Improved Collagen Organization Research Articles

PH-responsive hydrogel with dual-crosslinked network of polyvinyl alcohol/boric acid for controlled release of salvianolic acid B: novel pro-regenerative mechanisms in scar inhibition and wound healing.

This study investigates a novel pH-responsive hydrogel composed of polyvinyl alcohol (PVA) and boric acid (BA) designed for the controlled release of salvianolic acid B (SAB), addressing the critical challenge of scar formation and skin regeneration. The dual-crosslinked network architecture of the hydrogel exhibits remarkable pH sensitivity, enabling it to achieve a peak SAB release within 48 hours in the acidic microenvironment characteristic of early-stage wound healing. In vitro assessments demonstrated that the PVA-BA-SAB hydrogel significantly inhibits fibroblast activation and mitigates abnormal collagen deposition, effectively preventing excessive scar formation. Transcriptome sequencing reveals the potential role of PVA-BA-SAB hydrogel in balancing TGF-β and Wnt signaling pathways. Furthermore, in vivo studies revealed enhanced tissue regeneration, characterized by improved collagen organization and increased vascularization, as well as the promotion of mature hair follicle development. The hydrogel's biocompatibility, mechanical robustness and adhesive properties were also thoroughly evaluated, confirming its suitability for clinical applications. These findings suggest that the PVA-BA-SAB hydrogel fully exerts the excellent characteristics of biomaterials and maximizes the pharmacological effect of SAB. Our innovative drug delivery system not only facilitates enhanced wound healing but also offers a strategic approach to minimize scarring. This research provides valuable insights into innovative therapeutic strategies for effective wound management and tissue repair.

Current Concepts in Orthobiologics for Achilles Tendon Injuries: A Critical Analysis Review.

» Platelet-rich plasma and hyaluronic acid are low-risk and potentially high-reward treatments for Achilles tendinopathy, although clinical studies have yielded mixed results with questionable methodological quality» Case series and reports have reported that bone marrow aspirate, stem cells, and amniotic membrane products can improve functional outcomes, alleviate pain, and facilitate return to sport and activities, but high-level evidence studies are lacking» Exosomes are a promising novel biologic with laboratory studies showing improved collagen organization and cell proliferation, greater tendon mechanical properties, and prevention of extracellular matrix breakdown.» Standardization of protocols with clear reporting is necessary for future studies evaluating orthobiologic therapies for Achilles tendon injuries.

3D bioprinting of multilayered scaffolds with spatially differentiated ADMSCs for rotator cuff tendon-to-bone interface regeneration

Regeneration of the gradient structure of the tendon-to-bone interface is still a significant clinical challenge. This study reports a novel therapeutic method combining three-dimensional (3D) bioprinting and melt electrospinning writing techniques to regenerate a functional tendon-to-bone interface. We generated biomimetic multilayered scaffolds with 3D-bioprinted pre-differentiated autologous adipose-derived mesenchymal stem cells (ADMSC), which recapitulated compositional and cellular structures of the interface. The hydrogel-based bioinks offered high cell viability and proliferative capability for rabbit ADMSCs. The hydrogels with pre-differentiated (into tenogenic, chondrogenic, and osteogenic lineages) or undifferentiated rabbit ADMSCs were 3D-bioprinted into zonal-specific constructs to mimic the structure of the tendon-to-bone interface. These scaffolds were tested in a rabbit rotator cuff injury model and the histological, radiological, and biomechanical changes were analyzed. The in vivo studies demonstrated that the scaffold with spatially differentiated autologous ADMSCs had a superior histological score and improved collagen organization when compared to acellular scaffolds and similar T2 value as the normal interface tissue. The biomechanical characterization demonstrated that the application of multilayered scaffolds improved the biomechanical properties of the tendon-to-bone interface at 12 weeks after rotator cuff reconstruction surgery, but the incorporation of autologous ADMSCs within the multilayered scaffolds showed a limited contribution. Thus, our work provides a 3D-bioprinting-based strategy with the application of autologous ADMSCs to reconstruct massive rotator cuff tendon tears.

