Tendon Transection Research Articles

Obesity/Type II diabetes alters macrophage polarization resulting in a fibrotic tendon healing response.

Type II Diabetes (T2DM) dramatically impairs the tendon healing response, resulting in decreased collagen organization and mechanics relative to non-diabetic tendons. Despite this burden, there remains a paucity of information regarding the mechanisms that govern impaired healing of diabetic tendons. Mice were placed on either a high fat diet (T2DM) or low fat diet (lean) and underwent flexor tendon transection and repair surgery. Healing was assessed via mechanical testing, histology and changes in gene expression associated with collagen synthesis, matrix remodeling, and macrophage polarization. Obese/diabetic tendons healed with increased scar formation and impaired mechanical properties. Consistent with this, prolonged and excess expression of extracellular matrix (ECM) components were observed in obese/T2DM tendons. Macrophages are involved in both inflammatory and matrix deposition processes during healing. Obese/T2DM tendons healed with increased expression of markers of pro-inflammatory M1 macrophages, and elevated and prolonged expression of M2 macrophages markers that are involved in ECM deposition. Here we demonstrate that tendons from obese/diabetic mice heal with increased scar formation and increased M2 polarization, identifying excess M2 macrophage activity and matrix synthesis as a potential mechanism of the fibrotic healing phenotype observed in T2DM tendons, and as such a potential target to improve tendon healing in T2DM.

The Effect of Adipose-derived Mesenchymal Stem Cells on Healing of Massive Chronic Rotator Cuff Tear in Rodent Model

Objectives:Mesenchymal stem cells (MSCs) may promote restoration of the structure and function of the injured tendon-bone interface of the healing rotator cuff following repair. As compared to acute tears, chronic massive rotator cuff tears are a challenging healing microenvironment, with degenerative changes present in the muscle, tendon, and bone. Therefore, the purpose of this study was to explore the efficacy of MSCs in restoring the tendon-bone interface following repair of massive chronic rotator cuff tears in a rodent model.Methods:Adipose-derived mesenchymal stem cells (ADSCs) were isolated from inguinal fat of adult Lewis rats (n= 8) and expanded to passage 2 (P2). To induce degenerative changes in a rat modeling a massive chronic rotator cuff tear, the supraspinatus and infraspinatus tendons were transected bilaterally near their bony insertion and the corresponding muscle bellies were injected with 2.5 U botulinum toxin A (botox). Eight weeks later, tendons were repaired with or without augmentation at the tendon-bone interface. In particular, there were 5 groups ; (1) no repair (defect), (2) repair, (3) repair + fibrin, (4) repair + fibrin + ADSCs, (5) repair + fibrin + ADSCs + TGF-β3 (n=12 shoulders per condition). An equivalent cohort of animals received acute tendon transection (without botox injection) and immediate repair with or without augmentation. At 4 weeks following repair, animals were euthanized and the shoulders were harvested for analysis of bone morphometry, histology, and mechanical properties. Bone morphometry of the humeral head including; bone mineral density, bone volume percentage, and trabeculae number, thickness, and spacing was determined using micro-computed tomography. Statistical analyses included 2-way ANOVA with Tukey post-hoc tests. Analyses of histology and mechanical properties are ongoing.Results:ADSCs at P2 demonstrated a spindle-shaped morphology and were capable of forming colonies (Fig. 1A) and undergoing trilineage differentiation (Fig. 1B). Compared to healthy and acutely injured shoulders, muscles in the chronic condition exhibited atrophy, fibrosis, and fatty infiltration, with corresponding reductions in wet weights (Fig. 1C). All groups in the acute and chronic conditions possessed significantly reduced bone mineral density and bone volume percentage compared to healthy controls (p < 0.05), while only groups in the chronic condition exhibited compromised trabecular number and spacing. (p < 0.05). Augmentation of repairs with ADSCs (with or without TGF-β3) in the chronic condition significantly increased bone mineral density over defect controls (p < 0.05).Conclusion:Two-tendon transection and chemical paralysis of rotator cuff muscles in a rodent model mimics the degenerative changes seen in human shoulders, including decrements in bone quality. The efficacy of ADSCs in restoring structure and function of the tendon-bone interface likely depends on tear chronicity and size and may have greater benefit in chronic tears, as suggested by improvements in parameters of bone morphometry. This study will help further clarify the involvement of adult stem cell therapy in enhancing healing of the rotator cuff enthesis in the setting of degenerative tissue. Future studies would investigate the potential of adult stem cells to promote muscle regeneration in chronic rotator cuff tears and augment structural healing at the enthesis.

Science to Practice: Quantitative US Elastography Can Be Used to Quantify Mechanical and Histologic Tendon Healing in a Rabbit Model of Achilles Tendon Transection.

