The effect of mechanical stimulation on the maturation of TDSCs-poly(L-lactide-co-e-caprolactone)/collagen scaffold constructs for tendon tissue engineering

Mechanical stimulation plays an important role in the development and remodeling of tendons. Tendon-derived stem cells (TDSCs) are an attractive cell source for tendon injury and tendon tissue engineering. However, these cells have not yet been fully explored for tendon tissue engineering application, and there is also lack of understanding to the effect of mechanical stimulation on the maturation of TDSCs-scaffold construct for tendon tissue engineering. In this study, we assessed the efficacy of TDSCs in a poly(L-lactide-co-ε-caprolactone)/collagen (P(LLA-CL)/Col) scaffold under mechanical stimulation for tendon tissue engineering both in vitro and in vivo, and evaluated the utility of the transplanted TDSCs-scaffold construct to promote rabbit patellar tendon defect regeneration. TDSCs displayed good proliferation and positive expressed tendon-related extracellular matrix (ECM) genes and proteins under mechanical stimulation in vitro. After implanting into the nude mice, the fluorescence imaging indicated that TDSCs had long-term survival, and the macroscopic evaluation, histology and immunohistochemistry examinations showed high-quality neo-tendon formation under mechanical stimulation in vivo. Furthermore, the histology, immunohistochemistry, collagen content assay and biomechanical testing data indicated that dynamically cultured TDSCs-scaffold construct could significantly contributed to tendon regeneration in a rabbit patellar tendon window defect model. TDSCs have significant potential to be used as seeded cells in the development of tissue-engineered tendons, which can be successfully fabricated through seeding of TDSCs in a P(LLA-CL)/Col scaffold followed by mechanical stimulation.     

The utilization of decellularized tendon slices to provide an inductive microenvironment for the proliferation and tenogenic differentiation of stem cells

The extracellular matrix (ECM) microenvironment for the stem cell niches, including but not limited to the biochemical composition, matrix topography, and stiffness, is crucial to stem cell proliferation and differentiation. The purpose of this study was to explore the capacity of the decellularized tendon slices (DTSs) to induce stem cell proliferation and tenogenic differentiation. Rat adult stem cells, including tendon-derived stem cells (TDSCs) and bone marrow-derived stem cells (BMSCs), were identified to have universal stem cell characteristics. The DTSs were found to retain the native tendon ECM microenvironment cues, including the inherent surface topography, well-preserved tendon ECM biochemical composition and similar stiffness to native tendon. When the TDSCs and BMSCs were cultured on the DTSs respectively, the LIVE/DEAD assay, alamarBlue^® assay, scanning electron microscopy examination and qRT-PCR analysis demonstrated that the DTSs have the capacity to support these stem cells homogeneous distribution, alignment, significant proliferation and tenogenic differentiation. Taken together, the findings of this study indicate that the DTSs can provide a naturally inductive microenvironment for the proliferation and tenogenic differentiation of TDSCs and BMSCs, supporting the use of decellularized tendon ECM as a promising and valuable approach for tendon repair/reconstruction.     

Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration

Tendon injuries are often associated with significant dysfunction and disability due to tendinous tissue's very limited self-repair capacity and propensity for scar formation. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material present an alternative therapeutic option for tendon repair/regeneration that may be advantageous compared to other current treatment modalities. The MSC delivery vehicle is the principal determinant for successful implementation of MSC-mediated regenerative therapies. In the current study, a co-delivery system based on TGF-β3-loaded RGD-coupled alginate microspheres was developed for encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs). The capacity of encapsulated dental MSCs to differentiate into tendon tissue was investigated in vitro and in vivo. Encapsulated dental-derived MSCs were transplanted subcutaneously into immunocompromised mice. Our results revealed that after 4 weeks of differentiation in vitro, PDLSCs and GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited high levels of mRNA expression for gene markers related to tendon regeneration (Scx, DCn, Tnmd, and Bgy) via qPCR measurement. In a corresponding in vivo animal model, ectopic neo-tendon regeneration was observed in subcutaneous transplanted MSC-alginate constructs, as confirmed by histological and immunohistochemical staining for protein markers specific for tendons. Interestingly, in our quantitative PCR and in vivo histomorphometric analyses, PDLSCs showed significantly greater capacity for tendon regeneration than GMSCs or hBMMSCs (P < 0.05). Altogether, these findings indicate that periodontal ligament and gingival tissues can be considered as suitable stem cell sources for tendon engineering. PDLSCs and GMSCs encapsulated in TGF-β3-loaded RGD-modified alginate microspheres are promising candidates for tendon regeneration.     

