Glycine improves the remodeling process of tenocytes in vitro.

Tendinitis changes the biochemical and morphological properties of the tendon, promoting an increase of activity of metalloproteinases and disorganization of collagen bundles. Tenocytes, the primary cells in tendon, are scattered throughout the collagenic fibers, and are responsible of tendon remodeling and tissue repair in pathological condition. In vivo, glycine, component of the typical Gly-X-Y collagen tripeptide, showed beneficial effects in biochemical and biomechanical properties of Achilles tendon with tendinitis. In this study, we analyzed the effect of glycine in tenocytes subjected to inflammation. Tenocytes from Achilles tendon of rats were treated with TNF-α (10 ng/mL) with and without previous treatment with glycine (20 mM). Cell proliferation and migration were evaluated, as well as the expression of matrix molecules such as glycosaminoglycans, metalloproteinases (MMPs), TIMPs, and collagen I. Glycine can revert the inflammation due to the action of TNF-α by controlling the MMPs quantity and activity. These data indicated that the molecules involved to remodeling process of extracellular matrix are modulated both by TNF-α and the availability of collagen precursors; in fact, this study indicates the glycine can be useful for treatment of inflammation and for modulating tenocytes metabolism in tendons.

Inhibitory effect of red LED irradiation on fibroblasts and co-culture of adipose-derived mesenchymal stem cells.

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm2/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-ß1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-ß1, whereas TGF-ß1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-ß1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair.

Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells.

Tissue engineering and cell-based therapy combine techniques that create biocompatible materials for cell survival, which can improve tendon repair. This study seeks to use a new fibrin sealant (FS) derived from the venom of Crotalus durissus terrificus, a biodegradable three-dimensional scaffolding produced from animal components only, associated with adipose-derived stem cells (ASC) for application in tendons injuries, considered a common and serious orthopedic problem. Lewis rats had tendons distributed in five groups: normal (N), transected (T), transected and FS (FS) or ASC (ASC) or with FS and ASC (FS + ASC). The in vivo imaging showed higher quantification of transplanted PKH26-labeled ASC in tendons of FS + ASC compared to ASC on the 14th day after transection. A small number of Iba1 labeled macrophages carrying PKH26 signal, probably due to phagocytosis of dead ASC, were observed in tendons of transected groups. ASC up-regulated the Tenomodulin gene expression in the transection region when compared to N, T and FS groups and the expression of TIMP-2 and Scleraxis genes in relation to the N group. FS group presented a greater organization of collagen fibers, followed by FS + ASC and ASC in comparison to N. Tendons from ASC group presented higher hydroxyproline concentration in relation to N and the transected tendons of T, FS and FS + ASC had a higher amount of collagen I and tenomodulin in comparison to N group. Although no marked differences were observed in the other biomechanical parameters, T group had higher value of maximum load compared to the groups ASC and FS + ASC. In conclusion, the FS kept constant the number of transplanted ASC in the transected region until the 14th day after injury. Our data suggest this FS to be a good scaffold for treatment during tendon repair because it was the most effective one regarding tendon organization recovering, followed by the FS treatment associated with ASC and finally by the transplanted ASC on the 21st day. Further investigations in long-term time points of the tendon repair are needed to analyze if the higher tissue organization found with the FS scaffold will improve the biomechanics of the tendons.

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5.

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats' gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.