Examining the Potential of Vitamin C Supplementation in Tissue-Engineered Equine Superficial Digital Flexor Tendon Constructs.

Because equine tendinopathies are slow to heal and often recur, therapeutic strategies are being considered that aid tendon repair. Given the success of utilizing vitamin C to promote tenogenesis in other species, we hypothesized that vitamin C supplementation would produce dose-dependent improvements in the tenogenic properties of tendon proper (TP) and peritenon (PERI) cells of the equine superficial digital flexor tendon (SDFT). Equine TP- and PERI-progenitor-cell-seeded fibrin three-dimensional constructs were supplemented with four concentrations of vitamin C. The gene expression profiles of the constructs were assessed with 3'-Tag-Seq and real-time quantitative polymerase chain reaction (RT-qPCR); collagen content and fibril ultrastructure were also analyzed. Moreover, cells were challenged with dexamethasone to determine the levels of cytoprotection afforded by vitamin C. Expression profiling demonstrated that vitamin C had an anti-inflammatory effect on TP and PERI cell constructs. Moreover, vitamin C supplementation mitigated the degenerative pathways seen in tendinopathy and increased collagen content in tendon constructs. When challenged with dexamethasone in two-dimensional culture, vitamin C had a cytoprotective effect for TP cells but not necessarily for PERI cells. Future studies will explore the effects of vitamin C on these cells during inflammation and within the tendon niche in vivo.

Tendon Cells Root Into (Instead of Attach to) Humeral Bone Head via Fibrocartilage-Enthesis.

Large joints are composed of two closely linked cartilages: articular cartilage (AC; rich in type II collagen, a well-studied tissue) and fibrocartilaginous enthesis (FE; rich in type I collagen, common disorder sites of enthesopathy and sporting injuries, although receiving little attention). For many years, both cartilages were thought to be formed by chondrocytes, whereas tendon, which attaches to the humeral bone head, is primarily considered as a completely different connective tissue. In this study, we raised an unconventional hypothesis: tendon cells directly form FE via cell transdifferentiation. To test this hypothesis, we first qualitatively and quantitatively demonstrated distinct differences between AC and FE in cell morphology and cell distribution, mineralization status, extracellular matrix (ECM) contents, and critical ECM protein expression profiles using comprehensive approaches. Next, we traced the cell fate of tendon cells using ScxLin (a tendon specific Cre ScxCreERT2; R26R-tdTomato line) with one-time tamoxifen induction at early (P3) or young adult (P28) stages and harvested mice at different development ages, respectively. Our early tracing data revealed different growth events in tendon and FE: an initial increase but gradual decrease in the ScxLin tendon cells and a continuous expansion in the ScxLin FE cells. The young adult tracing data demonstrated continuous recruitment of ScxLin cells into FE expansion during P28 and P56. A separate tracing line, 3.2 Col 1Lin (a so-called "bone-specific" line), further confirmed the direct contribution of tendon cells for FE cell formation, which occurred in days but FE ECM maturation (including high levels of SOST, a potent Wnt signaling inhibitor) took weeks. Finally, loss of function data using diphtheria toxin fragment A (DTA) in ScxLin cells demonstrated a significant reduction of ScxLin cells in both tendons and FE cells, whereas the gain of function study (by stabilizing ß-catenin in ScxLin tendon cells via one-time injection of tamoxifen at P3 and harvesting at P60) displayed great expansion of both ScxLin tendon and FE mass. Together, our studies demonstrated that fibrocartilage is an invaded enthesis likely originating from the tendon via a quick cell transdifferentiation mechanism with a lengthy ECM maturation process. The postnatally formed fibrocartilage roots into existing cartilage and firmly connects tendon and bone instead of acting as a simple attachment site as widely believed. We believe that this study will stimulate more intense exploring in this understudied area, especially for patients with enthesopathy and sporting injuries.

Texturing Hierarchical Tissues by Gradient Assembling of Microengineered Platelet-Lysates Activated Fibers.

