Cultured Tendon Fibroblasts Research Articles

Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro.

IntroductionMacrophage-driven inflammation is a key feature of the early period following tendon repair, but excessive inflammation has been associated with poor clinical outcomes. Modulation of the inflammatory environment using molecular or cellular treatments may provide a means to enhance tendon healing.MethodsTo examine the effect of pro-inflammatory cytokines secreted by macrophages on tendon fibroblasts (TF), we established in vitro models of cytokine and macrophage-induced inflammation. Gene expression, protein expression, and cell viability assays were used to examine TF responses. In an effort to reduce the negative effects of inflammatory cytokines on TFs, adipose-derived mesenchymal stromal cells (ASCs) were incorporated into the model and their ability to modulate inflammation was investigated.ResultsThe inflammatory cytokine interleukin 1 beta (IL-1β) and macrophages of varying phenotypes induced up-regulation of pro-inflammatory factors and matrix degradation factors and down-regulation of factors related to extracellular matrix formation by TFs in culture. ASCs did not suppress these presumably negative effects induced by IL-1β. However, ASC co-culture with M1 (pro-inflammatory) macrophages successfully suppressed the effects of M1 macrophages on TFs by inducing a phenotypic switch from a pro-inflammatory macrophage phenotype to an anti-inflammatory macrophage phenotype, thus resulting in exposure of TFs to lower levels of pro-inflammatory cytokines (e.g., IL-1β, tumor necrosis factor alpha (TNFα)).ConclusionsThese findings suggest that IL-1β and M1 macrophages are detrimental to tendon healing and that ASC-mediated modulation of the post-operative inflammatory response may be beneficial for tendon healing.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0059-4) contains supplementary material, which is available to authorized users.

P38 MAPK signaling in postnatal tendon growth and remodeling.

Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration

Pentadecapeptide BPC 157, composed of 15 amino acids, is a partial sequence of body protection compound (BPC) that is discovered in and isolated from human gastric juice. Experimentally it has been demonstrated to accelerate the healing of many different wounds, including transected rat Achilles tendon. This study was designed to investigate the potential mechanism of BPC 157 to enhance healing of injured tendon. The outgrowth of tendon fibroblasts from tendon explants cultured with or without BPC 157 was examined. Results showed that BPC 157 significantly accelerated the outgrowth of tendon explants. Cell proliferation of cultured tendon fibroblasts derived from rat Achilles tendon was not directly affected by BPC 157 as evaluated by MTT assay. However, the survival of BPC 157-treated cells was significantly increased under the H(2)O(2) stress. BPC 157 markedly increased the in vitro migration of tendon fibroblasts in a dose-dependent manner as revealed by transwell filter migration assay. BPC 157 also dose dependently accelerated the spreading of tendon fibroblasts on culture dishes. The F-actin formation as detected by FITC-phalloidin staining was induced in BPC 157-treated fibroblasts. The protein expression and activation of FAK and paxillin were determined by Western blot analysis, and the phosphorylation levels of both FAK and paxillin were dose dependently increased by BPC 157 while the total amounts of protein was unaltered. In conclusion, BPC 157 promotes the ex vivo outgrowth of tendon fibroblasts from tendon explants, cell survival under stress, and the in vitro migration of tendon fibroblasts, which is likely mediated by the activation of the FAK-paxillin pathway.

Radix Dipsaci does not improve tendon healing in a rat model of patellar tendon donor site injury

To explore whether Radix Dipsaci (RD) exhibits beneficial effects on tendon healing. An attempt was made to explore the in vitro effects of a hot water extract of RD on gene expression of procollagen Type I (COL1A1), procollagen Type III (COL3A1) and decorin in cultured tendon fibroblasts, and its in vivo effects in a well-established rat model of patellar tendon donor site injury. It was found that gene expression of COL3A1 and decorin in cultured tendon fibroblasts was significantly increased by RD, but that COL1A1 was not affected. In vivo studies showed that RD increased blood vessels in the wound but did not significantly affect the expression of COL1A1, COL3A1 and decorin at day 14 post-injury. The ultimate tensile stress of the healing tendon was not significantly improved by either local injection or oral administration of hot water extract of RD (P > 0.05). The present findings imply that RD per se does not significantly improve tendon healing. Further investigation of RD in a herbal formula may be necessary to test its efficacy in tendon injuries.