Tenocytes form a 3-D network and are connected via nanotubes

Cells use different cell adhesion and communication structures to promote tissue development, maintenance of tissue integrity as well as repair and regenerative processes. Another recently discovered way of information exchange is long-distance thin cellular processes called nanotubes (NTs), mainly studied in vitro. Information on the existence and relevance of NTs in vivo is sparse. Building on two references which hint at the potential existence of longitudinally directed cell processes resembling NTs, we investigated tendons from young (3 weeks) and adult (9 weeks, 4 and 8 months) Fisher rats. Whole mounts of rat tail tendon fascicles (RTTfs) and sections of Achilles, flexor, extensor and patellar tendons were stained with Deep Red plasma membrane and DAPI nuclear stain and immunolabelled with Connexin43 (Cx43). In addition, 3-D reconstruction of serial semithin sections and TEM was used to verify the presence of NTs. We were able to demonstrate NTs as straight thin longitudinal processes (Ø 100–500 nm) reaching up to several 100 μm in length, mainly originating from lateral sheet-like cell processes or cell bodies in all tendon types investigated. NTs were observed to distend between tenocyte rows at the same level but also connect cells of different rows, thus leading to a complex 3-D cellular scaffold. Shorter NTs connected lateral cell sheets of tenocytes in the same row, omitting one or two cells. In addition, we detected links or potential branching of NTs. Cx43 immunostaining for the detection of gap junctions revealed Cx43-positive foci at the end-to-end contacts of tenocyte cell bodies as well as along their contacting sheet-like processes. Only rarely, we found clear Cx43 signals at their potential contact points between NTs and tendon cells as well as along the course of NTs, and most NTs appeared completely devoid of Cx43 signals. Therefore, we conclude that NTs in tendons could have a twofold function: long-distance communication as well as stabilization of a mechanically challenged tissue. From in vitro studies it is known that NTs allow intercellular transmission of various cell components, offering potential protective effects for the respective tissue. Further studies on functional properties of NTs in tendons under changing mechanical loading regimens are required in the future. The fact that NTs are present in tendons may necessitate the reconsideration of our traditional understanding of cell-to-cell communication.     