Regeneration of the rotator cuff tendon-to-bone interface using umbilical cord-derived mesenchymal stem cells and gradient extracellular matrix scaffolds from adipose tissue in a rat model

Regeneration of the gradient structure of the tendon-to-bone interface (TBI) is a crucial goal after rotator cuff repair. The purpose of this study was to investigate the efficacy of a biomimetic hydroxyapatite-gradient scaffold (HA-G scaffold) isolated from adipose tissue (AD) with umbilical cord derived mesenchymal stem cells (UC MSCs) on the regeneration of the structure of the TBI by analyzing the histological and biomechanical changes in a rat repair model. As a result, the HA-G scaffold had progressively increased numbers of hydroxyapatite (HA) particles from the tendon to the bone phase. After seeding UC MSCs to the scaffold, specific matrices, such as collagen, glycoaminoglycan, and calcium, were synthesized with respect to the HA density. In a rat repair model, compared to the repair group, the UC MSCs seeded HA-G scaffold group had improved collagen organization and cartilage formation by 52% at 8 weeks and 262.96% at 4 weeks respectively. Moreover, ultimate failure load also increased by 30.71% at 4 weeks in the UC MSCs seeded HA-G scaffold group compared to the repair group. Especially, the improved values were comparable to values in normal tissue. This study demonstrated that HA-G scaffold isolated from AD induced UC MSCs to form tendon, cartilage and bone matrices similar to the TBI structure according to the HA density. Furthermore, UC MSC-seeded HA-G scaffold regenerated the TBI of the rotator cuff in a rat repair model in terms of histological and biomechanical properties similar to the normal TBI.Statement of SignificanceWe found specific extracellular matrix (ECM) formation in the biomimetic-hydroxyapatite-gradient-scaffold (HA-G-scaffold) in vitro as well as improved histological and biomechanical results of repaired rotator cuff after the scaffold implantation in a rat model. This study has four strengths; An ECM scaffold derived from human adipose tissue; only one-layer used for a gradient scaffold not a multilayer used to mimic the unique structure of the gradient tendon-to-bone-interface (TBI) of the rotator cuff; UC-MSCs as a new cell source for TBI regeneration; and the UC-MSCs synthesized specific matrices with respect to the HA density without any other stimuli. This study suggested that the UC-MSC seeded HA-G-scaffold could be used as a promising strategy for the regeneration of rotator cuff tears.

Aesculus hippocastanum L. Extract Does Not Induce Fibroblast to Myofibroblast Conversion but Increases Extracellular Matrix Production In Vitro Leading to Increased Wound Tensile Strength in Rats.

The ability of horse chestnut extract (HCE) to induce contraction force in fibroblasts, a process with remarkable significance in skin repair, motivated us to evaluate its wound healing potential in a series of experiments. In the in vitro study of the ability of human dermal fibroblasts to form myofibroblast-like cells was evaluated at the protein level (Western blot and immunofluorescence). The in vivo study was conducted on male Sprague-Dawley rats with inflicted wounds (one open circular and one sutured incision) on their backs. Rats were topically treated with two tested HCE concentrations (0.1% and 1%) or sterile water. The control group remained untreated. The incisions were processed for wound tensile strength (TS) measurement whereas the open wounds were subjected to histological examination. On the in vitro level the HCE extract induced fibronectin-rich extracellular matrix formation, but did not induced α-smooth muscle actin (SMA) expression in dermal fibroblasts. The animal study revealed that HCE increased wound TS and improved collagen organization. In conclusion, the direct comparison of both basic wound models demonstrated that the healing was significantly increased following HCE, thus this extract may be found useful to improve healing of acute wounds. Nevertheless, the use of an experimental rat model warrants a direct extrapolation to the human clinical situation.

