Tendon Sheath Fibroblasts Research Articles

Mannose-6-Phosphate Reduces Expression of Transforming Growth Factor β and Receptors in Rabbit Flexor Tendon Tenocytes

Transforming growth factor β (TGF-β) plays an important role in tendon healing and adhesion formation. Inhibiting expression of TGF-β and receptors might prevent adhesion after tendon injury and/or opening surgeries. Here, we aimed to examine the effects of mannose-6-phosphate (M6P), an inhibitor of TGF-β as a natural plant compound, on expression of TGF-β and receptors in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes that were isolated from rabbit flexor toes. We enriched tendon sheath fibroblasts, epitenon tenocytes, as well as endotenon tenocytes from rabbit flexor tendons, and subsequently cultured these cell types individually. All 3 cell types were grouped into 2 groups randomly: the experiment group supplemented with M6P (the M6P Group), and the Control Group treated with vehicle. Expression of TGF-β and receptors was assessed with enzyme-linked immunosorbent assays. TGF-β1 expression in the mRNA level was also assessed by in situ hybridization and immunohistochemistry. Expression of TGF-β and receptors in the M6P Group was remarkably lower than that in the Control Group across all 3 cell types (P < 0.05). In the M6P Group, the expression ratio of TGF-β1 mRNA and the expression level of intracellular TGF-β1 mRNA in 3 cell types were significantly decreased than those in the Control Group (P < 0.05). Therefore, expression of TGF-β1 was significantly lower in all three types of tendon cell treated with M6P. Collectively, M6P is a negative regulator of TGF-β and receptors in cultured rabbit tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes.

Cell therapies for tendons: old cell choice for modern innovation.

Although tissue engineering and cell therapies are becoming realistic approaches for medical therapeutics, it is likely that musculoskeletal applications will be among the first to benefit on a large scale. Cell sources for tissue engineering and cell therapies for tendon pathologies are reviewed with an emphasis on small defect tendon injuries as seen in the hand which could adapt well to injectable cell administration. Specifically, cell sources including tenocytes, tendon sheath fibroblasts, bone marrow or adipose-derived stem cells, amniotic cells, placenta cells and platelet-derivatives have been proposed to enhance tendon regeneration. The associated advantages and disadvantages for these different strategies will be discussed and evolving regulatory requirements for cellular therapies will also be addressed. Human progenitor tenocytes, along with their clinical cell banking potential, will be presented as an alternative cell source solution. Similar cell banking techniques have already been described with other progenitor cell types in the 1950's for vaccine production, and these "old" cell types incite potentially interesting therapeutic options that could be improved with modern innovation for tendon regeneration and repair.

Inhibition Effect of Mannose-6-Phosphate on Expression of Transforming Growth Factor Beta Receptor in Flexor Tendon Cells

Transforming growth factor beta (TGF-β) has an important role in tendon healing and adhesion formation. Inhibiting TGF-β and its receptor expression may prevent adhesions after tendon open. The goal of this study was to examine the effects of mannose-6-phosphate, a natural inhibitor of TGF-β, on TGF-β and its receptor production in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes of rabbit flexor tendons. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. The cells were divided into 2 groups at random: an experiment group supplemented with mannose-6-phosphate and a control group without mannose-6-phosphate. The expression of TGF-β and TGF-β receptor was quantified with enzyme-linked immunosorbent assay. The luciferase assay measured TGF-β bioactivity. Transforming growth factor beta expression in the experimental group was not decreased compared with the control group, with no significant difference (P>.05) Transforming growth factor beta receptor expression in the experiment group was significantly lower than that in control group (P<.05). Mannose-6-phosphate significantly decreased the expression of TGF-β receptor and TGF-β bioactivity. Modulation of mannose-6-phosphate levels may provide a means of modulating the effects of TGF-β on adhesion formation in flexor tendon wound healing.