Compression-based ultrasonographic (US) elastography is associated with time-dependent mechanical and histologic changes of the healing tendon in a transected rabbit model of the Achilles tendon. This finding will lead to continued development of quantitative US, which can be used to objectively assess a diseased or healing tendon. With advances in the method used, clinical translation of tendon elastography may enable clinicians to diagnose tendon damage and track healing, which should improve both treatment and outcome.

Abstract 2: Macrophage-specific TGF-B is a Targetable Cytokine to Prevent Heterotopic Ossification

PURPOSE: Transforming growth factor beta (TGFβ) signaling is central to both normal and pathologic wound healing. However the source of TGFβ ligand during wound healing remains unknown. Heterotopic ossification (HO) is the pathologic formation of endochondral bone in soft tissue commonly occurring after severe trauma. HO is a SMAD-signaling dependent process typically occurring in response to an acute inflammatory insult. Traditionally targeted therapeutics for treatment of HO have focused on mesenchymal cell progenitors. However, macrophages which are integral orchestrators of the initial inflammatory response and are primary agents of cytokine secretion in wound regeneration, have received little attention. Here we identify a role of macrophage production of TGFβ in HO and demonstrate a role for macrophage targeted therapies in the treatment and prevention of this debilitating condition. METHODS: Wild type and macrophage-specific TGFβ knockout mice (LysM-cre/Tgfbfl/fl) mice underwent hindlimb Achilles’ tendon transection and dorsal 30% TBSA burn. Mice were euthanized at 2, 5, and 12days, and 3 and 9 weeks after injury. A concurrent cohort of wild type animals was treated with i.v. trabectedin (0.15 mg/kg), a macrophage-depleting agent, administered twice or vehicle control to assess the effect on HO. The tenotomy site of wild-type mice harvested at 2-, 5-, and 12-days were digested to a single-cell suspension, stained with antibodies against macrophage markers (Ly6G, Ly6C, F4/80, CD11b) and TGF-β for flow cytometric analysis. MicroCT images were obtained at 9 weeks after injury among wild type and TGFβ knockout mice, and among trabectedin and control-treated mice. In vitro and in vivo studies were performed to validate a novel PLGA microparticle system with preferential uptake by macrophages. RESULTS: Flow cytometry of tissue from wild type mice demonstrated enrichment of TGFβ in F4/80+ macrophages at all time-points with macrophages present as the dominant inflammatory population by 12-days post-injury (76%; p<0.05). LysM-cre/TGFβ mice similarly demonstrated a significant reduction in tHO volume vs. wild type controls (7.49 v. 1.25 mm3, p=.007). Macrophage depletion via trabectedin led to a 12.9-fold decrease in tHO volume when compared with control-treated mice (p<0.008). Histologic analysis at 3-weeks confirmed diminished cartilage presence in both mutant and trabectedin-treated mice when compared with respective controls. Finally, the PLGA microparticle demonstrated preferential uptake by macrophages both in vitro and in the wound site in vivo. CONCLUSION: We demonstrate that TGFβ is present in macrophages at the tenotomy site and that these macrophages comprise an increasingly large portion of the inflammatory infiltrate through 12-days post-injury. Global inhibition via trabectedin therapy is effective in preventing tHO. Importantly, genetic loss of TGFβ among macrophages was sufficient to significantly reduce HO. Our findings are strengthened by the development of a novel microparticle which exhibits preferential uptake by macrophages for drug delivery.

Abstract 128: A Translational Strategy Targeting Type I BMP Receptors to Prevent Heterotopic Ossification

