Promote Tendon Healing Research Articles

Lactoferrin and parathyroid hormone are not harmful to primary tenocytes in vitro, but PDGF may be.

Recently, bone-active factors such as parathyroid hormone and lactoferrin, have been used in pre-clinical models to promote tendon healing. How-ever, there is limited understanding of how these boneactive factors may affect the cells of the ten-don themselves. Here, we present an in vitro study assessing the effects of parathyroid hor-mone and lactoferrin on primary tendon cells (tenocytes), and compare their responses to the tenogenic factors, PDGF, IGF-1 and TGF-β. Tenocyte proliferation and collagen production were assessed by alamarBlue® and Sirius red as-says, respectively. To assess tenocyte trans-differentiation, changes in the expression of genes important in tenocyte, chondrocyte and osteoblast biology were determined using real-time PCR. Parathyroid hormone and lactoferrin had no effect on tenocyte growth or collagen production, with minimal changes in gene expression and no detrimental effects observed to suggest trans-differentiation away from tendon cell behaviour. Tenogenic factors PDGF, IGF-1 and TGF all increasetenocyte collagen production, however, the gene expression data suggests that PDGF promotes severe de-differentiation of the tenocytes. Our findings suggest that using parathyroid hormone or lactoferrin as a singular factor to promote tendon healing may not be of benefit, but for use in tendon-bone healing there would be no detrimental effect on the tendon itself.

The effect of platelet-rich plasma on Achilles tendon healing in a rabbit model

ObjectiveThe aim of the present study was to evaluate the effects of PRP on Achilles tendon healing in rabbits during the inflammatory, proliferative, and remodeling phases by histological examination and quantitative assessments. MethodsFifty mature male Japanese albino rabbits with severed Achilles tendons were divided into two equal groups and treated with platelet-rich plasma (PRP) or left untreated. Tendon tissue was harvested at 1, 2, 3, 4, and 6 weeks after treatment, and sections were stained with hematoxylin-eosin and monoclonal antibodies against CD31 and type I collagen. ResultsCollagen fibers proliferated more densely early after severance, and subsequent remodeling of the collagen fibers and approximation of normal tendinous tissue occurred earlier in the PRP group than in the control group. The fibroblast number was significantly higher in the PRP group than in the control group at 1 and 2 weeks. Similarly, the area ratio of CD31-positive cells was significantly higher in the PRP group than in the control group at 1 and 2 weeks. Positive staining for type I collagen was more intense in the PRP group than in the control group after 3 weeks, indicating tendon maturation. ConclusionAdministration of PRP shortened the inflammatory phase and promoted tendon healing during the proliferative phase.

Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR‐29b‐3p and activating TGF‐β1 signaling

Tendon injures are common orthopedic conditions, but tendon development and the pathogenesis of tendon injures, such as tendinopathy, remain largely unknown and have limited the development of clinical therapy. Studies on tenogenic differentiation at the molecular level may help in developing novel therapeutic strategies. As novel regulators, long noncoding RNAs (lncRNAs) have been found to have widespread biological functions, and emerging evidence demonstrates that lncRNAs may play important regulatory roles in cell differentiation and tissue regeneration. In this study, we found that lncRNA H19 stimulated tenogenesis of human tendon-derived stem cells. Stable overexpression of H19 significantly accelerated TGF-β1-induced tenogenic differentiation in vitro and accelerated tendon healing in a mouse tendon defect model. H19 directly targeted miR-29b-3p, which is considered to be a negative regulator of tenogenesis. Furthermore, miR-29b-3p directly suppressed the expression of TGF-β1 and type I collagen, thereby forming a novel regulatory feedback loop between H19 and TGF-β1 to mediate tenogenic differentiation. Our study demonstrated that H19 promotes tenogenic differentiation both in vitro and in vivo by targeting miR-29b-3p and activating TGF-β1 signaling. Regulation of the TGF-β1/H19/miR-29b-3p regulatory loop may be a new strategy for treating tendon injury.-Lu, Y.-F., Liu, Y., Fu, W.-M., Xu, J., Wang, B., Sun, Y.-X., Wu, T.-Y., Xu, L.-L, Chan, K.-M., Zhang, J.-F., Li, G. Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR-29b-3p and activating TGF-β1 signaling.

