Verteporfin attenuates trauma-induced heterotopic ossification of Achilles tendon by inhibiting osteogenesis and angiogenesis involving YAP/ß-catenin signaling.

Heterotopic ossification occurs as a pathological ossification condition characterized by ectopic bone formation within soft tissues following trauma. Vascularization has long been established to fuel skeletal ossification during tissue development and regeneration. However, the feasibility of vascularization as a target of heterotopic ossification prevention remained to be further clarified. Here, we aimed to identify whether verteporfin as a widely used FDA-approved anti-vascularization drug could effectively inhibit trauma-induced heterotopic ossification formation. In the current study, we found that verteporfin not only dose dependently inhibited the angiogenic activity of human umbilical vein endothelial cells (HUVECs) but also the osteogenic differentiation of tendon stem cells (TDSCs). Moreover, YAP/ß-catenin signaling axis was downregulated by the verteporfin. Application of lithium chloride, an agonist of ß-catenin, recovered TDSCs osteogenesis and HUVECs angiogenesis that was inhibited by verteporfin. In vivo, verteporfin attenuated heterotopic ossification formation by decelerating osteogenesis and the vessels densely associated with osteoprogenitors formation, which could also be readily reversed by lithium chloride, as revealed by histological analysis and Micro-CT scan in a murine burn/tenotomy model. Collectively, this study confirmed the therapeutic effect of verteporfin on angiogenesis and osteogenesis in trauma-induced heterotopic ossification. Our study sheds light on the anti-vascularization strategy with verteporfin as a candidate treatment for heterotopic ossification prevention.

Endocrine modulation of brain-skeleton axis driven by neural stem cell-derived perilipin 5 in the lipid metabolism homeostasis for bone regeneration.

Factors released from the nervous system always play crucial roles in modulating bone metabolism and regeneration. How the brain-driven endocrine axes maintain bone homeostasis, especially under metabolic disorders, remains obscure. Here, we found that neural stem cells (NSCs) residing in the subventricular zone participated in lipid metabolism homeostasis of regenerative bone through exosomal perilipin 5 (PLIN5). Fluorescence-labeled exosomes tracing and histological detection identified that NSC-derived exosomes (NSC-Exo) could travel from the lateral ventricle into bone injury sites. Homocysteine (Hcy) led to osteogenic and angiogenic impairment, whereas the NSC-Exo were confirmed to restore it. Mecobalamin, a clinically used neurotrophic drug, further enhanced the protective effects of NSC-Exo through increased PLIN5 expression. Mechanistically, NSC-derived PLIN5 reversed excessive Hcy-induced lipid metabolic imbalance and aberrant lipid droplet accumulation through lipophagy-dependent intracellular lipolysis. Intracerebroventricular administration of mecobalamin and/or AAV-shPlin5 confirmed the effects of PLIN5-driven endocrine modulations on new bone formation and vascular reconstruction in hyperhomocysteinemic and high-fat diet models. This study uncovered a novel brain-skeleton axis that NSCs in the mammalian brain modulated bone regeneration through PLIN5-driven lipid metabolism modulation, providing evidence for lipid- or bone-targeted medicine development.

STING contributes to trauma-induced heterotopic ossification through NLRP3-dependent macrophage pyroptosis.

Trauma-induced heterotopic ossification (HO) is featured by aberrant bone formation at extra-skeletal site. STING is a master adaptor protein linking cellular damage to immune responses, while its role in HO remains elusive. A murine burn/tenotomy model was used to mimic trauma-induced HO in vivo. We demonstrated elevated STING expression in macrophages in inflammatory stage after burn/tenotomy, and STING inhibition significantly alleviated HO formation. Activated NLRP3-dependent macrophage pyroptosis was also found in inflammatory stage after burn/tenotomy. Either STING or NLRP3 suppression reduced mature HO by weakening macrophage pyroptotic inflammation, while protective effects of STING were abolished by NLRP3 overexpression. Further, in vitro, we also found a prominent STING level in pyroptotic BMDMs. STING suppression relieved macrophage pyroptotic inflammation, while abolished by NLRP3 overexpression. Our results reveal that STING poses regulatory effects on trauma-induced HO formation, via modulating NLRP3-dependent macrophage pyroptosis. Targeting STING-NLRP3 axis represents an attractive approach for trauma-induced HO prevention.