PURPOSE: Trauma-induced heterotopic ossification (tHO) is the aberrant growth of ectopic bone in soft tissue, which develops in patients following severe musculoskeletal trauma. Much of HO literature focuses on a related pathology known as fibrodysplasia ossificans progressiva (FOP), which is caused by a hyperactivating mutation in the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1 (ACVR1 R206H). Consequently, emphasis has been placed on developing inhibitors with improved specificity for ACVR1. However, patients who develop tHO do not harbor known ACVR1 mutations, and it is unclear whether emphasis on ACVR1-specific inhibition is beneficial for the treatment of tHO. Here investigate whether any single T1-BMPR is required for tHO, or whether these receptors perform overlapping roles during tHO development. We further evaluate the efficacy of the BMP ligand trap, Alk3-Fc, as a broad-spectrum inhibitor of T1-BMP receptors in the treatment and prevention tHO. METHODS: Wild-type, tamoxifen-inducible Acvr1 knockout (Acvr1 tmKO: Ub.creERT/Acvr1fl/fl), tamoxifen-inducible Bmpr1a knockout (Bmpr1a tmKO: Ub.creERT/Bmpr1afl/fl), and Bmpr1b knockout (Bmpr1b-/-) mice receivedhindlimb Achille’s tendon transection and dorsal 30% total body surface area partial-thickness burn. A second cohort of wild-type mice received daily i.p. injections of Alk3-fc (2 mg/kg) or saline for 6-weeks post-injury or during weeks 0–2 (inflammation), weeks2-4 (chondrogenesis), or weeks 4–6 (ossification) after injury. Mice receiving abbreviated treatment with Alk3-fc received intraperitoneal saline injections for the remainder of their 6-week treatment period. HO was quantified by microCT 9-weeks post-injury, and cartilage formation was assessed via pentachrome staining. RESULTS: Genetic loss of Acvr1 (p>0.05), Bmpr1a (p>0.05), or Bmpr1b (p>0.05) alone was unable to significantly or substantially reduce tHO after injury. However, genetic loss of both Acvr1 and Bmpr1a (Acvr1;Bmpr1a tmKO: Ub.creERT /Acvr1fl/fl;Bmpr1afl/fl) led to a substantial and significant reduction in tHO volume (16.7-fold decrease vs. wild-type; p<0.05). Based on these findings we studied the effect of Alk3-Fc, a BMP ligand trap which inhibits BMP receptor signaling capable of broadly T1-BMPR inhibition at different points during tHO development. Mice treated daily with Alk3-Fc for 6-weeks post-injury demonstrated a significant and substantial reduction in tHO volume (2.9-fold; p<0.05). Mice treated daily with Alk3-Fc from 0–2 weeks demonstrated similar reductions in tHO volume (2.3-fold; p<0.05). This was confirmed histologically, with diminished cartilage content in 6-week and 0–2 week treated groups when compared with controls. CONCLUSIONS: Here we demonstrate that although BMP signaling is required for tHO, no single T1-BMPR (ACVR1/ALK2, BMPR1a/ALK3, or BMPR1b/ALK6) alone is necessary for this disease. This suggests that, unlike with genetic forms of HO such as FOP, these receptors have functional redundancy in the setting of tHO. Based on these data we developed an approach based on broad-spectrum T1-BMPR blockade using the ligand trap Alk3-Fc to prevent tHO formation. Our findings suggest that Alk3-Fc treatment during the earliest period after injury is sufficient to block tHO and provides a translational approach to targeting this disease process.

Abstract 96: Identification and Therapeutic Targeting of a Central DNA-Based Mechanism Through Which Movement Augments Inflammation

PURPOSE: Musculoskeletal trauma and surgery poses a unique challenge to surgeons: early mobilization facilitates rehabilitation but also promotes local inflammation and pathologic wound healing. However the mechanism by which extremity movement promotes local inflammation remains poorly characterized. Recently, discrete structural webs composed of DNA and histones, known as neutrophil extracellular traps (NETs), have been identified as key components of the inflammatory cascade elicited by neutrophils. Here we demonstrate that physically or chemically disrupted NETs are responsible for augmenting local inflammation by directly inducing NET formation (NETosis) yielding a therapeutic target to prevent inflammation caused by early movement. METHODS: Mice received dorsal hindlimb tendon transection +/- cast immobilization with subsequent treatments to destabilize NETs (+/- DNase I), inhibit NETosis (+/- Cl-Amidine), and/or inhibit NET-induced NETosis (+/- ODN-2088). MicroCT imaging was performed to evaluate In vitro experiments were performed to confirm that NETs produced by neutrophils in response to the inducing agent PMA are able to induce neutrophils to form NETs (NET-induced NETosis). Furthermore, in vitro experiments were performed to confirm that Cl-Amidine but not ODN-2088 inhibits PMA-induced NETosis. RESULTS: In vitro experiments confirmed that NETs are capable of inducing secondary NET formation (NET-induced NETosis). Both Cl-Amidine and the toll-like receptor 7/8/9 inhibitor ODN-2088 reduced the number of NETs formed through NET-induced NETosis (Cl-Amidine: 46.6 v. 9.1, p<0.05; ODN-2088: 46.6 v. 11.0, p<0.05). Cast-immobilization of mice after tendon transection eliminated ectopic cartilage and heterotopic ossification. Flow cytometry showed that cast-immobilization significantly reduced the normalized presence of neutrophils (1.0 v. 0.08, p<0.05) and macrophages (1.0 v. 0.13, p<005) 48 hours after injury. However, treatment of cast-immobilized mice with DNase to destabilize NETs led to a rebound increase in inflammation (neutrophils: 1.0 v. 6.39, p<0.05; macrophages: 1.0 v. 3.0, p<0.05) and caused ectopic cartilage and heterotopic ossification. Furthermore, Cl-Amidine and ODN-2088 both reduced early neutrophil presence in mice with the mobile hindlimb (Cl-Amidine: 1.0 v. 0.4, p<0.05; ODN-2088: 1.0 v. 0.27, p<0.05) and in DNase-treated mice with cast-immobilization (Cl-Amidine: 1.0 v. 0.27, p<0.05; ODN-2088: 1.0 v. 0.28, p<0.05). CONCLUSION: These results elucidate a central mechanism by which movement induces inflammation - NETs produced during the early inflammation are disrupted and further induce NETosis. These experiments identify a class of toll-like receptors such as ODN-2088, which are capable of reducing inflammation caused by movement by targeting NET-induced NETosis. These findings have immense value in preventing wound healing pathology associated with unchecked inflammation during movement, while allowing the initial inflammatory response to injury to proceed unimpaired.