EGR1 Regulates Transcription Downstream of Mechanical Signals during Tendon Formation and Healing.

BackgroundTendon is a mechanical tissue that transmits forces generated by muscle to bone in order to allow body motion. The molecular pathways that sense mechanical forces during tendon formation, homeostasis and repair are not known. EGR1 is a mechanosensitive transcription factor involved in tendon formation, homeostasis and repair. We hypothesized that EGR1 senses mechanical signals to promote tendon gene expression.Methodology/Principal findingsUsing in vitro and in vivo models, we show that the expression of Egr1 and tendon genes is downregulated in 3D-engineered tendons made of mesenchymal stem cells when tension is released as well as in tendon homeostasis and healing when mechanical signals are reduced. We further demonstrate that EGR1 overexpression prevents tendon gene downregulation in 3D-engineered tendons when tension is released. Lastly, ultrasound and microbubbles mediated EGR1 overexpression prevents the downregulation of tendon gene expression during tendon healing in reduced load conditions.Conclusion/SignificanceThese results show that Egr1 expression is sensitive to mechanical signals in tendon cells. Moreover, EGR1 overexpression prevents the downregulation of tendon gene expression in the absence of mechanical signals in 3D-engineered tendons and tendon healing. These results show that EGR1 induces a transcriptional response downstream of mechanical signals in tendon cells and open new avenues to use EGR1 to promote tendon healing in reduced load conditions.

The Use of Cocultured Mesenchymal Stem Cells with Tendon-Derived Stem Cells as a Better Cell Source for Tendon Repair.

The management of tendon tissue injury presents a significant clinical challenge due to the unique properties of tendons. Cell-based therapy provides a new alternative for regenerating functional tendons, such as in tendon rupture repair, but largely remains at the preclinical research stage. A cell source for graft preparation is essential for successful clinic application. In this study, a novel cell coculture system of bone marrow mesenchymal stem cells (BMSCs) and tendon-derived stem cells (TDSCs) was developed and investigated. BMSCs and TDSCs were cultured separately or in combination at ratios of 20:1, 10:1, 5:1, and 1:1 in vitro, and the cocultured cells showed an enhanced proliferation and collagenous protein production. The coculture system promoted tenogenic differentiation with enhanced tenogenic marker gene expression and collagen matrix production, particularly in the groups with a ratio of 1:1. Using a rat patellar tendon window injury model, we demonstrated that the cell sheets formed by cocultured cells promoted tendon healing significantly, compared to those with a single-cell source. Our study suggests that BMSCs and TDSCs cocultured at the 1:1 ratio may be an improved cell source/preparation for tendon tissue engineering.

Bioactive, Elastic, and Biodegradable Emulsion Electrospun DegraPol Tube Delivering PDGF‐BB for Tendon Rupture Repair

Healing of tendon ruptures represents a major challenge in musculoskeletal injuries and combinations of biomaterials with biological factors are suggested as viable option for improved healing. The standard approach of repair by conventional suture leads to incomplete healing or rerupture. Here, a new elastic type of DegraPol® (DP), a polyester urethane, is explored as a delivery device for platelet-derived growth factor-BB (PDGF-BB) to promote tendon healing. Using emulsion electrospinning as an easy method for incorporation of biomolecules within polymers, DegraPol® supports loading and release of PDGF-BB. Morphological, mechanical and delivery device properties of the bioactive DP scaffolds, as well as differences arising due to different electrospinning parameters are studied. Emulsion electrospun DP scaffolds result in thinner fibers than pure DP scaffolds and experience decreased strain at break [%], but high enough for successful surgeon handling. PDGF-BB is released in a sustained manner from emulsion electrospun DP, but not completely, with still large amount of it being inside the polymeric fibers after 30 d. In vitro studies show that the bioactive scaffolds promote tenocyte proliferation in serum free and serum(+) conditions, demonstrating the potential of this surgeon-friendly bioactive delivery device to be used for tendon repair.

Effect of transforming growth factor-β3 on the expression of Smad3 and Smad7 in tenocytes.