The heterogeneity of hierarchical tissues requires designing multipart engineered constructs as suitable tissue replacements. Herein, the incorporation of platelet lysate (PL) within an electrospun fiber core is proposed aiming for the fabrication of functionally graded 3D scaffolds for heterotypic tissues regeneration, such as tendon-to-bone interfaces. First, anisotropic yarns (A-Yarns) and isotropic threads with nanohydroxyapatite (I-Threads/PL@nHAp) are fabricated to recreate the tendon- and bone-microstructures and both incorporated with PL using emulsion electrospinning for a sustained and local delivery of growth factors, cytokines, and chemokines. Biological performance using human adipose-derived stem cells demonstrates that A-Yarns/PL induce a higher expression of scleraxis, a tenogenic-marker, while in I-Threads/PL@nHAp, higher alkaline phosphatase activity and matrix mineralization suggest an osteogenic commitment without the need for biochemical supplementation compared to controls. As a proof-of-concept, functional 3D gradient scaffolds are fabricated using a weaving technique, resulting in 3D textured hierarchical constructs with gradients in composition and topography. Additionally, the precise delivery of bioactive cues together with in situ biophysical features guide the commitment into a phenotypic gradient exhibiting chondrogenic and osteochondrogenic profiles in the interface of scaffolds. Overall, a promising patch solution for the regeneration of tendon-to-bone tissue interface through the fabrication of PL-functional 3D gradient constructs is demonstrated.

The effect of the platelet-rich plasma and ozone therapy on tendon-to-bone healing in the rabbit rotator cuff repair model.

BACKGROUND: The aim of this study is to histologically and biomechanically investigate the effects of local PRP and ozone therapy (O2O3) on tendon-to-bone healing in a rabbit model of the supraspinatus tendon tear. METHODS: Four groups were formed to have seven rabbits in each group: repair, R; repair + PRP, RP; repair + ozone, RO; and repair + PRP + ozone, RPO. The supraspinatus tendon was detached by sharp dissection from the footprint and an acute tear pattern was created. Thereafter, tendon repair was performed with the transosseous technique. In the RP group, PRP, and in the RPO group, PRP + O2O3 mixture was injected to the tendon repair site. In the RO group, O2O3 gas mixture was injected into subacromial space three times a week for a total of 4 weeks. The study was ended at postoperative 6th week. RESULTS: When compared with the R group, a statistically significant increase was observed in the biomechanical strength of the RP and RPO groups. The highest increase in biomechanical strength was detected in the RPO group. The histology of the RO and RPO groups showed better collagen fiber continuity and orientation than the R and RP groups. CONCLUSIONS: The results obtained from this study show that the ozonized PRP can be used as biological support to increase tendon-to-bone healing. However, these results need to be supported by clinical studies.

Photobiomodulation modulates the expression of inflammatory cytokines during the compensatory hypertrophy process in skeletal muscle.

Compensatory hypertrophy (CH) occurs due to excessive mechanical load on a muscle, promoting an increase in the size of muscle fibers. In clinical practice, situations such as partial nerve injuries, denervation, and muscle imbalance caused by trauma to muscles and nerves or diseases that promote the loss of nerve conduction can induce CH in muscle fibers. Photobiomodulation (PBM) has demonstrated beneficial effects on muscle tissue during CH. The aim of the present study was to evaluate the effect of PBM on the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) as well as type 2 metalloproteinases (MMP-2) during the process of CH due to excessive load on the plantaris muscle in rats. Forty-five Wistar rats weighing 250 g were divided into three groups: control group (n = 10), hypertrophy (H) group (n = 40), and H + PBM group (n = 40). CH was induced through the ablation of synergist muscles of the plantaris muscle. The tendons of the gastrocnemius and soleus muscles were isolated and sectioned to enable the partial removal of each of muscle. The preserved plantaris muscle below the removed muscles was submitted to excessive functional load. PBM was performed with low-level laser (AsGaAl, λ = 780 nm; 40 mW; energy density: 10 J/cm2; 10 s on each point, 8 points; 3.2 J). Animals from each group were euthanized after 7 and 14 days. The plantaris muscles were carefully removed and sent for analysis of the gene and protein expression of IL-6 and TNF-α using qPCR and ELISA, respectively. MMP-2 activity was analyzed using zymography. The results were submitted to statistical analysis (ANOVA + Tukey's test, p < 0.05). The protein expression analysis revealed an increase in IL-6 levels in the H + PBM group compared to the H group and a reduction in the H group compared to the control group. A reduction in TNF-α was found in the H and H + PBM groups compared to the control group at 7 days. The gene expression analysis revealed an increase in IL-6 in the H + PBM group compared to the H group at 14 days as well as an increase in TNF-α in the H + PBM group compared to the H group at 7 days. Increases in MMP-2 were found in the H and H + PBM groups compared to the control group at both 7 and 14 days. Based on findings in the present study, it is concluded that PBM was able to modulate pro-inflammatory cytokines that are essential for the compensatory hypertrophy process. However, it has not shown a modulation effect directly in MMP-2 activity during the same period evaluated.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1ß Primed Tendon Cells.