Improvement with Fractional Ablative Laser and Topical Poly-L-Lactic Acid in an RDEB Patient

Recessive dystrophic epidermolysis bullosa (RDEB) results from mutations in the COL7A1 gene for type VII collagen. Affected patients demonstrate skin fragility and blistering with scarring. Associated morbidity is great, and there are limited therapeutic options. Fractional carbon dioxide (CO2) ablative laser (FABL) treatments have been shown to promote collagen remodeling and demonstrate synergy with topical poly-l-lactic acid (PLLA) applications. We report the case of a young woman with RDEB who experienced decreased blistering, decreased healing time, and improved quality of life in a local skin area treated with FABL followed by topical PLLA. In comparing biopsy specimens from treated and untreated skin, improved collagen organization and collagen maturity were observed by histopathological examination including with Herovici staining. Moreover, type VII collagen, absent in untreated skin, was detected in treated affected skin by immunostaining. This report illustrates a treatment response to FABL and topical PLLA in a patient with RDEB including enhanced healing time, collagen remodeling, and type VII collagen expression. The findings are promising and, with further study, may have broader implications for treating this disorder.

ACL Graft Signal Characteristics Measured by Quantitative MRI Are Significantly Correlated with Patient Reported Outcomes Two-Years after Hamstring Autograft ACL Reconstruction

Objectives: Following surgical reconstruction of the anterior cruciate ligament (ACL), the tendon graft undergoes a remodeling process of ligamentization. Collagen within the ACL graft becomes organized along the long-axis of the ligament and the proteoglycan content increases. Quantitative imaging sequences, specifically T2 and T1rho, allow for a non-invasive assessment of collagen orientation and proteoglycan content, respectively. The purpose of this study was to investigate the longitudinal progression of T2 and T1rho relaxation times of the graft after ACL reconstruction and the relationship between these quantitative imaging markers and patient-reported outcome measures. We hypothesized that T2 and T1rho would decrease significantly over time, reflecting improved collagen organization and ncreased proteoglycan content, and that T2 and T1rho relaxation times would be inversely correlated with Knee Osteoarthritis Outcome Scores (KOOS). Methods: Thirty-two patients (mean age 29.4±8.2 years, 12 females) were followed prospectively after arthroscopic ACL reconstruction with hamstring autograft. Patients provided informed consent and all procedures were IRB-approved. Exclusion criteria included allograft reconstruction, other ligamentous injury, meniscal tear requiring repair, prior knee surgery, or history of arthritis. Post-operative MR imaging was obtained for 31 patients at 6 months, 30 patients at 12 months, 26 patients at 24 months, and 23 patients at 36 months after surgery. T2 and T1rho relaxation times were simultaneously acquired with a combined 3D sagittal sequence using a 3 T MR system (GE Healthcare) with a 1Tx/8Rx knee coil. The intra-articular ACL graft was identified on a fat-suppressed sagittal high-resolution 3D fast spin echo images and manually segmented. The T2 and T1rho relaxation times for the graft were recorded. Patients completed the KOOS at each imaging time point. Repeated measures analysis of variance (ANOVA) tests with Tukey corrections were used to compare T2 and T1rho relaxation times between time points. The relationship between KOOS and T2 and T1rho values at 2 years post-operative was investigated with Spearman’s rank correlation. Significance was defined as p<0.05. Results: The T2 relaxation times of the ACL graft were significantly higher at 6 months relative to 12 months (p<0.001), 24 months (p<0.001,) and 36 months (p<0.001) after ACL reconstruction, as well as significantly higher at 12 months relative to 36 months (p<0.001) (Figure 1A). The T1rho relaxation times of the ACL graft were significantly higher at 6 months relative to 12 months (p<0.001), 24 months (p<0.001), and 36 months (p<0.001) (Figure 1B). The two-year T2 relaxation times and T1rho relaxation times were significantly correlated with the KOOS Sports, Pain, Symptoms, and Activities of Daily Living sub-scores. Conclusion: We observed significant changes in the tendon graft over time following ACL reconstruction with hamstring autograft, reflecting a higher concentration of proteoglycan and more longitudinally-organized collagen structure over time. Improved collagen organization (lower T2 values) and increased proteoglycan content (lower T1rho values) were correlated with better outcomes based on KOOS scores. Quantitative T2 and T1rho relaxation times of ACL graft may offer a non-invasive method for monitoring graft maturation that correlates with patient-reported knee function after ACL reconstruction. [Figure: see text][Table: see text]

Flexible bipolar nanofibrous membranes for improving gradient microstructure in tendon-to-bone healing.