Tendon adhesion is a common problem in the healing of injured tendons. The molecular mechanisms of the TGF-β/Smad signaling pathway have been determined, and the role of TGF-β has been well characterized in wound healing. However, the intracellular mechanism or downstream signals by which TGF-β3 modulates its effects on tendon healing have not been well elucidated. The aim of this study was to determine the effect of TGF‑β3 on the TGF-β/Smad signaling pathway in tenocytes. Quantitative polymerase chain reaction and western blot analysis were used to analyze the effect of TGF‑β3 on the regulation of the expression of Smad proteins in tenocytes. The results demonstrated that TGF‑β3 has no significant effect on the proliferation of tendon cells. The addition of TGF‑β3 to tenocytes can significantly downregulate the expression of Smad3 and upregulate the expression of Smad7 at the gene and protein levels. The results demonstrate that TGF‑β3 may regulate Smad3 and Smad7 proteins through the TGF-β/Smad signaling pathway to minimize extrinsic scarring. Thus, it may provide a novel approach to decrease tendon adhesion and promote tendon healing.

Release of celecoxib from a bi-layer biomimetic tendon sheath to prevent tissue adhesion

Posttraumatic tendon adhesion limits the motion of the limbs greatly. Biomimetic tendon sheaths have been developed to promote tendon healing and gliding. However, after introduction of these biomaterials, the associated inflammatory responses can decrease the anti-adhesion effect. Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) that can decrease inflammation responses. We blended hyaluronic acid and poly(l-lactic acid)–polyethylene glycol (PELA) with microgel electrospinning technology to form an inner layer of a bi-layer biomimetic sheath using sequential electrospinning of an outer celecoxib–PELA layer. Electrospun bi-layer fibrous membranes were mechanically tested and characterized by morphology, surface wettability, and drug release. The tensile strength showed a decreased trend and water contact angles were 114.7±3.9°, 103.6±4.4°, 116.3±5.1°, 122.8±4.7°, and 126.5±4.2° for the surface of PELA, hyaluronic acid–PELA, 2, 6, and 10% celecoxib–PELA electrospun fibrous membranes, respectively. In vitro drug release studies confirmed burst release and then sustained release from the fibrous membranes containing celecoxib for 20days. In a chicken model of flexor digitorum profundus tendon surgery, the outer celecoxib/PELA layer offered advanced anti-adhesion roles compared to the outer PELA layer and the inner hyaluronic acid-loaded PELA layer still offered tendon healing and gliding. Thus, celecoxib-loaded anti-adhesive tendon sheaths can continuously offer bi-layer biomimetic tendon sheath effects with celecoxib release from the outer layer to prevent tendon adhesion.

Curcumin improves tendon healing in rats: a histological, biochemical, and functional evaluation

ABSTRACTCurcumin, a compound extracted from the roots of Zingiberaceae, has been proposed as a treatment for tissue injury but studies are yet to be done on its effect on tendon healing. Therefore, we performed a series of experiments to test our hypothesis that curcumin has positive effects on tendon repair. Patellar tendon window defect was created in Sprague–Dawley rats and these were divided into two groups: (i) control and (ii) curcumin-treated. Curcumin (100 mg/kg body weight) was applied by oral gavage. Its potential for promoting tendon healing was assessed by histological evaluation, mRNA expression of tenocyte-related genes, malondialdehyde (MDA) levels, manganese-dependent superoxide dismutase (MnSOD) activity, quantification of hydroxyproline (HOPro), and biomechanical testing. In this tendon injury model, curcumin significantly improved the healing properties as evidenced by extensive deposition of well-organized collagen fibers, decreased MDA levels, and increase in the biomechanical properties and MnSOD activity of the regenerated tendon tissues. The current study showed that curcumin can improve the quality of tendon rupture healing, and thus represents a promising strategy in the management of injured tendon tissue.

Allogeneic tendon-derived stem cells promote tendon healing and suppress immunoreactions in hosts: in vivo model.