单轴、双轴循环拉力对小鼠肌腱源性干细胞肌腱样分化的影响

目的 探讨单轴、双轴循环拉力对小鼠肌腱源性干细胞(TDSCs)分化的影响,为临床肌腱损伤后的康复治疗提供理论基础。方法 取6~8周龄C57BL/6小鼠10只,无菌条件下暴露双侧后腿至脚掌,显微镜下解剖收集小鼠髌腱和跟腱组织块,体外分离培养细胞,观察第3代细胞的形态特点。(1)取传代至第3代的细胞,采用流式细胞术检测间充质干细胞标志物(CD44、CD90、Sca-1)、内皮细胞标志物(CD34、Flk-1)、造血细胞标志物(CD45),鉴定细胞是否符合TDSCs特点。(2)取传代至第3代的TDSCs进行成骨细胞、软骨细胞和脂肪细胞分化培养,分别采用茜素红、油红和阿尔新蓝染料对培养的三系细胞进行染色,鉴定细胞是否具有多向分化潜能。(3)取传代至第3代的TDSCs接种到硅胶底培养皿上,分为双轴循环拉力组、单轴循环拉力组、对照组3组。双轴循环拉力组细胞使用Flexcell^􀆿 FX-4000TM柔性基底拉伸加载系统,单轴循环拉力组细胞使用自制拉伸力生物反应器,对照组细胞无拉力。双轴循环拉力组、单轴循环拉力组施加机械负荷组的参数均设置为0.25 Hz、6%的循环拉力,在培养期间进行机械负荷加载,每天加载8 h,共加载6 d。第6天机械负荷刺激结束后,收集3组细胞进行实时荧光定量PCR (qPCR),检测肌腱、成骨、脂肪和软骨相关转录因子的表达。结果 显微镜下观察第3代TDSCs形态一致,呈梭形纤维状。(1)流式细胞技术检测结果显示,间充质干细胞标志物CD44、CD90和Sca-1表达阳性、内皮细胞标志物CD34和Flk-1表达阴性、造血细胞标志物CD45表达阴性,符合TDSCs标记鉴定特点。(2)三系分化细胞检测结果显示,提取的细胞成功分化为成骨细胞、脂肪细胞和软骨细胞,验证了提取的细胞具有向成骨细胞、软骨细胞和脂肪细胞分化的潜能。(3)对照组、单轴循环拉力组、双轴循环拉力组3组间比较,肌腱、成骨、软骨、脂肪相关转录因子的相对表达量差异均有统计学意义(P值均＜0.05)。组间两两比较:单轴循环拉力组与对照组比较,肌腱、成骨相关转录因子以及脂肪相关转录因子PPARγ的相对表达均增高,软骨相关转录因子的相对表达均降低,差异均有统计学意义(P值均＜0.05),而脂肪相关转录因子CEB/P的相对表达差异无统计学意义(P＞0.05);双轴循环拉力组与对照组比较,肌腱相关转录因子Scx、Mohawk的相对表达降低,成骨相关转录因子Runx2的相对表达增高、碱性磷酸酶(ALP)的相对表达降低,软骨相关转录因子Sox9相对表达增高、Col2a1的相对表达降低,脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均＜0.05);单轴循环拉力组与双轴循环拉力组比较,双轴循环拉力组肌腱相关转录因子Scx、Mohawk、Col1a1的相对表达均降低,成骨相关转录因子ALP的相对表达降低,软骨、脂肪相关转录因子的相对表达均增高,差异均有统计学意义(P值均＜0.05)。结论 单轴循环拉力诱导TDSCs向肌腱细胞、成骨细胞分化,而双轴循环拉力诱导TDSCs向成骨细胞、脂肪细胞、软骨细胞分化。单轴循环拉力的作用下可以促进体外TDSCs向肌腱细胞分化,有利于肌腱组织的再生和损伤后的修复,为临床肌腱损伤后的康复治疗提供了理论依据。     

A Poly(lactic-co-glycolic acid) Knitted Scaffold for Tendon Tissue Engineering: An In Vitro and In Vivo Study

We have designed a composite scaffold for potential use in tendon or ligament tissue engineering. The composite scaffold was made of a cellularized alginate gel that encapsulated a knitted structure. Our hypothesis was that the alginate would act as a cell carrier and deliver cells to the injury site while the knitted structure would provide mechanical strength to the composite construct. The mechanical behaviour and the degradation profile of the poly(lactic-co-glycolic acid) knitted scaffolds were evaluated. We found that our scaffolds had an elastic modulus of 750 MPa and that they lost their physical integrity within 7 weeks of in vitro incubation. Autologous rabbit mesenchymal stem cell seeded composite scaffolds were implanted in a 1-cm-long defect created in the rabbit tendon, and the biomechanical properties and the morphology of the regenerated tissues were evaluated after 13 weeks. The regenerated tendons presented higher normalized elastic modulus of (60%) when compared with naturally healed tendons (40%). The histological study showed a higher cell density and vascularization in the regenerated tendons.     