In this study, we generated a biomimetic dual-layer organic/inorganic flexible bipolar fibrous membrane by sequential electrospinning and in situ biomineralization, producing integrated bipolar fibrous membranes of PLLA fibrous membrane as the upper layer and nHA-PLLA fibrous membrane as the lower layer to mimic non-mineralized fibrocartilage and mineralized fibrocartilage in tendon-to-bone integration of enthesis. Flexible bipolar nanofibrous membranes could be easily fabricated with gradient microstructure for enthesis regeneration in rotator cuff tears.

Healing improvement after rotator cuff repair using gelatin-grafted poly(L-lactide) electrospun fibrous membranes

BackgroundRotator cuff tears (RCTs) are a common cause of shoulder pain and disability in middle and older age. Despite improvements in the understanding of this disease process and advances in surgical treatment, rotator cuff (RC) repair failure rates remain high. Insufficient healing capacity is likely the main factor for failure of reconstruction. Materials and methodsWe fabricated implantable biodegradable gelatin-grafted poly(L-lactide) (PLLA) fibrous membranes using electrospinning technology and evaluated them using in vitro cell proliferation assays. Then, we established chronic rat RCT models and randomly assigned rats into one of three groups. In group 1 (n = 48), the detached supraspinatus tendon was repaired to its anatomic footprint (transosseous repair). In groups 2 and 3, the rats underwent transosseous repair and were implanted with either pure PLLA membranes (n = 48) or gelatin-PLLA membranes (n = 48) to augment the repairs. The animals were killed at 2, 4, and 8 wk postoperatively, which was followed by histomorphometric and biomechanical evaluation. ResultsHistologic observations revealed that gelatin-PLLA membranes have excellent biocompatibility and biodegradability. At 2, 4, and 8 wk postoperatively, the gelatin-PLLA membranes significantly increased the area of glycosaminoglycan staining at the tendon-bone interface compared with the control group (P < 0.05) and significantly improved collagen organization, as measured by birefringence under polarized light at the healing enthesis compared with the control and PLLA groups (P < 0.05). Biomechanical testing revealed that the gelatin-PLLA group had a greater ultimate load to failure and stiffness than the control group at 4 and 8 wk (P < 0.05). The gelatin-PLLA membranes had the highest stress of the healing enthesis. ConclusionsLocal application of gelatin-PLLA fibrous membranes to the healing tendon-bone interface after RC repair in a rat chronic RCT model was found to strengthen the healing enthesis, increase the area of fibrocartilage, and improve collagen organization compared with repair alone. Augmentation with gelatin-grafted PLLA may enhance healing after RC repair and might eventually lead to improvement of clinical surgical outcomes.

Effect of the Interposition of Calcium Phosphate Materials on Tendon-Bone Healing During Repair of Chronic Rotator Cuff Tear