The medium- to long-term healing effect and infiltration of inflammatory cells, after transplantation of allogeneic tendon-derived stem cell (TDSC) to the rat patellar tendon window wound, were examined. Allogeneic patellar TDSCs derived from a green fluorescent protein rat were used. The outcome of tendon healing and the infiltration of inflammatory cells were examined by histology and immunohistochemistry up to week 16 postinjury. The fate of the transplanted cells was examined by ex vivo fluorescent imaging and immunohistochemistry. Our results showed that the transplantation of allogeneic TDSCs promoted tendon healing with no increased risk of ectopic chondro-ossification up to week 16. A low infiltration of T cells, ED1 macrophages, ED2 macrophages, and mast cells in the window wound was obtained. The transplanted TDSCs were found in the window wound at week 1 and 2, but were absent after week 4 postinjury. In conclusion, allogeneic TDSCs promoted tendon repair in the medium to long term and exhibited weak immunoreactions and anti-inflammatory effects in the hosts after transplantation in a rat model. There was no increased risk of ectopic chondro-ossification after TDSC transplantation. The decrease in the number of transplanted cells with time suggested that allogeneic TDSCs did not promote tendon repair through direct differentiation.

Silver nanoparticles alter proteoglycan expression in the promotion of tendon repair

This study demonstrates a novel method of using silver nanoparticles for Achilles tendon injury healing. In vitro results indicated a stimulatory effect on cell proliferation and collagen synthesis with silver nanoparticles. Biomechanical test on the 42-day post operation Achilles tendon sample exhibited a significant improvement in tensile modulus when compared to the untreated group. Histology suggested that silver nanoparticles promoted cell alignment and proteoglycan synthesis. The collagen deposition was also improved. An alleviation of tumor necrosis factor α, and an increase in fibromodulin and proliferating cell nuclear antigen expression were seen in silver nanoparticles group by immunohistochemistry. This study further corroborates the finding of our previous study that silver nanoparticles help to restore the functionality of injured connective tissues. We believe that the anti-inflammatory nature of silver nanoparticles has an important role in accelerating the healing process and reducing scarring, leading to better functional outcome. From the Clinical EditorTendon healing after surgeries remains a slow and tedious process, typically requiring several weeks of recovery time and gradual introduction of physical therapy. There are no currently utilized methods that could promote tendon healing. In this study, silver nanoparticles are reported to facilitate Achilles tendon repair in a model system, through increased proteoglycan and collagen synthesis, paving the way to potential clinical applications in the future.

Efficacy of tendon stem cells in fibroblast-derived matrix for tendon tissue engineering

Background aimsAfter injury, tendons often heal with poor tissue quality and inferior mechanical properties. Tissue engineering using tendon stem cells (TSCs) is a promising approach in the repair of injured tendon. Tenogenic differentiation of TSCs needs an appropriate environment. More recently, the acellular extracellular matrix (ECM) generated from fibroblasts has been used to construct various engineering tissues. In this study, we successfully developed an engineered tendon tissue formed by seeding TSCs in de-cellularized fibroblast-derived matrix (dFM). MethodsPatellar TSCs and dermal fibroblast were isolated and cultured. Using the method of osmotic shock, dFM was obtained from dermal fibroblast. ECM proteins in dFM were examined. TSCs at passage 3 were seeded in dFM for 1 week. Proliferative capacity and characterization of TSCs cultured in dFM were determined by population doubling time, immunofluorescence staining and quantitative reverse transcriptase polymerase chain reaction. Engineered tendon tissue was prepared with dFM and TSCs. Its potentials for neo-tendon formation and promoting tendon healing were investigated. ResultsdFM is suitable for growth and tenogenic differentiation of TSCs in vitro. Neo-tendon tissue was formed with tendon-specific protein expression when TSCs were implanted together with dFM. In a patellar tendon injury model, implantation of engineered tendon tissue significantly improved the histologic and mechanical properties of injured tendon. ConclusionsThe findings obtained from our study provide a basis for potential use of engineered tendon tissue containing dFM and TSCs in tendon repair and regeneration.

Development of vitamin C irrigation saline to promote graft healing in anterior cruciate ligament reconstruction