Hypoxia-mediated efficient expansion of human tendon-derived stem cells in vitro

Tendons regenerate and repair slowly and inefficiently after injury. Tendon-derived stem cells (TDSCs) have been isolated recently and have been shown to promote tendon repair. The ability to achieve sufficient numbers of cells for transplantation is essential for their clinical application. In this study, we aimed to study the effect of low oxygen (O(2)) tension (2%) on the clonogenicity, metabolic rate, DNA incorporation, population doubling time, β-galactosidase activity, immunophenotypes, multilineage differentiation potential, and tenocyte-like properties of human TDSCs (hTDSCs). hTDSCs were isolated from patellar tendon and characterized according to their adherence to plastic; colony-forming ability; multilineage differentiation potential; and high expression level of CD44, CD73, CD 90, and CD105 but low CD34, CD45, CD146, and Stro-1 at 20% O(2) tension. Low O(2) tension increased DNA incorporation but not metabolic rate of hTDSCs. It increased cell number 25% and the number of colonies but reduced the osteogenic, adipogenic, and chondrogenic differentiation potential of hTDSCs. The reduction in differentiation potential was associated with lower messenger RNA (mRNA) expression ratios of some lineage-related markers, including BGLAP, ALP, C/EBPα, PPARγ2, ACAN, and SOX9; the expression of a tendon-related marker, TNMD, was greater. There was no significant difference in the production of collagenous to noncollagenous protein ratio; the immunophenotypes and β-galactosidase activity were similar at 2% and 20% O(2) tension. Hypoxia-preconditioned hTDSCs could successfully differentiate at 20% O(2) tension, as shown by the return of the mRNA expression ratios of lineage-related markers to levels comparable to cells pre-incubated and differentiated at 20% O(2) tension. In conclusion, hypoxia is advantageous for efficient expansion of hTDSCs in vitro for tendon tissue engineering.     

Stem Cell Competition for Niche Occupancy: Emerging Themes and Mechanisms

Tissue-specific adult stem cells are responsible for generating a range of various differentiated cells during their life time; these cells are intimately associated with niche for maintenance and function. Recent studies of germline stem cell niches in Drosophila gonad suggest that stem cells within a niche constantly compete with each other for niche occupancy. Competition within a niche occurs between same type of stem cells as well as different types. In both cases, cell adhesion molecules are critical in mediating the competition.     

修复运动性肌腱损伤组织工程化肌腱及种子细胞和支架材料

背景：获得大规模、具有再生活力的种子细胞以及具有与正常人体肌腱组织相接近的力学性能的理想支架材料是当前组织工程化肌腱研究面临的最为关键的限制性因素。 目的：总结和分析组织工程肌腱研究中的种子细胞和支架材料的研究进展。 方法：查阅近年来肌腱组织工程研究的相关文献,综合国内外最新研究成果,就肌腱组织工程中合适的种子细胞来源、研究更为理想的支架材料及组织相容性等方面的进展进行概述。 结果与结论：肌腱组织工程中常用的种子细胞有间充质干细胞、肌腱干细胞及胚胎干细胞等,可以向骨、软骨和脂肪分化,修复肌腱损伤的理想细胞。肌腱组织工程支架材料有天然材料及人工合成材料等,肌腱组织工程支架材料应有良好的生物相容性和适度的机械性能,复合材料将是肌腱组织工程支架材料研究的重点。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程      

Localized Rotator Cuff Tendon Degeneration for Cadaveric Shoulders with and Without Tears Isolated to the Supraspinatus Tendon

Localized differences in tissue degeneration throughout intact and torn rotator cuff tendons have not been well quantified. The objective of this study was to investigate histological differences in localized degeneration in tendons with and without rotator cuff tears isolated to the supraspinatus tendon. Four intact shoulders and four shoulders with rotator cuff tears isolated to the supraspinatus tendon were dissected down to the infraspinatus and supraspinatus tendons. Biopsies were taken throughout the tendon insertion, mid-substance, myotendinous junction, and around the tear if present. Samples were stained with hematoxylin and eosin and tendon degeneration was graded based on collagen fiber organization, nuclei shape, cellularity, and lipoid degeneration. Comparisons in degeneration parameters were made based on the tendon type (supraspinatus vs. infraspinatus), location within the tendon, and presence of a tear. Supraspinatus tendons exhibited more degeneration than the infraspinatus tendon (P < 0.05). Significant increases in lipoid degeneration were found near the myotendinous junction compared to the rest of the tendon (P < 0.001). Tendons with rotator cuff tears showed greater amounts of lipoid degeneration compared to intact tendons (P = 0.03). A strong negative correlation was found between lipoid degeneration and collagen fiber organization (r = −0.922, P = 0.001). No differences in degeneration were found between medial, anterior, and posterior edges of the tear. The study highlights specific factors of tendon degeneration contributing to the local differences in tendon degeneration. By understanding local differences in tendon degeneration, surgical protocols for repair can be improved. Clin. Anat., 33:1007–1013, 2020. © 2019 Wiley Periodicals, Inc.     