Background: The current nature of tendon-bone healing after rotator cuff (RC) repair is still the formation of granulation tissue at the tendon-bone interface rather than the formation of fibrocartilage, which is the crucial structure in native tendon insertion and can be observed after knee ligament reconstruction. The interposition of calcium phosphate materials has been found to be able to enhance tendon-bone healing in knee ligament reconstruction. However, whether the interposition of these kinds of materials can enhance tendon-bone healing or even change the current nature of tendon-bone healing after RC repair still needs to be explored. Hypothesis: The interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current nature of granulation tissue formation into a more favorable process. Study Design: Controlled laboratory study. Methods: A total of 144 male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon, followed by delayed repair after 3 weeks. The animals were allocated into 1 of 3 groups: (1) repair alone, (2) repair with Ca5(PO4)2SiO4 (CPS) bioceramic interposition, or (3) repair with hydroxyapatite (HA) bioceramic interposition at the tendon-bone interface. Animals were sacrificed at 2, 4, or 8 weeks postoperatively, and microcomputed tomography (micro-CT) was used to quantify the new bone formation at the repair site. New fibrocartilage formation and collagen organization at the tendon-bone interface was evaluated by histomorphometric analysis. Biomechanical testing of the supraspinatus tendon-bone complex was performed. Statistical analysis was performed using 1-way analysis of variance. Significance was set at P < .05. Results: The micro-CT analysis demonstrated remarkable osteogenic activity and osteoconductivity to promote new bone formation and ingrowth of CPS and HA bioceramic, with CPS bioceramic showing better results than HA. Histological observations indicated that CPS bioceramic had excellent biocompatibility and biodegradability. At early time points after the RC repair, CPS bioceramic significantly increased the area of fibrocartilage at the tendon-bone interface compared with the control and HA groups. Moreover, CPS and HA bioceramics had significantly improved collagen organization. Biomechanical tests indicated that the CPS and HA groups have greater ultimate load to failure and stiffness than the control group at 4 and 8 weeks, and the CPS specimens exhibited the maximum ultimate load to failure, stiffness, and stress of the healing enthesis. Conclusion: Both CPS and HA bioceramics aid in cell attachment and proliferation and accelerate new bone formation, and CPS bioceramic has a more prominent effect on tendon-to-bone healing. Clinical Relevance: Local application of CPS and HA bioceramic at the tendon-bone interface shows promise in improving healing after rotator cuff tear repair.

Biological augmentation of rotator cuff repair using bFGF-loaded electrospun poly(lactide-co-glycolide) fibrous membranes.

Clinically, rotator cuff tear (RCT) is among the most common shoulder pathologies. Despite significant advances in surgical techniques, the re-tear rate after rotator cuff (RC) repair remains high. Insufficient healing capacity is likely the main factor for reconstruction failure. This study reports on a basic fibroblast growth factor (bFGF)-loaded electrospun poly(lactide-co-glycolide) (PLGA) fibrous membrane for repairing RCT. Implantable biodegradable bFGF–PLGA fibrous membranes were successfully fabricated using emulsion electrospinning technology and then characterized and evaluated with in vitro and in vivo cell proliferation assays and repairs of rat chronic RCTs. Emulsion electrospinning fabricated ultrafine fibers with a core-sheath structure which secured the bioactivity of bFGF in a sustained manner for 3 weeks. Histological observations showed that electrospun fibrous membranes have excellent biocompatibility and biodegradability. At 2, 4, and 8 weeks after in vivo RCT repair surgery, electrospun fibrous membranes significantly increased the area of glycosaminoglycan staining at the tendon–bone interface compared with the control group, and bFGF–PLGA significantly improved collagen organization, as measured by birefringence under polarized light at the healing enthesis compared with the control and PLGA groups. Biomechanical testing showed that the electrospun fibrous membrane groups had a greater ultimate load-to-failure and stiffness than the control group at 4 and 8 weeks. The bFGF–PLGA membranes had the highest ultimate load-to-failure, stiffness, and stress of the healing enthesis, and their superiority compared to PLGA alone was significant. These results demonstrated that electrospun fibrous membranes aid in cell attachment and proliferation, as well as accelerating tendon–bone remodeling, and bFGF-loaded PLGA fibrous membranes have a more pronounced effect on tendon–bone healing. Therefore, augmentation using bFGF–PLGA electrospun fibrous membranes is a promising treatment for RCT.

Cell‐based tissue engineering augments tendon‐to‐bone healing in a rat supraspinatus model

Rotator cuff pathology causes substantial pain/disability and health care costs. Cell-based tissue engineering offers promise for improved outcomes in tendon to bone healing. Cells from the tendon-bone interface were used here to amplify surgical defect healing in a rat model. Cells from tendon-to-bone interface of the rotator cuff were seeded in sponges and implanted into critical rotator cuff defects: Group I, control; II, surgical defect only; III, suture-repaired defect; IV, surgical defect, repair with sponge only; V, surgical defect, repair with sponge with cells. Three, 6-, and 12-week results were assessed for histologic features. At 3 weeks, histologic indices in Group V were significantly increased versus other treatment groups. Group V (12 weeks) showed significantly improved collagen organization versus other treatment groups; there was no difference in collagen organization in Group I versus V. In summary, increased cellularity, inflammation, vascularity, and collagen organization were present at 3 weeks; increased collagen organization at 12 weeks in Group V provides evidence for improved healing with cells. Data further support the utility of tendon-bone interface cells in rotator cuff healing.