Human osteoprogenitor cells obtained from traumatic heterotopic ossification samples showed enhanced osteogenic differentiation potential and ERK/hedgehog signaling than that from normal bone.

Traumatic heterotopic ossification (HO) refers to the abnormal ectopic osteogenesis following trauma, causing limb dysfunction and seriously lowering the life quality of patients. Aberrant osteogenic behavior of progenitor cells that ectopically accumulated within the soft tissues are believed to be responsible for HO formation. However, the detailed mechanism still remained to be clarified. Here in this study, we successfully isolated osteoprogenitors from human heterotopic ossification tissues (HO-ops) and identified their stemness and multi-directional differentiation potential. Using alkaline phosphatase staining together with alizarin red staining, we confirmed that the HO-ops in the heterotopic ossified tissues gained greater osteogenic potential than the normal human bone marrow mesenchymal stem cells (HBMSCs). RT-qPCR also indicated that HO-ops obtained more gene transcriptions of critical osteogenic determinators than HBMSCs. In addition, through Western blot, we proved that ERK signaling pathway and hedgehog signaling pathway were significantly activated in the HO-ops. When U0126 and cyclopamine were used to inhibit ERK signaling and hedgehog signaling respectively, the osteogenic potential of HO-ops decreased significantly. The hedgehog signaling and ERK signaling also showed cross-talk in HO-ops during osteogenic differentiation in HO-ops during osteogenic differentiation. The elevated ERK signaling and hedgehog signaling were further confirmed in the human traumatic HO sample sections by immunohistochemical staining. In sum, our results showed that the activation of ERK and hedgehog signaling pathway jointly enhanced the osteogenic potential of HO-ops to induce the formation of traumatic HO, which provides novel insights into the molecular basis of HO formation and offers promising targets for future therapeutic strategy.

Long-term outcomes of open arthrolysis combined with radial head arthroplasty for post-traumatic elbow stiffness: results are durable over 8 years.

BACKGROUND: Post-trauma elbow stiffness (PTES) is a common complication after elbow trauma that causes severe upper limb disability. Open elbow arthrolysis (OEA) with radial head arthroplasty (RHA) is an effective method to treat PTES with rotation limitation, or persistent pain/instability after radial head resection. However, no long-term results have been reported for this technique. This study aimed to show the clinical and radiographic outcomes of OEA with RHA over 8 years and compare its efficacy at 3 years (short-term). METHODS: Patients with PTES treated by OEA with RHA between September 2010 and December 2012 were retrospectively reviewed. Seventeen patients were followed up over 8 years (range, 100-106 months). A bipolar prosthesis of RHA was performed during OEA. Preoperative, 3-year, and 8-year elbow and forearm motion, upper limb function, radiographic outcomes, and complications were recorded. RESULTS: Clinically important improvements in elbow motion and forearm rotation were obtained, from 34° and 58° preoperatively, to 109° and 135° at 3 years, which were maintained over 8 years, to 113° (P = .262) and 134° (P = .489). The Mayo Elbow Performance Index had clinically important increases from the preoperative level of 58 to 94 points at 3 years, and was maintained over 8 years (95 points, P = .422), with 100% reporting excellent to good outcomes. Pain and nerve symptoms were also improved. Complications consisted of new-onset ulnar nerve symptoms in 1 patient, nonclinically significant heterotopic ossification recurrence in 3, humeroulnar arthritis exacerbation in 4, and periprosthetic lucency in 8. CONCLUSIONS: OEA with RHA yielded satisfactory short-term outcomes for PTES at 3 years, with substantial improvements in elbow mobility and function, and the results were durable over the long term (8 years).

Inhibition of IL-17 prevents the progression of traumatic heterotopic ossification.