Infrapatellar fat pad may be with tendon repairing ability and closely related with the developing process of patella Baja

Patella Baja, as a common complication of knee trauma or knee surgery, was very difficult to deal with, and scarring and shortening of the patellar tendon were looked on as the most important reason for it. Infrapatellar fat pad, also known as Hoffa’s fat pad was traditionally regarded as with only buffering and lubricating functions in knee joints, which can limit the knee’s excessive activities, absorb shocks from the anterior knee and reduces friction between the patellar tendon and the tibia, it should be with direct protection function and avoid the damage of patella tendon. Recently, a large number of studies had shown that adipose tissue was an accessible and abundant source of mesenchymal stem cells for tissue engineering. Adipose derived stem cells (ADSCs) can be induced to differentiate into adipocytes, osteoblasts, nerve cells, tendon cells and so on. In addition, interestingly, infrapatellar fat pad were just located behind the patellar tendon, and SDF-1 (stromal cell-derived factor-1), as a powerful cytokine that regulates inflammatory cell recruitment and stem cell homing, was unregulated after ligament injury, so there may be a certain correlation between ADSCs from infrapatellar fat pad and injured tendons, maybe ADSCs from infrapatellar fat pad can biologically repair injured tendons when patella tendon was damaged. We hypothesized injured patella tendon repairing should include not only the self-repairing of the tendon, but also the biological repairing from ADSCs in infrapatellar fat pad. When both of the repairing failed to repair the damage of the tendon, patellar tendon may begin to shrink and scar, which will result in patella Baja. In our opinion, healthy infrapatellar fat pad was with direct protection function of patella tendon and full of rich ADSCs, which may play an important part in patella tendon repairing and the developing process of patella Baja.     

A device to measure in vivo forces in human flexor tendons during carpal tunnel operations

An apparatus has been designed to measure the in vivo forces transmitted along human hand flexor tendons during carpal tunnel release procedures. The tendon will be run through three hooks, the central one of which is attached to a load cell. The rationale is that once these forces are known this will aid in the design of repair techniques and rehabilitation regimens. As a first stage, an in vitro validation study is presented using cord subjected to varying forces to mimic an in vivo flexor tendon under varying conditions of use. Our results show that we can accurately and reproducibly measure the force in the cord.     

An in vitro scratch tendon tissue injury model: effects of high frequency low magnitude loading

The healing process of ruptured tendons is suboptimal, taking months to achieve tissue with inferior properties to healthy tendon. Mechanical loading has been shown to positively influence tendon healing. However, high frequency low magnitude (HFLM) loads, which have shown promise in maintaining healthy tendon properties, have not been studied with in vitro injury models. Here, we present and validate an in vitro scratch tendon tissue injury model to investigate effects of HFLM loading on the properties of injured rat tail tendon fascicles (RTTFs). A longitudinal tendon tear was simulated using a needle aseptically to scratch a defined length along individual RTTFs. Tissue viability, biomechanical, and biochemical parameters were investigated before and 7 days after culture . The effects of static, HFLM (20 Hz), and low frequency (1 Hz) cyclic loading or no load were also investigated. Tendon viability was confirmed in damaged RTTFs after 7 days of culture, and the effects of a 0.77 ± 0.06 cm scratch on the mechanical property (tangent modulus) and tissue metabolism in damaged tendons were consistent, showing significant damage severity compared with intact tendons. Damaged tendon fascicles receiving HFLM (20 Hz) loads displayed significantly higher mean tangent modulus than unloaded damaged tendons (212.7 ± 14.94 v 92.7 ± 15.59 MPa), and damaged tendons receiving static loading (117.9 ± 10.65 MPa). HFLM stimulation maintained metabolic activity in 7-day cultured damaged tendons at similar levels to fresh tendons immediately following damage. Only damaged tendons receiving HFLM loads showed significantly higher metabolism than unloaded damaged tendons (relative fluorescence units —7021 ± 635.9 v 3745.1 ± 641.7). These validation data support the use of the custom-made in vitro injury model for investigating the potential of HFLM loading interventions in treating damaged tendons.     