HGF depress the production of α-SMA and extracellular matrix induced by TGF-β1 in fibroblasts of the flexor tendon sheath

Objective To examine the effectiveness of HGF in blocking TGF-β1 induced α-SMA and extracellular matrix production in fibroblasts of the flexor tendon sheath. Methods Seven adult male New Zealand white rabbits (3.75-4.00 kg) were used for this study. Both of their front feet were sterilised and the middle digit flexor digitorum profundus tendon equivalents were identified and isolated. These specimens were used to establish primary cell cultures. Sheath fibroblasts were obtained from rabbit flexor tendons. After the cells reached confluence, cells were detached with trypsin/ethylenediamine tetra-acetic acid. All experiments were performed using the cells at the third passage. At 70% confluence the medium was supplemented with 5 ng/ml of TGF-β1 along with increasing doses of HGF (10-40 ng/ml). After 72 hours incubation, the productions of α-SMA were assayed by Western-Blot. The productions of collagen Ⅰ and fibronectin in supernatants culture were examined using ELISA. Results Evaluation of protein expression revealed that TGF-β1 markedly induced α-SMA expression in cultured rabbit flexor tendon sheath fibroblasts. TGF-β1 treated fibroblasts expressed 1.8-fold more protein compared to non-treated fibroblasts (P ＜ 0.05). However, simultaneous incubation of HGF significantly abrogated TGF-β1 induced α-SMA expression in a dose-dependent manner (P＜ 0.05). Treatment with TGF-β1 significantly stimulated collagen Ⅰ and fibronectin production in flexor tendon sheath fibroblasts (P ＜ 0.01). Remarkably, the addition of HGF reduced productions of all components induced by TGF-β1 in a dose-dependent manner (P ＜ 0.05). Conclusion HGF antagonizes TGF-β1 induced α-SMA, collagen Ⅰ, and fibronectin production in flexor tendon sheath fibroblasts. The findings provide a cellular and molecular basis for HGF's acting as a therapeutic agent for adhesions in flexor tendons. Key words: Hepatocyte growth factor; Transforming growth factor-β; Flexor tendon; Fibroblast; α-smooth muscle actin

Effects of Antisense Transforming Growth Factor-β1 Gene Transfer on the Biological Activities of Tendon Sheath Fibroblasts

Recent studies have shown the importance of transforming growth factor-beta (TGF-beta) in flexor tendon wound healing. Decreased adhesion formation and increased range of motion after the administration of TGF-beta antibodies after tendon repair have been shown. But TGF-beta antibodies have a short biologic half-life, and continuous supplementation of exogenous TGF-beta antibodies is not practical. Transfer of growth factor genes to tenocytes provides an alternative to protein therapeutics, and a gene therapy approach will prolong the availability of therapeutic proteins.We investigated the biological activities effects of rabbit tendon sheath fibroblasts transfected by antisense TGF-beta1 gene. Tendon sheath fibroblasts were isolated from New Zealand white rabbits and transfected by antisense TGF-beta1 gene with Lipofectin (Invitrogen, Carlsbad, California). Reverse transcription polymerase chain reaction was used to measure collagen I, collagen III, and TGF-beta1 expression, and Western blot was used to measure collagen protein I expression in tendon sheath fibroblasts after being transfected by antisense TGF-beta1 gene. Reverse transcription polymerase chain reaction displayed that tendon sheath fibroblasts transfected with antisense TGF-beta1 gene showed marked decrease collagen I, collagen III, and TGF-beta1 mRNA expression. Western blot showed that tendon sheath fibroblasts transfected with antisense TGF-beta1 gene showed marked decrease expression of collagen I protein, and there was significant difference compared with the untransfected and empty transfected groups (P<.01). Tendon sheath fibroblasts can transfect with antisense TGF-beta1 gene successfully and can decrease production of collagen I, collagen III, and TGF-beta1, which were factors of tendon adhere formation.

Effects of antisense transforming growth factor-β1 gene transfer on the biological activities of ten-don sheath fibroblasts

Objective To investigate the biological activity changes of rabbit tendon sheath fibro-blasts transfected by antisense TGF-β1 gene so as to provide a novel approach to prevention of postopera-tive tendon adhesion. Methods Tendon sheath fibroblasts were isolated from the New Zealand white rabbits and transfected by antisense TGF-β1 gene with Lipofectin. RT-PCR was used to measure mRNA expressions of collagen Ⅰ, collagen Ⅲ and TGF-β1 expressing and Western blot to measure protein ex-pression of collagen Ⅰ in tendon sheath fibroblasts. Results RT-PCR displayed marked decrease of mRNA expressions of collagen Ⅰ, collagen Ⅲ and TGF-β1 in tendon sheath fibroblasts transfected with antisense TGF-β1 gene, with statistical difference compared with untransfected group or blank transfected group (P<0.01). Western blot showed marked decrease of expression of collagen Ⅰ protein in tendon sheath fibroblasts transfected with antisense TGF-β1 gene, with statistical difference compared with un-transfected group or blank transfected group (P<0.01). Conclusions Tendon sheath fibroblasts can successfully transfect antisense transforming growth factor-β1 gene, decrease production of collagen Ⅰ,collagen Ⅲ and TGF-β1 that are factors for tendon adhesion and may play a certain role in prevention of tendon adhesion after tendon injuries. Key words: Tendon injuries; Oligoribonucleotides,antisense; Transforming growth factor beta; Collagen

Journal of Hand Surgery

Long-term outcomes of closed reduction and percutaneous pinning for the treatment of distal radius fractures. Glickel SZ, Catalano LW, Raia FJ, Barron OA, Grabow R, Chia B. J Hand Surg Am. 2008;33:1700–1705.