Hyaluronan Modulates Proliferation and Migration of Rabbit Fibroblasts Derived From Flexor Tendon Epitenon and Endotenon

There is a growing body of evidence supporting the use of hyaluronan (HA) for treatment of injured tendons, although the mechanism of the healing effect has not yet been clarified. We therefore investigated the effects of HA on the proliferation and migration of tendon fibroblasts derived from rabbit flexor tendon epitenon and endotenon. From explanted rabbit intrasynovial flexor tendons (n = 5), we cultured tendon fibroblasts derived from the epitenon and endotenon. CD44 expression on the tendon fibroblasts was detected by flow cytometric analysis. Various concentrations of HA (0.1-5.0 mg/mL) were added to monolayer-cultured tendon fibroblasts. We evaluated cell proliferation by recording changes in cell number, and measured cell migration by wound-healing assay. Flow cytometric analysis detected CD44 expression on the tendon fibroblasts. Treatment with HA at various concentrations notably and dose dependently inhibited cell proliferation and promoted cell migration. Hyaluronan modulates the proliferation and migration of rabbit fibroblasts derived from the flexor tendon epitenon and endotenon.

Involvement of Na+/Ca2+ exchanger in migration and contraction of rat cultured tendon fibroblasts

In response to injury and inflammation of tendons, tendon fibroblasts are activated, migrate to the wound, and eventually induce contraction of the extracellular matrices to repair the tissue. Under such conditions, Ca(2+) signalling is involved in motility and contractility of tendon fibroblasts. Using cultured tendon fibroblasts isolated from rat Achilles tendons, we investigated functional expression of Na(+)/Ca(2+) exchangers (NCX). The fluorometric study showed that the intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased by reducing extracellular Na(+) concentration ([Na(+)](o)) in tendon fibroblasts. Selective NCX inhibitors, KB-R7943 and SEA0400, both attenuated [Na(+)](o)-dependent [Ca(2+)](i) elevation and the resting [Ca(2+)](i) in tendon fibroblasts. RT-PCR, Western blots and sequence analyses revealed that NCX1.3 and NCX1.7 were expressed in cultured tendon fibroblasts. NCX2 mRNA was undetected. NCX3 expression was negligibly low. Immunofluorescence microscopy indicated that NCX1 protein localized in the plasma membrane especially at the microspikes of tendon fibroblasts. In the wound-healing scratch assay, the cells migrated toward the space created by a scratch and almost completely filled the space within 48 h. This phenomenon was significantly suppressed by KB-R7943 and SEA0400. Furthermore, the NCX inhibitors abrogated the tendon fibroblast-mediated collagen-matrix contractions. Two types of siRNAs for NCX1 also suppressed the migration and contraction of tendon fibroblasts. We conclude that NCX is expressed and mediates Ca(2+) influx in cultured tendon fibroblasts. Since the pharmacological inhibitors and siRNA for NCX1 suppressed motility and contractility of tendon fibroblasts, NCX may play an important role in the function of tendon fibroblasts in the wound healing.

Expression of transforming growth factor β isoforms and their roles in tendon healing

Transforming growth factor beta (TGF-beta) plays active roles in tendon healing. However, the differential effects of TGF-beta isoforms on tendon healing have not been investigated. In cultured tendon fibroblasts, we tested the effects of TGF-beta1, beta2, and beta3 on the mRNA levels of COL1A1 and COL3A1 by quantitative real-time polymerase chain reaction. We also investigated the expression of TGF-beta isoforms, TGF-beta receptors, procollagen Type I and Type III in a rat model of tendon healing. We found that TGF-beta3 exhibited the highest potency in stimulating COL1A1 and COL3A1. TGF-beta1 exerted antagonistic effects to TGF-beta2 and beta3. All TGF-beta isoforms and procollagen Type I were confined to the edges of the healing tendon at day 28 postinjury. Our results indicated that interaction of TGF-beta isoforms exist in the regulation of collagen synthesis in tendon fibroblasts. Their effects may be further complicated by uneven spatial distribution of TGF-beta and TGF-beta receptors in healing tendons.

Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF-beta1, was compared by quantitative real-time RT-PCR. The expression of proliferation cell nuclear antigen (PCNA) and alpha-smooth muscle actin (alpha-SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF-beta1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher alpha-SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing.

Role of TGF-beta1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue.