Effect of recombinant human platelet-derived growth factor-BB-coated sutures on Achilles tendon healing in a rat model: A histological and biomechanical study

Purpose: Repairing tendon injuries with recombinant human platelet-derived growth factor-BB has potential for improving surgical outcomes. Augmentation of sutures, a critical component of surgical tendon repair, by coating with growth factors may provide a clinically useful therapeutic device for improving tendon repair. Therefore, the purpose of this study was to (a) coat Vicryl sutures with a defined dose of recombinant human platelet-derived growth factor-BB without additional coating excipients (e.g. gelatin), (b) quantify the recombinant human platelet-derived growth factor-BB released from the suture, and (c) use the recombinant human platelet-derived growth factor-BB-coated sutures to enhance tendon repair in a rat Achilles tendon transection model. Methods: Vicryl sutures were coated with 0, 0.3, 1.0, and 10.0 mg/mL concentrations of recombinant human platelet-derived growth factor-BB using a dip-coating process. In vitro release was quantified by an enzyme-linked immunosorbent assay. Acutely transected rat Achilles tendons were repaired using one of the four suture groups (n = 12 per group). Four weeks following repair, the tensile biomechanical and histological (i.e. collagen organization and angiogenesis) properties were determined. Results: A dose-dependent bolus release of recombinant human platelet-derived growth factor-BB occurred within the first hour in vitro, followed by a gradual release over 48 h. There was a significant increase in ultimate tensile strength (p < 0.01) in the two highest recombinant human platelet-derived growth factor-BB dose groups (1.9 ± 0.5 and 2.1 ± 0.5 MPa) relative to controls (1.0 ± 0.2 MPa). The modulus significantly increased (p = 0.031) with the highest recombinant human platelet-derived growth factor-BB dose group (7.2 ± 3.8 MPa) relative to all other groups (control: 3.5 ± 0.9 MPa). No significant differences were identified for the maximum load or stiffness. The histological collagen and angiogenesis scores were comparable in all groups, although there was a trend for improved collagen organization in the recombinant human platelet-derived growth factor-BB-treated groups (p = 0.054). Conclusions: The results of this study suggest that recombinant human platelet-derived growth factor-BB can be used to reproducibly coat Vicryl sutures and improve remodeling in a rat Achilles tendon transection model by significantly decreasing the resulting cross-sectional area, thus improving the material properties of the repaired tendon.

Calcium-Phosphate Matrix With or Without TGF-β3 Improves Tendon-Bone Healing After Rotator Cuff Repair