Abstract 95: Inhibition of Scleraxis Signaling Provides a Target to Reduce Mesenchymal Cell Inflammation During Wound Healing

PURPOSE: Inflammation following trauma is a critical step during both normal and pathologic wound healing. Recently, we have identified Scleraxis, a transcription factor with conserved topoisomerase homology, as a mediator of pathologic wound healing. Here we demonstrate that ciprofloxacin, a known topoisomerase inhibitor, reduces exhibits anti-inflammatory properties during injury through a reduction in Scleraxis signaling. METHODS: Mesenchymal cells were isolated from mice with genetic loss of Scleraxis (Prx-cre/Scxfl/fl) and wild type mice. Separately, wild type cells were treated with ciprofloxacin (10 mg/kg) or vehicle control. In vitro assays were performed to quantify proliferation and Scleraxis signaling. Furthermore, mutant or wild type mice underwent hindlimb tendon transection and subsequent flow cytometry and histologic analysis during the first week after injury. This was similarly performed for mice treated with ciprofloxacin or vehicle control. RESULTS: Flow cytometry demonstrated that genetic loss of scleraxis among mesenchymal cells significantly reduced the presence of macrophages (F4/80+) and neutrophils (Cd11b+Ly6G+) at the injury site within 48 hours after injury. The presence of PDGFRa+ mesenchymal cells was also significantly reduced based on both flow cytometry and histologic analysis. These findings were confirmed with ciprofloxacin treatment. Furthermore, genetic loss of Scleraxis and ciprofloxacin both corresponded with a significant reduction in mesenchymal cell proliferation. Ciprofloxacin treatment led to reduced chondrogenic differentiation and aggrecan expression. Genetic loss of Scleraxis reduced ectopic cartilage formation when compared with wild type controls. CONCLUSION: These findings indicate that Scleraxis is a potent target to prevent mesenchymal cell proliferation and inflammation. Ciprofloxacin, an FDA-approved drug, has therapeutic efficacy as an anti-inflammatory agent with translational potential to prevent pathologic wound healing.

Effect of platelet-rich plasma on rat Achilles tendon healing is related to microbiota

Background and purpose — In 3 papers in Acta Orthopaedica 10 years ago, we described that platelet-rich plasma (PRP) improves tendon healing in a rat Achilles transection model. Later, we found that microtrauma has similar effects, probably acting via inflammation. This raised the suspicion that the effect ascribed to growth factors within PRP could instead be due to unspecific influences on inflammation. While testing this hypothesis, we noted that the effect seemed to be related to the microbiota.Material and methods — We tried to reproduce our old findings with local injection of PRP 6 h after tendon transection, followed by mechanical testing after 11 days. This failed. After fruitless variations in PRP production protocols, leukocyte concentration, and physical activity, we finally tried rats carrying potentially pathogenic bacteria. In all, 242 rats were used.Results — In 4 consecutive experiments on pathogen-free rats, no effect of PRP on healing was found. In contrast, apparently healthy rats carrying Staphylococcus aureus showed increased strength of the healing tendon after PRP treatment. These rats had higher levels of cytotoxic T-cells in their spleens.Interpretation — The failure to reproduce older experiments in clean rats was striking, and the difference in response between these and Staphylococcus-carrying rats suggests that the PRP effect is dependent on the immune status. PRP functions may be more complex than just the release of growth factors. Extrapolation from our previous findings with PRP to the situation in humans therefore becomes even more uncertain.

Perivascular Stem Cells Diminish Muscle Atrophy Following Massive Rotator Cuff Tears in a Small Animal Model.