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.

A Textile Platform Using Continuous Aligned and Textured Composite Microfibers to Engineer Tendon-to-Bone Interface Gradient Scaffolds.

Tendon-to-bone interfaces exhibit a hierarchical multitissue transition. To replicate the progression from mineralized to nonmineralized tissue, a novel 3D fibrous scaffold is fabricated with spatial control over mineral distribution and cellular alignment. For this purpose, wet-spun continuous microfibers are produced using polycaprolactone (PCL)/ gelatin and PCL/gelatin/hydroxyapatite nano-to-microparticles (HAp). Higher extrusion rates result in aligned PCL/gelatin microfibers while, in the case of PCL/gelatin/HAp, the presence of minerals leads to a less organized structure. Biological performance using human adipose-derived stem cells (hASCs) demonstrates that topography of PCL/gelatin microfibers can induce cytoskeleton elongation, resembling native tenogenic organization. Matrix mineralization on PCL/gelatin/HAp wet-spun composite microfibers suggest the production of an osteogenic-like matrix, without external addition of osteogenic medium supplementation. As proof of concept, a 3D gradient structure is produced by assembling PCL/gelatin and PCL/gelatin/HAp microfibers, resulting in a fibrous scaffold with a continuous topographical and compositional gradient. Overall, the feasibility of wet-spinning for the generation of continuously aligned and textured microfibers is demonsrated, which can be further assembled into more complex 3D gradient structures to mimic characteristic features of tendon-to-bone interfaces.

Tenogenic differentiation protocol in xenogenic-free media enhances tendon-related marker expression in ASCs.

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFß-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFß3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells.

Mechanical strain determines the site-specific localization of inflammation and tissue damage in arthritis.

Many pro-inflammatory pathways leading to arthritis have global effects on the immune system rather than only acting locally in joints. The reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Here we show that biomechanical loading acts as a decisive factor in the transition from systemic autoimmunity to joint inflammation. Distribution of inflammation and erosive disease is confined to mechano-sensitive regions with a unique microanatomy. Curiously, this pathway relies on stromal cells but not adaptive immunity. Mechano-stimulation of mesenchymal cells induces CXCL1 and CCL2 for the recruitment of classical monocytes, which can differentiate into bone-resorbing osteoclasts. Genetic ablation of CCL2 or pharmacologic targeting of its receptor CCR2 abates mechanically-induced exacerbation of arthritis, indicating that stress-induced chemokine release by mesenchymal cells and chemo-attraction of monocytes determines preferential homing of arthritis to certain hot spots. Thus, mechanical strain controls the site-specific localisation of inflammation and tissue damage in arthritis.

Housekeeping Gene Stability in Human Mesenchymal Stem and Tendon Cells Exposed to Tenogenic Factors.

The use of biochemical inducers of mesenchymal stem cell (MSC) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. Bone morphogenetic protein 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes. To date, no published studies have evaluated the stability of housekeeping genes in MSCs during tenogenic induction. Three candidate housekeeping genes (YWHAZ, RPL13A, and GAPDH) in human MSCs from bone marrow (BMSCs), adipose tissue (ASCs), and tendon cells (TCs) supplemented with BMP-12 or AA and bFGF in comparison with control untreated cells for 3 and 10 days were evaluated. GeNorm, NormFinder, and BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability and the overall ranking was determined by using by the RefFinder algorithm. In all culture conditions, YWHAZ was the most stable gene and RPL13A was the second choice. YWHAZ and RPL13A were the two most stable genes also for ASCs and BMSCs, regardless of the time point analyzed, and for TCs at 10 days of tenogenic induction. Only for TCs at 3 days of tenogenic induction were GAPDH and YWHAZ the best performers. In conclusion, our findings will be useful for the proper selection of housekeeping genes in studies involving MSCs cultured in the presence of tenogenic factors, to obtain accurate and high-quality data from quantitative gene expression analysis.

Tenogenic Differentiation of Human Embryonic Stem Cells.