Traumatic heterotopic ossification (HO) is the abnormal formation of bone in soft tissues as a consequence of injury. However, the pathological mechanisms leading to traumatic HO remain unknown. Here, we report that aberrant expression of IL-17 promotes traumatic HO formation by activating ß-catenin signalling in mouse model. We found that elevated IL-17 and ß-catenin levels are correlated with a high degree of HO formation in specimens from patients and HO animals. We also show that IL-17 initiates and promotes HO progression in mice. Local injection of an IL-17 neutralizing antibody attenuates ectopic bone formation in a traumatic mouse model. IL-17 enhances the osteoblastic differentiation of mesenchymal stem cells (MSCs) by activating ß-catenin signalling. Moreover, inhibition of IL-17R or ß-catenin signalling by neutralizing antibodies or drugs prevents the osteogenic differentiation of isolated MSCs and decreases HO formation in mouse models. Together, our study identifies a novel role for active IL-17 as the inducer and promoter of ectopic bone formation and suggests that IL-17 inhibition might be a potential therapeutic target in traumatic HO.

MicroRNA engineered umbilical cord stem cell-derived exosomes direct tendon regeneration by mTOR signaling.

BACKGROUND: Exosomes are extracellular vesicles of nano-structures and represent an emerging nano-scale acellular therapy in recent years. Tendon regeneration is a sophisticated process in the field of microsurgery due to its poor natural healing ability. To date, no successful long-term solution has been provided for the healing of tendon injuries. Functional recovery requires advanced treatment strategies. Human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos) are considered as promising cell-free therapeutic agents. However, few studies reported their potential in the tendon repair previously. In this study, we explored the roles and underlying mechanisms of HUMSC-Exos in the tendon regeneration. RESULTS: Expression of tendon-specific markers in, and collagen deposition by, tendon-derived stem cells (TDSCs) treated with HUMSC-Exos increased in vitro. In a rat Achilles tendon injury model, treatment with HUMSC-Exos improved the histological structure, enhanced tendon-specific matrix components, and optimized biomechanical properties of the Achilles tendon. Findings in miRNA sequencing indicated a significant increase in miR-29a-3p in HUMSC-Exo-treated Achilles tendons. Next, luciferase assay in combination with western blot identified phosphatase and tensin homolog (PTEN) as the specific target of miR-29a-3p. Furthermore, we applied a miR-29a-3p-specific agonist to engineer HUMSC-Exos. These HUMSC-Exos overexpressing miR-29a-3p amplified the gain effects of HUMSC-Exos on tendon healing in vivo. To explore the underlying mechanisms, a transforming growth factor-ß1 (TGF-ß1) inhibitor (SB-431542), mTOR inhibitor (rapamycin), and engineered HUMSC-Exos were employed. The results showed that TGF-ß1 and mTOR signaling were involved in the beneficial effects of HUMSC-Exos on tendon regeneration. CONCLUSION: The findings in our study suggest that PTEN/mTOR/TGF-ß1 signaling cascades may be a potential pathway for HUMSC-Exos to deliver miR-29a-3p for tendon healing and implicate a novel therapeutic strategy for tendon regeneration via engineered stem cell-derived exosomes.

Celecoxib-Loaded Electrospun Fibrous Antiadhesion Membranes Reduce COX-2/PGE2 Induced Inflammation and Epidural Fibrosis in a Rat Failed Back Surgery Syndrome Model.

To date, failed back surgery syndrome (FBSS) remains a therapy-refractory clinical condition after spinal surgery. The antiadhesion membrane is applied to prevent FBSS by isolating fibrosis; however, the inflammation stimulated by the foreign body and surgical trauma needs to be further resolved simultaneously. Therefore, we developed new electrospun polycaprolactone (PCL) fibrous membranes loaded with celecoxib (CEL) to prevent fibrosis and inflammation associated with FBSS. The CEL-loaded PCL fibers were randomly distributed, and the drug was released over two weeks. Fluorescence micrographs revealed that the fibroblasts proliferated less on the PCL-CEL fibrous membranes than in the PCL group and the blank control. In the rat laminectomy model after 4 weeks, magnetic resonance imaging of epidural fibrosis was least in the PCL-CEL group. Expression of COX-2 and PGE2 was lower in the PCL-CEL group. It concluded that the CEL-loaded PCL membrane could reduce fibrosis and inflammation in a rat model of FBSS via COX-2/PGE2 signaling pathways.