Rotator cuff tears are a common cause of shoulder pain and often necessitate operative repair. Muscle atrophy, fibrosis, and fatty infiltration can develop after rotator cuff tears, which may compromise surgical outcomes. This study investigated the regenerative potential of 2 human adipose-derived progenitor cell lineages in a murine model of massive rotator cuff tears. Ninety immunodeficient mice were used (15 groups of 6 mice). Mice were assigned to 1 of 3 surgical procedures: sham, supraspinatus and infraspinatus tendon transection (TT), or TT and denervation via suprascapular nerve transection (TT + DN). Perivascular stem cells (PSCs) were harvested from human lipoaspirate and sorted using fluorescence-activated cell sorting into pericytes (CD146 CD34 CD45 CD31) and adventitial cells (CD146 CD34 CD45 CD31). Mice received no injection, injection with saline solution, or injection with pericytes or adventitial cells either at the time of the index procedure ("prophylactic") or at 2 weeks following the index surgery ("therapeutic"). Muscles were harvested 6 weeks following the index procedure. Wet muscle weight, muscle fiber cross-sectional area, fibrosis, and fatty infiltration were analyzed. PSC treatment after TT (prophylactic or therapeutic injections) and after TT + DN (therapeutic injections) resulted in less muscle weight loss and greater muscle fiber cross-sectional area than was demonstrated for controls (p < 0.05). The TT + DN groups treated with pericytes at either time point or with adventitial cells at 2 weeks postoperatively had less fibrosis than the TT + DN controls. There was less fatty infiltration in the TT groups treated with pericytes at either time point or with adventitial cells at the time of surgery compared with controls. Our findings demonstrated significantly less muscle atrophy in the groups treated with PSCs compared with controls. This suggests that the use of PSCs may have a role in the prevention of muscle atrophy without leading to increased fibrosis or fatty infiltration. Improved muscle quality in the setting of rotator cuff tears may increase the success rates of surgical repair and lead to superior clinical outcomes.

Quantitative US Elastography Can Be Used to Quantify Mechanical and Histologic Tendon Healing in a Rabbit Model of Achilles Tendon Transection.

Purpose To determine the time-dependent change in strain ratios (SRs) at the healing site of an Achilles tendon rupture in a rabbit model of tendon transection and to assess the correlation between SRs and the mechanical and histologic properties of the healing tissue. Materials and Methods Experimental methods were approved by the institutional animal care and use committee. The Achilles tendons of 24 New Zealand white rabbits (48 limbs) were surgically transected. The SRs of Achilles tendons were calculated by using compression-based quantitative ultrasonographic elastography measurements obtained 2, 4, 8, and 12 weeks after transection. After in vivo elastography, the left Achilles tendon was harvested for mechanical testing of ultimate load, ultimate stress, elastic modulus, and linear stiffness, and the right tendons were harvested for tissue histologic analysis with the Bonar scale. Time-dependent changes in SRs, mechanical parameters, and Bonar scale scores were evaluated by using repeated-measures analysis of variance. The correlation between SRs and each measured variable was evaluated by using the Spearman rank correlation coefficient. Results Mean SRs and Bonar scale values decreased as a function of time after transection, whereas mechanical parameters increased (P < .001). SR correlated with ultimate stress (ρ = 0.68, P <.001,) elastic modulus (ρ = 0.74, P <.001), and the Bonar scale (ρ = 0.87, P <.001). Conclusion Quantitative elastography could be a useful method with which to evaluate mechanical and histologic properties of the healing tendon. © RSNA, 2017 Online supplemental material is available for this article.

Cox-2 inhibition and the composition of inflammatory cell populations during early and mid-time tendon healing.

During early tendon healing, the cells within the regenerating tissue are, to a large part, inflammatory leukocytes (CD45+). In a rat Achilles tendon healing model, the inflammation resolves between 5 and 10 days. In the same model, Cox inhibitors (NSAIDs) impair healing when given during the first 5 days, but have a positive effect if given later. We tested the hypothesis that a Cox inhibitor would exert these effects by influencing inflammation, and thereby the composition of the inflammatory cell subpopulations. Achilles tendon transection was performed in 44 animals. Animals were randomized to either parecoxib or saline injections. Healing was evaluated by mechanical testing day 7 after surgery and by flow cytometry day 3 and 10. Cross-sectional area, peak force and stiffness were reduced by parecoxib 31, 33, and 25% respectively (p=0.005, p=0.002, and p=0.005). By flow cytometry, there was a strong effect of time (p<0.001) on virtually all inflammatory cell subpopulations (CD45, CD11b, CD68, CCR7, CD163, CD206, CD3, CD4), but no significant effect of parecoxib at any time point. The results suggest that the negative effects of Cox inhibitors on tendon healing might be exerted mainly via mechanisms not directly related to inflammatory cells.

Knocking-out matrix metalloproteinase-13 exacerbates rotator cuff muscle fatty infiltration.