The role of engineered tendon matrix in the stemness of tendon stem cells in vitro and the promotion of tendon-like tissue formation in vivo

When injured, tendons tend to heal but with poor structure and compromised function. Tissue engineering is a promising approach to enhancing the quality of healing tendons. Our group and others have identified tendon stem cells (TSCs), a type of tendon-specific stem cells which may be optimal for cellular interventions seeking to restore normal structure and function to injured tendons. However, in vitro expanding of TSCs on regular plastic cell culture dishes only yields a limited number of TSCs before they lose the stemness, i.e., the self-renewal capability and multipotency. In this study, we developed a substrate material for TSCs, engineered tendon matrix (ETM) from decellularized tendon tissues. We showed that ETM in vitro was able to stimulate TSC proliferation and better preserve the stemness of TSCs than plastic culture surfaces. In vivo, implantation of ETM-TSC composite promoted tendon-like tissue formation whereas implantation of TSCs alone led to little such tissue formation. Together, the findings of this study indicate that ETM may be used to effectively expand TSCs in vitro and with TSCs, to enhance repair of injured tendons in vivo.     

Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair

Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. Platelet-derived growth factor BB (PDGF-BB), along with adipose-derived mesenchymal stem cells (ASCs), were incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly(lactic-co-glycolic acid) (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently labeled ASCs and the scaffold were found at the repair site 9 days post-operatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner.     

Novel fiber-based pure chitosan scaffold for tendon augmentation: biomechanical and cell biological evaluation

One possibility to improve the mechanical properties after tendon ruptures is augmentation with a scaffold. Based on wet spinning technology, chitosan fibres were processed to a novel pure high-grade multifilament yarn with reproducible quality. The fibres were braided to obtain a 3D tendon scaffold. The CS fibres and scaffolds were evaluated biomechanically and compared to human supraspinatus (SSP) tendons. For the cytobiological characterization, in vitro cell culture experiments with human mesenchymal stem cells (hMSC) were performed. Three types of 3D circular braided scaffolds were fabricated. Significantly, higher ultimate stress values were measured for scaffold with larger filament yarn, compared to scaffold with smaller filament yarn. During cultivation over 28 days, the cells showed in dependence of isolation method and/or donor a doubling or tripling of the cell number or even a six-fold increase on the CS scaffold, which was comparable to the control (polystyrene) or in the case of cells obtained from human biceps tendon even higher proliferation rates. After 14 days, the scaffold surface was covered homogeneously with a cell layer. In summary, the present work demonstrates that braided chitosan scaffolds constitute a straightforward approach for designing tendon analogues, maintaining important flexibility in scaffold design and providing favourable mechanical properties of the resulting construct.     

Ovarian Stem Cell Niche and Follicular Renewal in Mammals

Stem cell niche consists of perivascular compartment, which connects the stem cells to the immune and vascular systems. During embryonic period, extragonadal primordial germ cells colonize coelomic epithelium of developing gonads. Subsequently, ovarian stem cells (OSC) produce secondary germ cells under the influence of OSC niche, including immune system‐related cells and hormonal signaling. The OSC in fetal and adult human ovaries serve as a source of germ and granulosa cells. Lack of either granulosa or germ cell niche will result in premature ovarian failure in spite of the presence of OSC. During perinatal period, the OSC transdifferentiate into fibroblast‐like cells forming the ovarian tunica albuginea resistant to environmental threats. They represent mesenchymal precursors of epithelial OSC during adulthood. The follicular renewal during the prime reproductive period (PRP) ensures that there are fresh eggs available for a healthy progeny. End of PRP is followed by exponentially growing fetal genetic abnormalities. The OSC are present in adult, aging, and postmenopausal ovaries, and differentiate in vitro into new oocytes. During in vitro development of large isolated oocytes reaching 200 μm in diameter, an ancestral mechanism of premeiotic nurse cells, which operates during oogenesis in developing ovaries from invertebrates to mammalian species, is utilized. In vitro developed eggs could be used for autologous IVF treatment of premature ovarian failure. Such eggs are also capable to produce parthenogenetic embryos like some cultured follicular oocytes. The parthenotes produce embryonic stem cells derived from inner cell mass, and these cells can serve as autologous pluripotent stem cells. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Tgif1 and SnoN modified chondrocytes or stem cells for tendon–bone insertion regeneration