Tendon healing is complex to manage because of the limited regeneration capacity of tendon tissue; stem cell-based tissue engineering approaches may provide alternative healing strategies. We sought to determine whether human embryonic stem cells (hESC) could be induced to differentiate into tendon-like cells by the addition of exogenous bone morphogenetic protein (BMP)12 (growth differentiation factor[GDF]7) and BMP13 (GDF6). hESC (SHEF-1) were maintained with or without BMP12/13 supplementation, or supplemented with BMP12/13 and the Smad signaling cascade blocking agent, dorsomorphin. Primary rat tenocytes were included as a positive control in immunocytochemistry analysis. A tenocyte-like elongated morphology was observed in hESC after 40-days continuous supplementation with BMP12/13 and ascorbic acid (AA). These cells displayed a tenomodulin expression pattern and morphology consistent with that of the primary tenocyte control. Analysis of tendon-linked gene transcription in BMP12/13 supplemented hESC demonstrated consistent expression of COL1A2, COL3A1, DCN, TNC, THBS4, and TNMD levels. Conversely, when hESCs were cultured in the presence of BMP12/13 and dorsomorphin COL3A1, DCN, and TNC gene expression and tendon matrix formation were inhibited. Taken together, we have demonstrated that hESCs are responsive to tenogenic induction via BMP12/13 in the presence of AA. The directed in vitro generation of tenocytes from pluripotent stem cells may facilitate the development of novel repair approaches for this difficult to heal tissue.

Effects of Redox Modulation on Cell Proliferation, Viability, and Migration in Cultured Rat and Human Tendon Progenitor Cells.

Tendon healing is slow and usually results in inferior fibrotic tissue formation. Recently, application of tendon derived stem cells (TDSCs) improved tendon healing in animal studies. In a chicken model, local injection of antioxidants reduced tendon adhesion after tendon injury. An in vitro study demonstrated that supplementation of H2O2 reduced tenogenic marker expression in TDSCs. These findings suggested that the possibility of TDSCs is involved in tendon healing and the cellular activities of TDSCs might be affected by oxidative stress of the local environment. After tendon injury, oxidative stress is increased. Redox modulation might affect healing outcomes via affecting cellular activities in TDSCs. To study the effect of oxidative stress on TDSCs, the cellular activities of rat/human TDSCs were measured under different dosages of vitamin C or H2O2 in this study. Lower dose of vitamin C increased cell proliferation, viability and migration; H2O2 affected colony formation and suppressed cell migration, cell viability, apoptosis, and proliferation. Consistent with previous studies, oxidative stresses (H2O2) affect both recruitment and survival of TDSCs, while the antioxidant vitamin C may exert beneficial effects at low doses. In conclusion, redox modulation affected cellular activities of TDSCs and might be a potential strategy for tendon healing treatment.

Thermal Preconditioning May Prevent Tendon Adhesion by Up-Regulating HSP72 in Rats.

BACKGROUND/AIMS: The study aims to determine the effects of thermal preconditioning on tendon adhesion by regulating the expression of heat shock protein 72 (HSP72) in rat models. METHODS: Sixty male Wistar rats were collected and randomly assigned into the thermal preconditioning and control groups. During the 4th and 8th weeks following surgery, 15 rats were sacrificed in each period respectively, and their tendon adhesion was observed and evaluated. Biomechanical testing was performed to measure the tensile strength and gliding distance of tendons. Hematoxylin-eosin (HE) was used to observe the morphological structure of the tendons. Immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the HSP72, fibroblast growth factor-2 (FGF-2), fibroblast growth factor receptor-1 (FGFR-1), ß-catenin, epithelial cell adhesion molecule (EPCAM), Tenomodulin and scleraxis protein expressions. Pearson correlation analysis was applied to analyze the correlation between HSP72 expression and tendon adhesion. RESULTS: At the 4th week after surgery, we found no differences in the tendon adhesion scores or mRNA and protein expressions of HSP72 between the thermal preconditioning and control groups. However, after the 8th week after surgery, the thermal preconditioning group had a lower tendon adhesion score and higher mRNA and protein expressions of HSP72 than the control group. During the same period, we found longer gliding distance and higher expression levels of FGF-2, FGFR-1, ß-catenin, Tenomodulin and scleraxis, but lower EPCAM expression in the thermal preconditioning group. Pearson correlation analysis indicated that HSP72 mRNA and protein expression levels were negatively correlated with tendon adhesion. CONCLUSIONS: These findings provide evidence that thermal preconditioning may alleviate tendon adhesions via upregulation of HSP72 expression.