Rotator cuff (RC) tears are common tendon injuries. Clinically, both muscle atrophy and fatty infiltration have generally been attributed to poor functional outcomes. Matrix metalloproteinase-13 plays a crucial role in extracellular matrix remodeling in many physiological and pathological processes. Nevertheless, its role in rotator cuff muscle atrophy and fatty infiltration remains unknown. The purpose of this study is to define the functional role of MMP-13 in rotator cuff muscle atrophy and fatty infiltration using a mouse RC tears model. Unilateral complete supraspinatus and infraspinatus tendon transection and suprascapular nerve transection was performed on nine of MMP-13 (-/-) knockout and nine of MMP-13 (+/+) wildtype mice at 3 months old. Mice were sacrificed 6 weeks after surgery. Supraspinatus (SS) and infraspinatus (IS) muscles were harvested for histology and gene expression analysis with RT-PCR. Six weeks after RC surgery, no significant difference in muscle atrophy and fibrosis between MMP-13 knockout and wild type mice was observed. However, there was a significant increase in the amount of fatty infiltration in MMP-13 knockout mice compared to the wild types. Muscles from MMP-13 knockout mice have significantly higher expression of fatty infiltration related genes. Results from this study suggest that MMP-13 plays a crucial role in rotator cuff muscle fatty degeneration. This novel finding suggests a new molecular mechanism that governs RC muscle FI and MMP-13 may serve as a target for therapeutics to treat muscle FI after RC tears.

Prenatal myonuclei play a crucial role in skeletal muscle hypertrophy in rodents.

Multinucleated muscle fibers are formed by the fusion of myogenic progenitor cells during embryonic and fetal myogenesis. However, the role of prenatally incorporated myonuclei in the skeletal muscle fibers of adult animals is poorly understood. We demonstrated, using muscle-specific reporter mice, that the prenatal myonuclei remained in the adult soleus muscle, although cardiotoxin injection caused the loss of prenatal myonuclei. Overloading by the tendon transection of synergists failed to induce compensatory hypertrophy in regenerated soleus muscle fibers of adult rats, whereas unloading by tail suspension normally induced the fiber atrophy. Loss of hypertrophying function correlated with the lowered histone acetylation at the transcription start site of Igf1r gene, which was one of the genes that did not respond to the overloading. These parameters were improved by the transplantation of cells harvested from the juvenile soleus muscles of neonatal rats in association with enhanced histone acetylation of Igf1r gene. These results indicated that the presence of prenatal myonuclei was closely related to the status of histone acetylation, which could regulate the responsiveness of muscle fibers to physiological stimuli.

Lymphatic Contribution to the Cellular Niche in Heterotopic Ossification

The objective of this study was to determine the contribution of lymphatic tissue to heterotopic ossification (HO). HO is the pathologic development of ectopic bone within soft tissues often following severe trauma. Characterization of the tissue niche supporting HO is critical to identifying therapies directed against this condition. Lymphangiogenesis is upregulated during incidents of trauma, thereby coincident with the niche supportive of HO. We hypothesized that lymphatic tissues play a critical role in HO formation. Mice underwent hindlimb Achilles' tendon transection and dorsal burn injury (burn/tenotomy) to induce HO. The popliteal and inguinal lymph nodes were excised ipsilateral to the tenotomy site. Flow cytometry and immunostaining were used to quantify and localize lymphoendothelium. MicroCT was used to quantify HO. Enrichment of mature lymphatic tissues was noted 2 weeks after injury at the tendon transection sites when compared with the contralateral, intact tendon based on LYVE1+ tubules (10.9% vs 0.8%, P < 0.05). Excision of the inguinal and popliteal nodes with draining popliteal lymphatic vessel significantly decreased the presence of mature lymphoendothelium 2 weeks after injury (10.9% vs 3.3%, P < 0.05). Bone-cartilage-stromal progenitor cells (CD105+/AlphaV+/Tie2-/CD45-/CD90-/BP1-) were also significantly decreased after lymph node excision (10.2% vs 0.5%, P < 0.05). A significant decrease was noted in the volume of de novo HO present within the soft tissues (0.12 mm vs 0.02 mm). These findings suggest that lymphatic vessels are intimately linked with the de novo formation bone within soft tissues following trauma, and their presence may facilitate bone formation.

Surgical Excision of Heterotopic Ossification Leads to Re-Emergence of Mesenchymal Stem Cell Populations Responsible for Recurrence.