Tendon–bone insertion injuries are a common occurrence but rarely heal, despite the many strategies that have been employed. The tendon–bone insertion consists of four types of tissues: tendon, fibrocartilage, mineral fibrocartilage and bone, making it hard to regenerate. The key to reconstructing the tendon enthesis is to rebuild the gradations of cell type, collagen type, mineral content and collagen fiber orientation. Chondrocytes were found to be able to differentiate into tendon and bone tissues upon special stimulation, which offers promise for tendon enthesis regeneration. Tgif1 is a key factor that represses the expression of the cartilage master gene Sox9, which is induced by TGFβs, and changes the expression rate of Sox9 versus Scx, eventually promoting fibrogenesis. SnoN is a key factor that is induced by TGFβs to inhibit the hypertrophy of chondrocytes and therefore bone formation. It appears that the induction of Tgif1 and the repression of SnoN can cause chondrocytes to differentiate into tendon and bone tissues. Moreover, a gradation of the expression levels of Tgif1 and SnoN in chondrocytes may create a gradation of the tissue from tendon to fibrocartilage to bone. Consequently, we propose that a gradation of gene-modified chondrocytes (Tgif1-inducing cells, primary cells, SnoN-repressing cells) or stem cells that arise from a gradation of stimulation (Tgif1 induction and SnoN repression) will aid in the regeneration of the tendon–bone insertion.     

Age-Related Changes in the Cellular,Mechanical, and Contractile Properties of Rat Tail Tendons

Tendon laxity following injury, cyclic creep, or repair has been shown to alter the normal homeostasis of tendon cells, which can lead to degenerative changes in the extracellular matrix. While tendon cells have been shown to have an inherent contractile mechanism that gives them some ability to retighten lax tendons and reestablish a homeostatic cellular environment, the effect of age on this process is unknown. To determine the effect of aging on cell number, cell shape, and tensile modulus on tendons as well as the rate of cell-mediated contraction of lax tendons, tail tendon fascicles from 1-, 3-, and 12-month-old rats were analyzed. Aging results in a decrease (p < 0.001) in cell number per mm²: 1 m (981 ± 119), 3 m (570 ± 108), and 12 m (453 ± 23), a more flattened (p < 0.001) cell nuclei shape and a higher (p < 0.001) tensile modulus (MPa) of the tendons: 1 m (291 ± 2), 3 m (527 ± 38), and 12 m (640 ± 102). Both the extent and rate of contraction over 7 days decreased with age (p = 0.007). This decrease in contraction rate with age correlates to the observed changes seen in aging tendons [increased modulus (r² = 0.95), decreased cell number (r² = 0.89)]. The ability of tendons to regain normal tension following injury or exercise-induced laxity is a key factor in the recovery of tendon function. The decreased contraction rate as a function of age observed in the current study may limit the ability of tendon cells to retighten lax tendons in older individuals. This, in turn, may place these structures at further risk for injury or altered function.     

In vitro and in vivo differentiation of mesenchymal stem cells in the cardiomyocyte direction

The possibility of mesenchymal stem cell differentiation in the cardiomyocyte direction was studied on Wistar-Kyoto rats with myocardial infarction induced by ligation of the left coronary artery. In vitro treatment of mesenchymal stem cells with 5-azacitidine led to spontaneous contractions of about 15% cells in culture. Analysis of the expression of matrix RNA showed expression of fetal and functional markers of the myocardium in this cell culture. In vivo on day 21 after myocardial infarction and intravenous transplantation of mesenchymal stem cells into the periinfarction area, myocardial cells carrying donor label were detected. Immunohistochemical analysis showed that these cells were cardiomyocytes integrated into the myocardium. These cells can be a result of differentiation of transplanted mesenchymal stem cells or fusion of endogenous cardiomyocytes with exogenous mesenchymal stem cells. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 4, pp. 194–197, December, 2006