Project description:Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real-time RT-PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp-9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp-2 and Mmp-14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf-5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level.

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Keywords: time_series_design

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Computed

Project description:BackgroundWithout surgical repair, flexor tendon injuries do not heal and patients' ability to bend fingers and grip objects is impaired. However, flexor tendon repair surgery also requires optimal rehabilitation. There are currently three custom-made splints used in the rehabilitation of zone I/II flexor tendon repairs, each with different assumed harm/benefit profiles: the dorsal forearm and hand-based splint (long), the Manchester short splint (short), and the relative motion flexion splint (mini). There is, however, no robust evidence as to which splint, if any, is most clinical or cost effective. The Flexor Injury Rehabilitation Splint Trial (FIRST) was designed to address this evidence gap.MethodsFIRST is a parallel group, superiority, analyst-blind, multi-centre, individual participant-randomised controlled trial. Participants will be assigned 1:1:1 to receive either the long, short, or mini splint. We aim to recruit 429 participants undergoing rehabilitation following zone I/II flexor tendon repair surgery. Potential participants will initially be identified prior to surgery, in NHS hand clinics across the UK, and consented and randomised at their splint fitting appointment post-surgery. The primary outcome will be the mean post-randomisation score on the patient-reported wrist and hand evaluation measure (PRWHE), assessed at 6, 12, 26, and 52 weeks post randomisation. Secondary outcome measures include blinded grip strength and active range of movement (AROM) assessments, adverse events, adherence to the splinting protocol (measured via temperature sensors inserted into the splints), quality of life assessment, and further patient-reported outcomes. An economic evaluation will assess the cost-effectiveness of each splint, and a qualitative sub-study will evaluate participants' preferences for, and experiences of wearing, the splints. Furthermore, a mediation analysis will determine the relationship between patient preferences, splint adherence, and splint effectiveness.DiscussionFIRST will compare the three splints with respect to clinical efficacy, complications, quality of life and cost-effectiveness. FIRST is a pragmatic trial which will recruit from 26 NHS sites to allow findings to be generalisable to current clinical practice in the UK. It will also provide significant insights into patient experiences of splint wear and how adherence to splinting may impact outcomes.Trial registrationISRCTN: 10236011.

Project description:Background/objectiveStem cell–based therapy has been applied to accelerate the revitalization of allograft tendon into a viable and functional tendon. Although many authors have proposed different methods to help the seeded stem cell distribution in the decellularized allograft, limited success has been achieved as tendon is a high dense connective tissue. We hypothesized that bone marrow stromal cells (BMSCs), seeded through the lateral slit, can regenerate the decellularized tendon (DCT) graft. The cell proliferation, cell viability, and tendon-specific gene expression are increased with the seeded cell density.MethodsEighty-seven flexor digitorum profundus tendons were equally and randomly divided into 6 treatment groups that were seeded with low-density (2 × 107 cells/mL) and high-density (5 × 107 cells/mL) BMSCs through lateral slits cultured for 2 and 4 weeks, DCT without cells, and fresh live tendons. Tendons were evaluated for cell distribution, cell proliferation, cell viability, gene expression of Collagen I and Collagen III, tenogenic markers, and MMPs.ResultsHistologic evaluation revealed BMSCs distributed from the lateral slit to the whole DCT. BMSCs were proliferated and kept viable in lateral slit decellularized tendon (LSDCT) in both seeded cell density groups after 2 and 4 weeks of culture. However, no significant differences in the cell proliferation between both cell density groups at 2 and 4 weeks of culture were observed. The lowest cell viability was found in the high-density group after 4 weeks of culture. BMSCs in LSDCT showed a significant tendency of higher gene expression of Collagen I, Collagen III, tenascin C, MMP2, MMP9, and MMP13 compared to normal tendons in both cell density groups at 2 and 4 weeks of culture.ConclusionBMSCs proliferated and remained viable after 2 and 4 weeks of culture with distribution throughout the lateral slits. Lateral slit preparation allows for the effective delivery and maintenance of mesenchymal cells with proliferation and generating a tenogenic behaviour of DCT in both the low and high cell densities in an in vitro model.The translation potential of this articleRevitalizing the implanted decellularized allograft is important for clinical application. In this study, we demonstrated that the DCT, with lateral slits, could harbour the seeded stem cell and stimulate proliferation with collagen synthesis. This evidence was presented for clinical application of the lateral slit technique, in DCT grafts, which would repopulate the seeded BMSCs during tendon and ligament reconstruction.

Dataset Information

Serpine1 Knockdown Enhances MMP Activity after Flexor Tendon Injury in Mice: Implications for Adhesions Therapy.

Publications

Serpine1 Knockdown Enhances MMP Activity after Flexor Tendon Injury in Mice: Implications for Adhesions Therapy.

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