Trauma‐induced heterotopic ossification (HO) occurs after severe musculoskeletal injuries and burns, and presents a significant barrier to patient rehabilitation. Interestingly, the incidence of HO significantly increases with repeated operations and after resection of previous HO. Treatment of established heterotopic ossification is challenging because surgical excision is often incomplete, with evidence of persistent heterotopic bone. As a result, patients may continue to report the signs or symptoms of HO, including chronic pain, nonhealing wounds, and joint restriction. In this study, we designed a model of recurrent HO that occurs after surgical excision of mature HO in a mouse model of hind‐limb Achilles’ tendon transection with dorsal burn injury. We first demonstrated that key signaling mediators of HO, including bone morphogenetic protein signaling, are diminished in mature bone. However, upon surgical excision, we have noted upregulation of downstream mediators of osteogenic differentiation, including pSMAD 1/5. Additionally, surgical excision resulted in re‐emergence of a mesenchymal cell population marked by expression of platelet‐derived growth factor receptor‐α (PDGFRα) and present in the initial developing HO lesion but absent in mature HO. In the recurrent lesion, these PDGFRα+ mesenchymal cells are also highly proliferative, similar to the initial developing HO lesion. These findings indicate that surgical excision of HO results in recurrence through similar mesenchymal cell populations and signaling mechanisms that are present in the initial developing HO lesion. These results are consistent with findings in patients that new foci of ectopic bone can develop in excision sites and are likely related to de novo formation rather than extension of unresected bone. Stem Cells Translational Medicine 2017;6:799–806

A possible link between loading, inflammation and healing: Immune cell populations during tendon healing in the rat.

Loading influences tendon healing, and so does inflammation. We hypothesized that the two are connected. 48 rats underwent Achilles tendon transection. Half of the rats received Botox injections into calf muscles to reduce mechanical loading. Cells from the regenerating tissue were analyzed by flow cytometry. In the loaded group, the regenerating tissue contained 83% leukocytes (CD45+) day 1, and 23% day 10. The M1/M2 macrophage ratio (CCR7/CD206) peaked at day 3, while T helper (CD3+CD4+) and Treg cells (CD25+ Foxp3+) increased over time. With Botox, markers associated with down-regulation of inflammation were more common day 5 (CD163, CD206, CD25, Foxp3), and M1 or M2 macrophages and Treg cells were virtually absent day 10, while still present with full loading. The primary variable, CCR7/CD206 ratio day 5, was higher with full loading (p = 0.001) and the Treg cell fraction was lower (p < 0.001). Free cage activity loading is known to increase size and strength of the tendon in this model compared to Botox. Loading now appeared to delay the switch to an M2 type of inflammation with more Treg cells. It seems a prolonged M1 phase due to loading might make the tendon regenerate bigger.

Perivascular Stem Cells Diminish Muscle Atrophy and Retain Viability in a Rotator Cuff Tear Model

Objectives:Rotator cuff tears (RCTs) are a common cause of shoulder pain and often necessitate surgical repair. Muscle changes including atrophy, fibrosis, and fatty degeneration can develop after RCTs, which may compromise surgical repair and clinical outcomes. Lipoaspirate-derived human perivascular stem cells (PSCs) have demonstrated myogenic and angiogenic potential in other small animal models of muscle injury. We hypothesized that the administration of PSCs following massive RCTs may help to diminish these muscle changes in a small animal model.Methods:A total of 90 immunodeficient mice were used (15 groups, N=6). Each was assigned to one of three surgical groups: i) sham, ii) supraspinatus and infraspinatus tendon transection (TT), or iii) TT and suprascapular nerve denervation (TT+DN). PSCs were harvested from human lipoaspirate and sorted using fluorescence-activated cell sorting into small blood vessel residing pericytes (CD146+ CD34- CD45- CD31-) and large blood perivascular adventitial cells (CD146- CD34+ CD45- CD31-). Mice received either a) no injection, b) saline injection, c) pericyte injection, or d) adventitial cell injection at the time of the index procedure or at two weeks following index surgery. The supraspinatus muscles were harvested six weeks after the index procedure. Muscle atrophy was assessed by measuring percent wet muscle weight change for each sample. Muscle fiber cross-sectional area (CSA), fibrosis, and fatty degeneration were analyzed using Image J™. Additionally, pericytes and adventitial cells were transduced with a luciferase-containing construct. Animals were given injections of luciferin and imaged using IVIS to track in vivo bioluminescence following injections to assess cell viability.Results:Treatment with PSC injection after TT resulted in less wet weight loss and greater muscle fiber CSA than control groups (P<0.05). The TT+DN groups treated with PSC injections two weeks post-op also had less muscle weight loss and greater muscle fiber CSA than their respective control groups. The TT+DN groups treated with PSC injections at the time of surgery demonstrated no differences in weight loss, but had greater muscle fiber CSA than their respective controls. There was no difference in fibrosis between the TT groups. However, TT+DN groups treated with pericyte injections at both time points and adventitial cell injections two weeks post-op had less fibrosis than TT+DN controls. There was less fatty degeneration in the TT groups treated with pericyte injections at both time points and adventitial cells at the time of surgery compared to matched controls. There were no differences in the amount of fatty degeneration between the TT+DN groups. Bioluminescence imaging demonstrated viability of the injected cells at three weeks following injections (Figure 1).Conclusion:Our findings demonstrate significantly less muscle atrophy in the groups treated with PSC injections compared to respective controls for both TT and TT+DN procedures. These results suggest that the use of PSCs may have a role in the prevention of muscle atrophy by aiding in the maintenance of muscle bulk without leading to increased fibrosis or fatty infiltration. Additionally, bioluminescence data suggests that these cells maintain viability and engraft within the native muscle to improve muscle bulk. Improved muscle quality in the setting of rotator cuff tears may increase the success rates of rotator cuff repair and lead to superior clinical outcomes.