How effective is periarticular multimodal drug injection in open elbow arthrolysis? A prospective double-blind randomized controlled trial.

BACKGROUND: Evidence on the efficacy and safety of periarticular multimodal drug injection (PMDI) in open elbow arthrolysis (OEA) is limited. This study aimed to investigate differences in postoperative pain, blood loss, and range of motion (ROM) between PMDI vs. no injection among patients undergoing OEA, and the presence of PMDI-related complications. METHODS: This prospective, double-blind randomized controlled trial included 59 patients who underwent OEA. Patients randomly received PMDI (ropivacaine, epinephrine, ketoprofen) before wound closure or no injection. The primary outcomes were elbow pain over the first postoperative week at rest and during motion, measured using the visual analog scale (VAS). VAS scores were compared to attain the 20-mm threshold values for a minimum clinically important difference. Parecoxib consumption on OEA night and postoperative days (PODs) 1-3 and total consumption during the first postoperative week were recorded. Blood loss was recorded every 24 hours until POD 3. ROM during rehabilitation was measured daily from day 1 to day 7 after surgery, as well as at 3-month follow-up. Medication-related side effects were recorded prospectively. RESULTS: The mean VAS score showed clinically important differences between PMDI and control groups at rest on OEA night (mean difference [MD], 25 mm; P < .001) and first 3 PODs with motion (POD 1: MD, 28 mm, P < .001; POD 2: MD, 21 mm, P < .001; POD 3: MD, 21 mm, P < .001) but not in other postoperative assessments. Parecoxib consumption was lower in the PMDI group on OEA night and PODs 1-3. Total parecoxib consumption during the first postoperative week was lower in the PMDI group vs. the control group (MD, 148 mg; P < .001). Blood drainage was less in the PMDI group vs. the control group on POD 1 (MD, 38 mL; P = .016) but not on POD 2 (P = .950), POD 3 (P = .259), or total (P = .184). The PMDI group exhibited significantly better ROM during the first 4 PODs than the control group, whereas there was no difference at 3-month follow-up. No medication-related side effects were noted in the PMDI group. CONCLUSION: PMDI effectively relieves pain and reduces analgesic consumption for OEA patients, without an apparent increase in risks.

Intravenous tranexamic acid reduce postoperative drainage and pain after open elbow arthrolysis: a randomized controlled trial.

BACKGROUND: Open elbow arthrolysis (OEA), which has become an established treatment for post-traumatic elbow stiffness (PTES), requires complete release of contracture tissue and wide excision of ectopic bone, which results in extensive bleeding. The aim of the present study is to evaluate the efficacy of intravenous tranexamic acid (TXA) on postoperative drainage, calculated blood loss, and early clinical outcomes in patients undergoing OEA. METHODS: A double-blind, randomized, placebo-controlled trial including 96 patients undergoing OEA was undertaken. Patients received intravenously either 100 mL saline (placebo group, n = 48), or 100 mL saline plus 1 g TXA (TXA group, n = 48) before skin incision. The primary outcome was the drainage volume on postoperative days (PODs) 1-3. Secondary outcomes included the calculated blood loss, elbow pain score measured by visual analog scale (VAS), elbow function valued by Mayo Elbow Performance Score (MEPS), and rate of complications after OEA. RESULTS: Mean total postoperative drainage volume (TXA group: 182 mL vs. placebo group: 214 mL, P = .003) and mean calculated total blood loss (TXA group: 582 mL vs. placebo group: 657 mL, P = .004) were significantly lower in the TXA group. No transfusions were necessary in either group. Mean VAS pain scores in elbow motion showed marked differences between both groups on POD 1 (TXA: 5 vs. placebo: 6, P = .003) and POD 2 (TXA: 4 vs. placebo: 5, P = .023) but not in other postoperative time points. No differences were detected in complications, such as pin-related infection, hematoma, new or exacerbation of ulnar nerve symptoms, and recurrent heterotopic ossification. At the 6-month follow-up, no statistical differences were found between the 2 groups with respect to the elbow functions including range of motion, VAS score, and MEPS. CONCLUSION: Intravenous administration of TXA significantly decreased the postoperative drainage volume and the total estimated blood loss and alleviated the elbow pain with motion during early postoperative days in patients undergoing OEA.