Mechanical loading of tissue is known to influence local collagen synthesis, and microdialysis studies indicate that mechanical loading of human tendon during exercise elevates tendinous type I collagen production. Transforming growth factor-beta1 (TGF-beta1), a potent stimulator of type I collagen synthesis, is released from cultured tendon fibroblasts in response to mechanical loading. Thus TGF-beta1 could link mechanical loading and collagen synthesis in tendon tissue in vivo. Tissue levels of TGF-beta1 and type I collagen metabolism markers [procollagen I COOH-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen (ICTP)] were measured by microdialysis in peritendinous tissue of the Achilles' tendon in six male volunteers before and after treadmill running (1 h, 12 km/h, 3% uphill). In addition, blood levels of TGF-beta1, PICP, and ICTP were obtained. PICP levels increased 68 h after exercise (P < 0.05). Dialysate levels of TGF-beta1 changed from 303 +/- 46 pg/ml (at rest) to 423 +/- 86 pg/ml 3 h postexercise. This change was nonsignificant, but the decay of tissue TGF-beta1 after catheter insertion was markedly delayed by exercise compared with the decay seen in resting subjects. Plasma concentrations of TGF-beta1 rose 30% in response to exercise (P < 0.05 vs. pre). Our observations indicate an increased local production of type I collagen in human peritendinous tissue in response to uphill running. Although not conclusive, changes in circulating and local TGF-beta1, in response to exercise, suggest a role for TGF-beta1 in mechanical regulation of local collagen type I synthesis in tendon-related connective tissue in vivo.

Proteoglycan synthesis by fibroblasts from different regions of bovine tendon cultured in alginate beads

The ability of cell shape to modulate proteoglycan synthesis in tendon fibroblasts was investigated by placing freshly isolated tendon fibroblasts and chondrocytes into primary culture either as adherent cells on a polystyrene substratum or as rounded cells in alginate beads. Chondrocytes and cells from the compressed region of adult tendon synthesized predominantly large proteoglycan when maintained either as dense monolayers, where actin stress fibers in the cytoskeleton were prominent, or in alginate beads, where actin fibers could not be detected. After three rounds of proliferation as elongated adherent cells the synthesis of large proteoglycan was greatly reduced, i.e. the chondrocytic cells underwent `dedifferentiation'. Cells from the tensional region of adult tendon synthesized predominantly small proteoglycan when in primary culture as a monolayer, after proliferation on a flat substratum, or as round cells in alginate beads. Fibroblasts from the tensional region of newborn tendon showed no tendency toward increased synthesis of large proteoglycan when maintained as round cells in alginate beads for 7 weeks. In tendon there appears to be a mechanically induced developmental transition from fibroblastic to chondrocytic cells. However, neither the change to a rounded cell shape nor the lack of organized cytoskeletal actin fibers was sufficient to induce chondrocyte-like proteoglycan synthesis in differentiated tendon fibroblasts in culture.

Matrix Protein mRNA Levels in Canine Meniscus Cellsin Vitro

The resident cells of the meniscus synthesize a fibrocartilaginous extracellular matrixin vivocomposed predominantly of type I collagen fibers. To increase our understanding of matrix biosynthesis by meniscus cellsin vitro,we examined matrix protein mRNA levels in cultured meniscus cells isolated from skeletally mature dogs. The mRNA levels of five matrix protein genes (COL1A1, COL2A1, aggrecan, COL6A1, and fibronectin) were measured in meniscus cells by Northern blotting and compared with those of patellar tendon fibroblasts and femoral articular cartilage chondrocytes. In freshly isolated cells (Day 0 cells), COL1A1, COL2A1, and aggrecan mRNA levels were low or undetectable in both meniscus cells and tendon fibroblasts. In intact meniscus tissue, COL1A1 mRNA levels were also low or undetectable. COL2A1 and aggrecan mRNA transcripts were readily observed, however, in Day 0 articular chondrocytes. The levels of expression of COL6A1 and fibronectin mRNA transcripts in Day 0 meniscus cells were intermediate between higher articular chondrocyte levels and lower tendon fibroblast levels. After 1 week in monolayer culture (Day 7 cells), meniscus cells expressed readily detectable levels of COL1A1 mRNA transcripts, similar to that observed for cultured tendon fibroblasts. COL1A1 mRNA transcripts were either not detected or detected at very low levels in monolayer cultures of articular chondrocytes. COL2A1 and aggrecan mRNA transcripts were readily detected in cultured articular chondrocytes but not in meniscus cells or in tendon fibroblasts. All three types of cells continued to express COL6A1 and fibronectin mRNA transcripts after 1 week in culture. These results demonstrate that the patterns of expression of COL1A1 and COL2A1 mRNA transcripts by meniscus cells are similar to those of tendon fibroblasts and dissimilar to those of articular chondrocytes both in freshly isolated cells and in monolayer cultured cells. This mRNA expression pattern supports the idea that monolayer culture of meniscus cells results in the expression of a predominantly fibroblastic phenotype.