Summary Graft healing in anterior cruciate ligament (ACL) reconstruction is often slow and compromised. There are numerous treatment strategies targeting at the biological healing process in ACL reconstruction. Most of these treatments attempt to promote graft incorporation at the tendon to bone interface, although the intra-articular mid substance would be more problematic. As intraoperative injection of vitamin C has been reported to be able to promote tendon healing, we speculate that vitamin C supplementation may also work for graft healing enhancement in ACL reconstruction. The purpose of this study is to formulate a vitamin C-supplemented saline for intraoperative irrigation, in order to promote biological healing in ACL reconstruction. The study hypothesis was tested using 114 rats. During ACL reconstruction, the knee joint of the rats were irrigated with either normal saline, or saline supplemented with 3 mg/mL, 10 mg/mL, or 30 mg/mL vitamin C. Inflammatory response was measured by serum C-reactive protein enzyme-linked immunosorbent assay at Day 1, Day 4, and Day 7 postoperation. Functional recovery was evaluated by gait analysis at preinjury, and Week 4 and Week 6 postoperation. Knee samples were harvested at Week 6 postoperation for anterior–posterior (A–P) knee laxity test and graft pull-out test. Histology was performed at Day 1, Day 4, Day 7, and Day 42 for scoring. Vitamin C supplementation at all doses significantly reduced serum C-reactive protein level as compared to saline control at Day 1 ( p = 0.029) postoperation. At 6 weeks postoperation, ACL reconstruction with 3 mg/mL vitamin C irrigation led to a better restoration of A–P knee laxity as compared to saline irrigation ( p = 0.032), whereas no positive effect was observed in the groups with 10 mg/mL and 30 mg/mL vitamin C irrigation. Vitamin C irrigation did not affect ultimate load of the graft complex. Histological analysis revealed that at Week 6 postoperation, graft deterioration was significantly reduced in 3 mg/mL and 10 mg/mL vitamin C groups, but no significant difference was observed in graft incorporation among different groups at Day 42 postoperation. Although no significant difference was detected in the gait parameters among experimental groups, increased percentage of rats with asymmetric walking gait (with limb idleness index > 1.3) was found in the groups of 10 mg/mL or 30 mg/mL vitamin C irrigation. The results showed that intraoperative supplementation of low dose (3 mg/mL) vitamin C effectively reduced graft deterioration and improved restoration of A–P knee laxity at 6 weeks postoperation, but higher doses of vitamin C irrigation may have side effects that lead to a higher chance of gait asymmetry. This study reveals the potential use of surgical irrigation saline as a delivery method for bioactive agents to promote healing, which might be a cost-effective and translatable approach for biological modulation for orthopaedic surgeries.

Human acellular amniotic membrane prevents tendon adhesion after allogenic tendon transplantation to repair Achilles' tendon defects in rabbits

Objective To investigate the effect of human acellular amniotic membrane on tendon adhesion after allogenic tendon transplantation to repair Achilles' tendon defects in rabbits.Methods Thirty New Zealand white rabbits were randomly divided into 2 groups (n =15).In the experimental group the transplanted Achilles' tendon was covered with human acellular amniotic membrane while in the control group the transplanted Achilles' tendon was covered with nothing.The transplanted tissue samples were harvested at 2 and 4 weeks for histological observation.Peripheral blood samples were harvested for flow cytometry respectively at 2,4,and 6 weeks.The transplanted Achilles' tendons were harvested for gross observation,biomechanical tests and the hydroxyproline (Hyp) content determination at 2,4,and 6 weeks after operation.Results The histological observation showed the inflammatory reaction in the experimental group was slighter than in the control group at 2 and 4 weeks and the arrangement of collagen fibers in the former was denser and more orderly than in the latter.The flow cytometry showed that at 2,4,and 6 weeks the peripheral blood in the experimental group contained significantly fewer CD4 + and CD8 + T lymphocytes than in the control group,and the ratio of CD4+/CD8+T lymphocyte in the experimental group was significantly lower than in the control group (both P ＜ 0.05).At 2,4,and 6 weeks,the maximum tensile strength in the experimental group was significantly larger than in the control group,and the tensile adhesion power consumption in the experimental group was significantly smaller than in the control group (both P ＜ 0.05).The Hyp content in the experimental group was significantly higher than in the control group (P ＜ 0.05).Conclusions Human acellular amniotic membrane can prevent tendon adhesion,promote tendon healing and intensify strength effectively,and may suppress immunological rejection to a certain extent in the allogenic tendon transplantation. Key words: Amnion; Achilles; Transplantation, homologous; Achilles' tendon adhesion; Rabbits

PARot – assessing platelet-rich plasma plus arthroscopic subacromial decompression in the treatment of rotator cuff tendinopathy: study protocol for a randomized controlled trial