A new pathologic classification for elbow stiffness based on our experience in 216 patients.

BACKGROUND: Elbow stiffness commonly causes functional impairment and upper-limb disability. This study aimed to develop a new pathologic classification to further understand and standardize elbow arthrolysis from a new perspective, as well as to determine clinical outcomes. METHODS: Extension-flexion dysfunction was classified into 4 types: EFI, tethers alone; EFII, tethers with blocks; EFIII, articular malformation; and EFIV, bony ankylosis. Forearm rotation dysfunction was classified into 3 types: FRI, contracture alone; FRII, radial head malunion or nonunion; and FRIII, proximal radioulnar bony ankylosis. A total of 216 patients with elbow stiffness were prospectively included and categorized preoperatively. All surgical procedures were performed by the same chief surgeon; different types underwent specific procedures. Patient data, elbow motion, and functional scores were analyzed. RESULTS: Mean range of motion (ROM) increased from 40° preoperatively to 118° at final follow-up; 88% of patients regained ROM of 100° or greater. The forearm rotation arc (FRA) with forearm rotation dysfunction increased from a preoperative mean of 76° to 128°; 82% of patients regained an FRA of 100° or greater. The mean Mayo Elbow Performance Index (MEPI) increased from 63 to 91 points; the proportion of patients with good or excellent results was 95%. EFI patients had the best ROM (129°) and MEPI (93 points) and EFIV patients achieved the most-changed ROM (116°), whereas EFIII patients had the worst ROM (104°) and MEPI (84 points) and the least-changed ROM (64°). The FRA was best in FRI patients (142°), followed by FRII patients (118°), and worst in FRIII patients (82°); in contrast, the changed FRA was greatest in FRIII patients (82°), followed by FRII patients (64°), and least in FRI patients (37°). CONCLUSION: This study suggests that the proposed pathologic classification provides a new perspective on the understanding and standardization of elbow arthrolysis, providing satisfactory clinical outcomes.

What Range of Motion and Functional Results Can Be Expected After Open Arthrolysis with Hinged External Fixation For Severe Posttraumatic Elbow Stiffness?