Tendons and Ligaments: Connecting Developmental Biology to Musculoskeletal Disease Pathogenesis.

Tendons and ligaments provide connections between muscle and bone or bone and bone to enable locomotion. Damage to tendons and ligaments caused by acute or chronic injury or associated with aging and arthritis is a prevalent cause of disability. Improvements in approaches for the treatment of these conditions depend on a better understanding of tendon and ligament development, cell biology, and pathophysiology. This review focuses on recent advances in the discovery of transcription factors that control ligament and tendon cell differentiation, how cell and extracellular matrix homeostasis are altered in disease, and how this new insight can lead to novel therapeutic approaches. © 2017 American Society for Bone and Mineral Research.

Low level laser therapy accelerates the extracellular matrix reorganization of inflamed tendon.

In tendon lesions, inflammation indicates the beginning of tissue repair and influences cell proliferation and the remodeling of the extracellular matrix (ECM). Low level laser (LLL) therapy has been an important method to induce tissue repair, and several studies have sought to better understand the therapeutic possibilities of this modality. This study analyzed the effect of LLL on the ECM of rat tendons during the early phase of the inflammatory process. Wistar rats received an intratendinous application of carrageenan adjacent to the osteotendinous region in the right paw. The animals were divided into the following groups: G1-intact, G2-animals with no treatment after the inflammation induction, G3-animals treated with LLL 1 and 3h after induction of inflammation (4J/cm2 continuous). After 4h of application, the animals of the two groups were euthanized with isoflurane overdose. Our results demonstrate that LLL therapy can promote decrease in non-collagenous protein and glycosaminoglycans content, as well as an increase in metalloproteinases -9, which proved, for the first time, that LLL therapy promotes alterations in the inflamed tendons even when analyzed only four hours after this process occur and could be a useful tool to improve the balance in inflamed tissues.

The effect of estrogen on tendon and ligament metabolism and function.

Tendons and ligaments are crucial structures inside the musculoskeletal system. Still many issues in the treatment of tendon diseases and injuries have yet not been resolved sufficiently. In particular, the role of estrogen-like compound (ELC) in tendon biology has received until now little attention in modern research, despite ELC being a well-studied and important factor in the physiology of other parts of the musculoskeletal system. In this review we attempt to summarize the available information on this topic and to determine many open questions in this field.

Do Dietary Factors Influence Tendon Metabolism?

There is very little direct research to conclusively prove the relevance of diet in primary tendinopathies, however it seems prudent to ask whether our current knowledge about the impact of nutrition on collagen metabolism could be useful in assessing, preventing, or treating tendinopathy. The objective of this chapter is to discuss the potential impact (negative or positive) that nutrition may have on the metabolism of tendons by summarizing the related research. The chapter briefly discusses the roles that specific vitamins, amino acids, lipids, and antioxidants have in various processes of the body that may be directly or indirectly related to tenocyte metabolism.

Low-level laser therapy modulates pro-inflammatory cytokines after partial tenotomy.

Tendon injuries give rise to substantial morbidity, and current understanding of the mechanisms involved in tendon injury and repair is limited. This lesion remains a clinical issue because the injury site becomes a region with a high incidence of recurrent rupture and has drawn the attention of researchers. We already demonstrated that low-level laser therapy (LLLT) stimulates the synthesis and organization of collagen I, MMP-9, and MMP-2 and improved the gait recovery of the treated animals. The aim of this study was to evaluate the effects of LLLT in the nitric oxide and cytokines profile during the inflammatory and remodeling phases. Adult male rats were divided into the following groups: G1--intact, G2-- injured, G3--injured + LLLT (4 J/cm(2) continuous), G4--injured + LLLT (4 J/cm(2)-20 Hz--pulsed laser). According to the analysis, the animals were euthanized on different dates (1, 4, 8, or 15 days after injury). ELISA assay of TNF-α, IL-1ß, IL-10, and TGF-ß was performed. Western blotting of isoform of nitric oxide synthase (i-NOS) and nitric oxide dosage experiments was conducted. Our results showed that the pulsed LLLT seems to exert an anti-inflammatory effect over injured tendons, with reduction of the release of proinflammatory cytokines, such as TNF-α and the decrease in the i-NOS activity. Thanks to the pain reduction and the facilitation of movement, there was a stimulation in the TGF-ß and IL-1ß release. In conclusion, we believe that pulsed LLLT worked effectively as a therapy to reestablish the tendon integrity after rupture.

Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.