BackgroundPlatelet-rich plasma (PRP) is an autologous platelet concentrate. It is prepared by separating the platelet fraction of whole blood from patients and mixing it with an agent to activate the platelets. In a clinical setting, PRP may be reapplied to the patient to improve and hasten the healing of tissue. The therapeutic effect is based on the presence of growth factors stored in the platelets. Current evidence in orthopedics shows that PRP applications can be used to accelerate bone and soft tissue regeneration following tendon injuries and arthroplasty. Outcomes include decreased inflammation, reduced blood loss and post-treatment pain relief. Recent shoulder research indicates there is poor vascularization present in the area around tendinopathies and this possibly prevents full healing capacity post surgery (Am J Sports Med36(6):1171–1178, 2008). Although it is becoming popular in other areas of orthopedics there is little evidence regarding the use of PRP for shoulder pathologies. The application of PRP may help to revascularize the area and consequently promote tendon healing. Such evidence highlights an opportunity to explore the efficacy of PRP use during arthroscopic shoulder surgery for rotator cuff pathologies.Methods/DesignPARot is a single center, blinded superiority-type randomized controlled trial assessing the clinical outcomes of PRP applications in patients who undergo shoulder surgery for rotator cuff disease. Patients will be randomized to one of the following treatment groups: arthroscopic subacromial decompression surgery or arthroscopic subacromial decompression surgery with application of PRP.The study will run for 3 years and aims to randomize 40 patients. Recruitment will be for 24 months with final follow-up at 1 year post surgery. The third year will also involve collation and analysis of the data. This study will be funded through the NIHR Biomedical Research Unit at the Oxford University Hospitals NHS Trust.Trial registrationCurrent Controlled Trials: ISRCTN10464365

Engineered scaffold-free tendon tissue produced by tendon-derived stem cells

Most of the exogenous biomaterials for tendon repair have limitations including lower capacity for inducing cell proliferation and differentiation, poorer biocompatibility and remodeling potentials. To avoid these shortcomings, we intend to construct an engineered tendon by stem cells and growth factors without exogenous scaffolds. In this study, we produced an engineered scaffold-free tendon tissue (ESFTT) in vitro and investigated its potentials for neo-tendon formation and promoting tendon healing in vivo. The ESFTT, produced via tendon-derived stem cells (TDSCs) by treatment of connective tissue growth factor (CTGF) and ascorbic acid in vitro, was characterized by histology, qRT-PCR and immunohistochemistry methods. After ESFTT implanted into the nude mouse, the in vivo fluorescence imaging, histology and immunohistochemistry examinations showed neo-tendon formation. In a rat patellar tendon window injury model, the histology, immunohistochemistry and biomechanical testing data indicated ESFTT could significantly promote tendon healing. In conclusion, this is a proof-of-concept study demonstrating that ESFTT could be a potentially new approach for tendon repair and regeneration.

Survivorship of implanted bone marrow-derived mesenchymal stem cells in acute rotator cuff tear

This study examined whether a mesenchymal stem cells (MSCs)-seeded 3-dimensional construct into a tendon defect would promote cellular differentiation and matrix healing. Bone marrow was harvested from the iliac crests of 2 male New Zealand White rabbits. The MSCs were cultured, and an open-cell polylactic acid (OPLA) scaffold was encapsulated with these cells. The injury model was a 5-mm×5-mm-sized full-thickness window defect in the central part of each rotator cuff tendon. The defects on the right side were grafted with the autologous MSCs-seeded OPLA scaffold implant and a biodegradable suture. The same procedure was done on the left side, except a cell-free OPLA scaffold was used. Three rabbits were used as controls, without treatment of the tendon defect. Samples were harvested at 2, 4, and 6 weeks for analysis, which included evaluation of gross morphology, fluorescent analysis, histologic assessment, and immunohistochemistry studies. The expression of immunohistochemical stainings for collagen I was higher in the scaffold with MSCs than in the scaffold without MSCs. The expression of collagen II, however, was not different between the scaffolds with and without MSCs. Even though this is a short-term study, we demonstrated that many MSCs in the scaffold survived after implantation in an acute rabbit rotator cuff defect. Furthermore, the generation of type I collagen increased more in the scaffold with MSCs than it did in the scaffold without MSCs. MSCs are thought to promote tendon healing by producing type I collagen when they are applied at the tendon defect.