Arthroscopic Management of Popliteal Tendon Dysfunction in Total Knee Arthroplasty

Unexplainable knee pain may follow knee replacement surgery. The popliteal tendon should be evaluated as a possible cause of lateral knee pain and dysfunction after knee arthroplasty. The tendon can snap over or impinge on lateral osteophytes or prominent femoral components and produce mechanical symptoms and pain. Ultrasound-guided injections may confirm the diagnosis and provide symptomatic relief in some patients. Those who respond well to injections and in whom conservative management ultimately fails may be offered arthroscopic release of the popliteal tendon. We present the arthroscopic technique for popliteal tendon release in a symptomatic patient whose pain resolved immediately postoperatively. We recommend the use of an anterolateral viewing portal and an accessory lateral working portal to access the posterolateral knee. There can be extensive scar tissue overlying the popliteal tendon after arthroplasty. Arthroscopic tendon transection and excision comprise a reliable procedure for popliteal tendon impingement after knee arthroplasty without compromising knee stability.

Focal experimental injury leads to widespread gene expression and histologic changes in equine flexor tendons.

It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3cm regions) for histologic (scoring and immunohistochemistry) and gene expression (real time PCR) analysis of extracellular matrix changes. The histopathology score was significantly higher in transected tendons compared to control tendons in all regions except for the most distal (P ≤ 0.03) with no differences between overstressed (medial) and stress-deprived (lateral) tendon halves. Proteoglycan scores were increased by transection in all but the most proximal region (P < 0.02), with increased immunostaining for aggrecan, biglycan and versican. After correcting for location within the tendon, gene expression for aggrecan, versican, biglycan, lumican, collagen types I, II and III, MMP14 and TIMP1 was increased in transected tendons compared with control tendons (P < 0.02) and decreased for ADAMTS4, MMP3 and TIMP3 (P < 0.001). Aggrecan, biglycan, fibromodulin, and collagen types I and III expression positively correlated with all histopathology scores (P < 0.001), whereas lumican, ADAMTS4 and MMP14 expression positively correlated only with collagen fiber malalignment (P < 0.001). In summary, histologic and associated gene expression changes were significant and widespread six weeks after injury to the equine SDFT, suggesting rapid and active development of tendinopathy throughout the entire length of the tendon. These extensive changes distant to the focal injury may contribute to poor functional outcomes and re-injury in clinical cases. Our data suggest that successful treatments of focal injuries will need to address pathology in the entire tendon, and that better methods to monitor the development and resolution of tendinopathy are required.

Quantitative Assessment of Tendon Healing by Using MR T2 Mapping in a Rabbit Achilles Tendon Transection Model Treated with Platelet-rich Plasma.

To determine if magnetic resonance (MR) imaging T2 mapping can be used to quantify histologic tendon healing by using a rabbit Achilles tendon transection model treated with platelet-rich plasma (PRP). Experiments were approved by the Institutional Animal Care and Use Committee. The Achilles tendons of 24 New Zealand white rabbits (48 limbs) were surgically transected, and PRP (in the test group) or saline (in the control group) was injected into the transection site. The rabbits were sacrificed 2, 4, 8, and 12 weeks after surgery. Thereafter, T2 mapping and histologic evaluations were performed by using the Bonar scale. A mixed-model multivariate analysis of variance was used to test the effects of time and PRP treatment on the T2 value and Bonar grade, respectively. The correlation between the T2 value and Bonar grade was also assessed by using the Spearman correlation coefficient. The Bonar scale values decreased in both groups during tendon healing. The T2 value also shortened over time (P < .001 for both groups). The T2 values were positively correlated with the Bonar grade (ρ = 0.78, P < .001). Both the T2 value and Bonar scale value were lower in the PRP group than in the control group at 4, 8, and 12 weeks; however, there was no significant effect of PRP treatment on the T2 value or Bonar grade. The T2 value changes reflected histologic tendon healing. While T2 and Bonar grade were lower at all time points in tendons treated with PRP, there was no significant difference between the treatment and control tendons.