A Comparison of Tenocytes and Mesenchymal Stem Cells for Use in Flexor Tendon Tissue Engineering

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. 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. 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. 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.

Flexor tendon wound healing in vitro: lactate up-regulation of TGF-beta expression and functional activity.

Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

Flexor tendon wound healing in vitro: the effect of lactate on tendon cell proliferation and collagen production.

Flexor tendon repair in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. Lactate is an early mediator of wound healing known to play an important role in stimulation of collagen production after cellular injury. Little attention has been paid to the role of lactate in flexor tendon wound healing. In this study tendon and tendon sheath were excised from rabbit forepaws. We examined proliferation of tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes; collagen production by each of these 3 cell types; and effects of lactate on cell proliferation and collagen production. Three cell lines, tendon sheath, epitenon, and endotenon, were isolated and cultured. Tendon sheath fibroblasts showed the greatest proliferation. All 3 cell lines produced collagen I, II, and III. Lactate significantly increased collagen production by all 3 cell lines. We show that cells of the tendon sheath, epitenon, and endotenon produce collagen in vitro. Modulation of lactate levels may provide a means to modulate collagen production.

Molecular studies in flexor tendon wound healing: The role of basic fibroblast growth factor gene expression

Basic fibroblast growth factor (bFGF) is a cytokine that plays a fundamental role in angiogenesis. This study examines bFGF messenger RNA (mRNA) expression in a rabbit flexor tendon wound healing model. Thirty-four New Zealand white rabbit forepaws underwent transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals and analyzed by in situ hybridization and immunohistochemistry. Few tenocytes and tendon sheath cells expressed bFGF mRNA in unwounded tendons. In contrast, tendons subjected to transection and repair exhibited an increased signal for bFGF mRNA in both resident tenocytes concentrated along the epitenon and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal tenocytes and tendon sheath cells are capable of bFGF production, (2) bFGF mRNA is upregulated in the tendon wound environment, and (3) the upregulation of this angiogenic cytokine occurs in tenocytes as well as in tendon sheath fibroblasts and inflammatory cells.

Gene expression of transforming growth factor beta-1 in rabbit zone II flexor tendon wound healing: evidence for dual mechanisms of repair.

The postoperative outcome of hand flexor tendon repair can be complicated by adhesions between the repair site and surrounding tissue. To date, the biology of hand flexor tendon wound healing remains controversial--both intrinsic (resident tenocyte) and extrinsic (tendon sheath fibroblast and inflammatory cell) processes may contribute to repair. Transforming growth factor beta-1 is a cytokine that plays multiple roles in wound healing but is also implicated in the pathogenesis of excessive scar formation. This study examines the activation of transforming growth factor beta-1 mRNA in a rabbit zone II flexor tendon wound-healing model. Forty New Zealand White rabbit forepaws underwent complete transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals (1, 3, 7, 14, 28, and 56 days) and analyzed by in situ hybridization and immunohistochemistry to determine the expression patterns of transforming growth factor beta-1. A small number of tenocytes exhibited expression of transforming growth factor beta-1 mRNA at baseline in nonwounded control tendon specimens. The surrounding tendon sheath in these control specimens also revealed low numbers of fibroblasts and inflammatory cells expressing transforming growth factor beta-1 mRNA. In contrast, flexor tendons subjected to transection and repair exhibited increased signal for transforming growth factor beta-1 mRNA in both resident tenocytes and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal unwounded tenocytes and tendon sheath cells are capable of transforming growth factor beta-1 production, (2) this cytokine is activated in the tendon wound environment, as evidenced by mRNA upregulation, and (3) the upregulation of this cytokine in both "intrinsic" tenocytes and "extrinsic" tendon sheath fibroblasts and inflammatory cells supports dual mechanisms for tendon repair. Because transforming growth factor beta-1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that perioperative biochemical modulation of transforming growth factor beta-1 levels may help limit flexor tendon adhesion formation.