Platelet-Rich Plasma Stimulates Cell Proliferation and Enhances Matrix Gene Expression and Synthesis in Tenocytes From Human Rotator Cuff Tendons With Degenerative Tears

Background: Platelet-rich plasma (PRP) contains various growth factors and appears to have a potential to promote tendon healing, but evidence is lacking regarding its effect on human tenocytes from rotator cuff tendons with degenerative tears. Hypothesis: Platelet-rich plasma stimulates cell proliferation and enhances matrix gene expression and synthesis in tenocytes isolated from human rotator cuff tendons with degenerative tears. Study Design: Controlled laboratory study. Methods: Tenocytes were enzymatically isolated and cultured. To evaluate cell proliferation, tenocytes were cultured with 10% (vol/vol) platelet-poor plasma (PPP), PRP activated with calcium, and PRP activated with calcium and thrombin at platelet concentrations of 100, 200, 400, 800, 1000, 2000, 4000, 8000, and 16,000 × 103/µL for 14 days. Cell number was measured at days 7 and 14. To investigate matrix gene expression and synthesis, cells were cultured with a PPP or PRP gel (10% vol/vol) at a platelet concentration of 1000 × 103/µL for 14 days. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to determine the expressions of type I and III collagen, decorin, tenascin-C, and scleraxis, and measurements of total collagen and glycosaminoglycan (GAG) synthesis were conducted at days 7 and 14. Results: Platelet-rich plasma significantly increased cell proliferation at days 7 and 14 in a dose-dependent manner, and the addition of thrombin moved up the plateau of proliferation. Platelet-rich plasma significantly induced the gene expression of type I collagen at day 7 but not at day 14, while it significantly promoted that of type III both at days 7 and 14. The ratio of type III/I collagens did not change at days 7 and 14. The expressions of decorin and scleraxis significantly increased at day 14, whereas that of tenascin-C significantly increased at days 7 and 14. Platelet-rich plasma significantly increased total collagen synthesis at days 7 and 14 and GAG synthesis at day 14. Conclusion: Platelet-rich plasma promoted cell proliferation and enhanced gene expression and the synthesis of tendon matrix in tenocytes from human rotator cuff tendons with degenerative tears. Clinical Relevance: These findings suggest that PRP might be used as a useful biological tool for regenerative healing of rotator cuff tears by enhancing the proliferation and matrix synthesis of tenocytes from tendons with degenerative tears.

Platelet-released growth factors can accelerate tenocyte proliferation and activate the anti-oxidant response element

Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2-ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2-ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.

Growth differentiation factor-5 regulation of extracellular matrix gene expression in murine tendon fibroblasts

The synthesis and organization of extracellular matrix (ECM) of tendon, in resting and states of repair, are governed by fibroblasts. Growth differentiation factor-5 (GDF-5) may enhance the cellular response to tendon injury, thus improving the structural outcome of the regenerative tissue. This study was an attempt to identify potential mechanisms controlling the response of fibroblasts to injury and GDF-5, in the pursuit of improved tissue regeneration. There were two sets of experiments. Isolated mice Achilles tendon fibroblasts were treated with different concentrations of rGDF-5 (0-100 ng/ml) for 0-12 days in cell culture. The temporal effect of rGDF-5 on ECM gene expression was analysed for type I collagen and aggrecan expression. Microarray and gene expression analysis were performed on cells treated with 100 ng/ml for 4 days. Forty-five mice underwent bilateral mid-substance Achilles tendon tenotomy and suture repair. Repair sites were injected with 10 µg rGDF-5 or saline. Tendons were assessed histologically at 2, 4 and 6 weeks. Expression of ECM genes procollagen IX, aggrecan, matrix metalloproteinase 9 and fibromodulin were upregulated. Proinflammatory reaction genes were downregulated. rGDF-5 led to an increase in total DNA, glycosaminoglycan (GAG) and hydroxyproline (OHP). The OHP:DNA ratio of fibroblast cultures was increased over all time points, with increased GAG:DNA at day 12. rGDF-5 treatment showed improved collagen organization over controls. The results delineate the mode of action of rGDF-5 at the cellular and gene level. rGDF-5 could play a role in tendon repair and be used for future therapies that promote tendon healing.