCs(26.87%);细胞周期显示ASCs的增殖能力低于LMSCs;定量PR-PCR显示ASCs中HMGA2平均aQ值为1,远低于在LMSCs中的表达(1.79),两者差异具有统计学意义(P<0.01);免疫细胞化学结果:hTERT在ASCs和LMSCs中的累计吸光度A值分别为1 379.597±498.617和3 328.108±902.856,面积分别为132 390.27±35 568.945和238 000.53±49 264.289,平均吸光度A值分别为0.009±0.003和0.014±0.003,ASCs中hTERT表达远低于LMSCs.结论 体外培养的ASCs生物学特性与LMSCs存在显著差异,未发现其恶性转、化为肿瘤干细胞的证据.     

载骨髓间充质干细胞的磷酸钙骨水泥的制备

[目的]观察载骨髓间充质干细胞(Bone marrow mesenchymal stem cells,MSCs)明胶微球磷酸钙骨水泥(Calcium phosphate cement,CPC)的理化性质,探究新型CPC的制备。[方法]制备载MSCs的明胶微球后,分别以0%(A),2.5%(B),5%(C),10%(D)的质量比(w/w)与CPC复合。测定其抗压强度、初凝时间、孔隙率和大孔率等理化性质,进一步检测MSCs在CPC中的成长情况。[结果]在各组CPC中MSCs均生长良好,且与空白对照组比较,随着明胶微球比例的增大,各组CPC的抗压强度逐步降低,初凝时间延长,孔隙率增大,大孔率增高(P<0.05)。[结论]载MSCs的明胶微球与CPC的质量比为5%时,CPC的理化性质最为理想,细胞生长良好,此结果为下一步体内实验奠定了坚实的基础。     

骨髓间充质干细胞复合脱细胞血管基质构建组织工程血管的初步研究

目的：探讨骨髓间充质干细胞复合脱细胞血管基质构建组织工程血管的可行性。方法：采用胰蛋白酶、乙二胺四乙酸和曲拉通X-100对兔主动脉进行脱细胞处理，制备成脱细胞血管基质；体外分离和扩增人骨髓间充质千细胞，并将其作为种子细胞种植于脱细胞血管基质上，复合构建组织工程化人工血管。结果：制备的脱细胞血管基质由胶原、弹力纤维等组成，未见细胞成分残留；体外扩增的人骨髓间充质干细胞种植于脱细胞血管基质上可生长增殖。结论：骨髓间充质干细胞与脱细胞血管基质支架材料复合可成功构建组织工程血管，有望为血管缺损的修复提供一种全新的技术方法和手段。     

外周血来源间充质干细胞/内皮祖细胞复合细胞膜片的构建及其生物学特性初步研究

目的从外周血中分离获取外周血来源间充质干细胞(peripheral blood mesenchymal stem cells,PBMSC)和外周血内皮祖细胞(peripheral blood endothelial progenitor cells,PBEPC),构建复合细胞膜片并初步研究其生物学特性。方法抽取健康成年新西兰大白兔外周血,采用密度梯度离心法分离获取PBMSC和PBEPC,分别行形态学观察及鉴定。取第3代PBMSC和PBEPC以1∶1比例进行成膜诱导形成复合细胞膜片,取单纯第3代PBMSC同法制备单一细胞膜片。取两种细胞膜片行HE染色,观察细胞膜片内细胞分布情况;分别对两种细胞膜片行成骨诱导,收集诱导1、5、10 d的上清液,利用ELISA法检测两种细胞膜片上清液内ALP、骨钙素(osteocalcin,OCN)和VEGF的表达情况。结果PBMSC细胞形态单一,呈纺锤形或多角形,具备良好的成骨、成脂分化能力;PBEPC细胞形态单一,呈铺路石样,成管试验阳性。两种细胞膜片均呈白色半透明膜状物。HE染色示,与单一细胞膜片组相比,复合细胞膜片组膜片更厚,具备更多细胞层数及更高的细胞密度。ELISA法检测示,随诱导时间延长,两组细胞膜片上清液中ALP、OCN和VEGF表达量均有所增加。复合细胞膜片组诱导培养5、10 d OCN表达量显著高于单一细胞膜片组,10 d ALP表达量显著高于单一细胞膜片组,1、5、10 d VEGF表达量均显著高于单一细胞膜片组,差异均有统计学意义(P<0.05);其余各时间点两组间各蛋白表达量比较差异均无统计学意义(P>0.05)。结论PBMSC具备稳定的MSCs分化能力,因其取材微创具备良好的应用前景。通过与PBEPC共培养、成膜诱导等手段构建复合细胞膜片,为组织缺损的修复提供了一种新的思路与探索。