Characteristics of the In Vitro Interaction of a Small Proteoglycan (PG II) of Bovine Tendon with Type I Collagen

Binding of the small derma tan sulfate proteoglycan of bovine tendon (PG II type/decorin-like) to type I collagen was characterized in an in vitro fibril-forming assay, using native collagen prepared from bovine tendon by acid extraction and radiolabeled proteoglycans synthesized by bovine tendon fibroblasts in culture. Substantial binding to collagen was noted for both intact small proteoglycan and core protein from which the glycosaminoglycan chain was removed. However, binding to collagen was minimal for free glycosaminoglycan chains or large proteoglycans. Binding of the small proteoglycan was optimal at approximately physiological conditions of salt concentration and pH. Scatchard analysis showed a binding affinity constant of 3.3 X 107M-1 with 0.054 proteoglycan binding sites/collagen molecule, when about 0.25 -6 µg proteoglycan was combined with 100 µg collagen. Binding to preformed fibrils of native tendon collagen and to pepsin-treated bovine skin collagen was similar to binding to native tendon collagen. Binding occurred in non-ionic detergents at concentrations up to 1 % and once bound, the proteoglycan was not released by washing with up to 2 M NaCl. When both PG I and PG II small proteoglycans were added to collagen, only PG II was bound. This difference is not readily explained by differences in disulfide bond position. These studies indicate a strong, specific interaction between type I collagen fibrils and the core protein of the small (PC II) proteoglycan of tendon.

Effect of vitamin A and its derivatives on collagen production and chemotactic response of fibroblasts

Vitamin A and several other retinoids were added to fibroblast cultures in order to study possible alterations in biochemical properties and cellular responsiveness. The proliferation of cells was inhibited as the concentration of retinoids increased from 10(-9) to 10(-5) mol/l. Synthesis of non-collagenous proteins and production of both type I and type III collagen were decreased. The onset of type III collagen synthesis by tendon fibroblasts in culture was delayed. Furthermore, the chemotactic response of fibroblasts to fibroblast-conditioned medium was markedly reduced in the presence of retinoids (10(-6) to 10(-12) mol/l).

The biosynthesis of glycoproteins by cultured bovine tendon fibroblasts.

Confluent bovine fetal tendon fibroblasts maintained in a chemically defined medium incorporated L-[6-3H]fucose and L-[5-3H]proline in a linear manner into non-diffusible macromolecules for up to 48 hrs. Equilibrium CsCl density gradient centrifugation indicated that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. The [3H]fucose-labelled glycoproteins in the culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This technique demonstrated the presence of a number of high mol. wt. fucosylated components, the most notable of which was a glycoprotein of apparent mol. wt. 150,000. Immunological procedures allowed the tentative identification of four glycoproteins including fibronectin which was found in the cell medium and in extracts of the cell layer. Two of the glycoproteins (mol. wts. 150,000 and 270,000) released into the incubation medium were shown to be related to the microfibrillar components of elastic tissue. One or more of the newly synthesized [3H]fucose labelled molecules was shown to be immunologically related to a glycoprotein (mol. wt. 60,000) extracted from bovine Achilles tendon. These studies represent the first demonstration of the synthesis of microfibril-related and tendon glycoprotein-related macromolecules by tendon fibroblasts in culture.

Procollagen processing. Limited proteolysis of COOH-terminal extension peptides by a cathepsin-like protease secreted by tendon fibroblasts.

An enzymatic activity, capable of removing the COOH-terminal extensions of type I chick procollagen, has been demonstrated in embryonic chick tendons and in cultured tendon fibroblasts utilizing two new methods of analysis. The protease was purified by a combination of ultrafiltration concanavalin A affinity chromatography and gel filtration. The isolated protein has an apparent Mr of 43,000 by gel filtration and sodium dodecyl sulfate gel electrophoresis. The enzyme shows a major pH optimum at 4.2 and is susceptible to inhibitors such as pepstatin and leupeptin; it therefore seems related to the cathepsins. The possibility that this enzyme plays a role in the limited proteolytic processing of procollagen is discussed.