Background: Rotator cuff tendon heals by formation of an interposed zone of fibrovascular scar tissue. Recent studies demonstrate that transforming growth factor–beta 3 (TGF-β3) is associated with tissue regeneration and “scarless” healing, in contrast to scar-mediated healing that occurs with TGF-β1. Hypothesis: Delivery of TGF-β3 in an injectable calcium-phosphate matrix to the healing tendon-bone interface after rotator cuff repair will result in increased attachment strength secondary to improved bone formation and collagen organization and reduced scar formation of the healing enthesis. Study Design: Controlled laboratory study. Methods: Ninety-six male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon followed by acute repair using transosseous suture fixation. Animals were allocated into 1 of 3 groups: (1) repair alone (controls, n = 32), (2) repair augmented by application of an osteoconductive calcium-phosphate (Ca-P) matrix only (n = 32), or (3) repair augmented with Ca-P matrix + TGF-β3 (2.75 µg) at the tendon-bone interface (n = 32). Animals were euthanized at either 2 weeks or 4 weeks postoperatively. Biomechanical testing of the supraspinatus tendon-bone complex was performed at 2 and 4 weeks (n = 8 per group). Microcomputed tomography was utilized to quantitate bone microstructure at the repair site. The healing tendon-bone interface was evaluated with histomorphometry and immunohistochemical localization of collagen types I (COLI) and III (COLIII). Statistical analysis was performed using 2-way analysis of variance with significance set at P < .05. Results: There was significantly greater load to failure of the Ca-P matrix + TGF-β3 group compared with matrix alone or untreated controls at 4 weeks postoperatively (P = .04). At 2 weeks, microcomputed tomography revealed a larger volume of newly formed bone present at the healing enthesis in both experimental groups compared with the control group. By 4 weeks, this newly formed, woven bone had matured into calcified, lamellar bone. Histomorphometric analysis demonstrated significantly greater fibrocartilage and increased collagen organization at the healing tendon-bone insertion site in both experimental groups compared with the control group at 2 weeks (P = .04). Over time, TGF-β3 delivery led to greater COLI expression compared with COLIII at the healing enthesis, indicating a more favorable COLI to COLIII ratio with administration of TGF-β3. Conclusion: Augmentation with an osteoconductive Ca-P matrix at the tendon-bone repair site is associated with new bone formation, increased fibrocartilage, and improved collagen organization at the healing tendon-bone interface in the early postoperative period after rotator cuff repair. The addition of TGF-β3 significantly improved strength of the repair at 4 weeks postoperatively and resulted in a more favorable COLI/COLIII ratio. Clinical Relevance: The delivery of TGF-β3 with an injectable Ca-P matrix at the supraspinatus tendon footprint has promise to improve healing after soft tissue repair.

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.

Doxycycline-Mediated Inhibition of Matrix Metalloproteinases Improves Healing after Rotator Cuff Repair

Background Recent studies demonstrate a potentially critical role of matrix metalloproteinases (MMPs) and their inhibitors in the pathophysiology of rotator cuff tears. Hypothesis Doxycycline-mediated MMP inhibition after rotator cuff repair will improve tendon-to-bone healing. Study Design Controlled laboratory study. Methods Rats (n = 183) underwent acute detachment and repair of the supraspinatus tendon and the animals were divided into 4 groups: In controls (n = 66), the supraspinatus was repaired to its anatomical footprint. In experimental groups, an identical surgery was performed with doxycycline (130 mg/kg/d) administered orally at (1) preoperative day 1 (n = 66), (2) postoperative day (POD) 5 (n = 28), or (3) POD 14 (n = 23). Animals were sacrificed at 5 days, 8 days, 2 weeks, and 4 weeks. Tendon-bone interface was evaluated with histomorphometry. Enzyme-linked immunosorbent assay for local MMP-13 activity was performed at 8 days and 4 weeks. Biomechanical testing of the healing enthesis was performed at 8 days, 2 weeks, and 4 weeks. Serum doxycycline levels were measured at sacrifice. Statistical analysis was performed using unpaired t tests and 2-way analysis of variance (P < .05). Results Serum doxycycline levels were significantly higher in all treated groups compared with controls (1830 ± 835 vs 3 ± 3 ng/mL, respectively; P < .001). Doxycycline-treated animals demonstrated greater metachromasia and improved collagen organization at the healing enthesis at POD 5 (P < .06), POD 8 (P < .03), and 2 weeks (P < .04). The MMP-13 activity was significantly reduced in doxycycline-treated compared with control animals at POD 8 (6740 ± 2770 vs 10400 ± 2930 relative fluorescent units [RFU], respectively; P < .02) but not at 4 weeks (3600 ± 3280 vs 4530 ± 2720 RFU, respectively). The healing enthesis of animals started on doxycycline preoperatively or at POD 5 had an increased load to failure compared to controls at 2 weeks (13.6 ± 1.8 and 13.2 ± 1.94 N vs 9.1 ± 2.5 N, respectively; P < .01). Conclusion/Clinical Relevance Modulation of MMP-13 activity after rotator cuff repair may offer a novel biological pathway to augment tendon-to-bone healing.

Cell Permeant Peptide Analogues of the Small Heat Shock Protein, HSP20, Reduce TGF-β1-Induced CTGF Expression in Keloid Fibroblasts

A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.