BACKGROUND: The elbow is more susceptible to loss of motion after trauma than any other joint. Open arthrolysis often is performed for posttraumatic elbow stiffness if the stiffness does not improve with nonsurgical treatment, but the midterm results of this procedure and factors that may limit recovery have not been widely studied. QUESTIONS/PURPOSES: We reviewed patients who had undergone open arthrolysis with hinged external fixator for severe posttraumatic elbow stiffness (ROM ≤ 60°) with a minimum of 5 years followup to (1) analyze ROM gains; (2) assess functional improvement with the Mayo Elbow Performance Index (MEPI) and DASH, quality of life with the SF-36, pain with VAS, and ulnar nerve function with the Amadio rating scale and Dellon classification; and (3) identify complications and risk factors that might hinder mid-term elbow motion recovery after this procedure. METHODS: Between March 2011 and December 2012, we generally offered patients with elbow stiffness an open arthrolysis procedure when function did not improve with 6 months of nonoperative therapy, and no contraindications such as immature heterotopic ossification or complete destruction of articular cartilage were present. During that time, 161 patients underwent open arthrolysis for posttraumatic elbow stiffness at our institution; 49 of them satisfied the study inclusion criteria (adults with elbow ROM ≤ 60° as a result of trauma) and exclusion criteria (stiffness caused by burns or central nervous system injuries, causative trauma associated with nonunion or malunion of the elbow, severe articular damage that would have necessitated joint arthroplasty, or prior elbow release). In general, a combined medial-lateral approach to the elbow was performed to address the soft tissue tethers and any blocks to elbow motion, and a hinged external fixator was applied for 6 weeks to maintain elbow stability and improve the efficacy of postoperative rehabilitation. These patients were evaluated retrospectively at a mean followup period of 69 months (range, 62-83 months), and demographics, disease characteristics, arthrolysis details, pre- and postoutcome measures as noted, and complications were recorded via an electronic database. Multivariate regression analysis was performed to identify factors associated with ROM recovery. RESULTS: At final followup, total ROM increased from a preoperative mean of 27 ± 20° to a postoperative mean of 131 ± 11° (mean difference, 104°; 95% CI, 98°-111°; p < 0.001), and 98% (48 of 49) of patients achieved a functional ROM of 30° to 130°. Improvements were also found in functional scores (MEPI: 54 ± 12 to 95 ± 7, mean difference, 41 points; DASH: 48 ± 17 to 8 ± 8, mean difference, 40 points; both p < 0.001), life quality (physical SF-36: 46 ± 11 to 81 ± 12, mean difference, 35 points; mental SF-36: 43 ± 14 to 80 ± 9, mean difference, 37 points; both p < .001), pain (VAS: 2.5 ± 2.4 to 0.4 ± 0.8; mean difference, 2.0 points; p < 0.001), and ulnar nerve function (Amadio score: 7.8 ± 1.9 to 8.4 ± 0.8; mean difference, 0.6 points; p = 0.004). A total of 18% (nine of 49 patients) developed complications, including new-onset or exacerbated nerve symptoms (four patients), recurrent heterotopic ossification (two patients), and pin-related infections (three patients). No patients underwent subsequent surgery for any of the above complications. Lastly, the medium-term ROM was divided into ROM ≤ 120° (n = 9) and ROM > 120° (n = 40). After controlling for potential confounding variables such as duration of stiffness and tobacco use, we found that tobacco use was the only independent risk factor examined (odds ratio, 9; 95% CI, 2-47; p = 0.009) associated with recovery of ROM. CONCLUSIONS: Satisfactory medium-term results were found for open arthrolysis with hinged external fixation with our protocol in patients who had severe posttraumatic elbow stiffness. Appropriate and sufficient releases of tethered soft tissues and correction of any blocks that affect elbow motion intraoperatively, a dedicated team approach, and an aggressive and systematic postoperative rehabilitation program are the core steps for this procedure. Additionally, the importance of preoperative discontinuation of tobacco use should be emphasized. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Determining the effective timing of an open arthrolysis for post-traumatic elbow stiffness: a retrospective cohort study.

BACKGROUND: Following trauma, the elbow is the most susceptible to restricted motion among all joints. Open arthrolysis is often performed for post-traumatic elbow stiffness if that stiffness does not improve with non-operative management. However, the optimal timing for performing an open arthrolysis remains controversial. The purpose of this study was to compare the outcome (elbow motion and function) and the rate of complications among patients who had undergone early, median and late release procedures to establish an optimal time interval following the injury, after which, an effective open arthrolysis can be performed. METHODS: In this retrospective cohort study, we included total 133 patients, who had undergone open arthrolysis for post-traumatic elbow stiffness. The subjects were divided into 3 groups, with 31 patients in the early release group (arthrolysis performed at 6-10 months after injury), 78 patients in the median release group (at 11-20 months), and 24 patients in the late release group (at > 20 months). The release procedure in all patients was performed by the same surgeon, using the same technique. The general data, functional performance, and complications, if any, were retrospectively documented for all patients and statistically analysed. RESULTS: The demographic data and disease characteristics of all patients were comparable at baseline. Postoperatively, no significant differences were found among the three groups with respect to the range of motion (p = 0.067), Mayo Elbow Performance Score (p = 0.350) and its ratings (p = 0.329), visual analog scale score for pain (p = 0.227), Dellon classification for ulnar nerve symptoms (p = 0.497), and each discrete complication (all p values > 0.05). CONCLUSIONS: At the final follow-up, our results showed no significant difference in the postoperative elbow motion capacities, functional scores and the rates of complications among patients who had undergone an early, median, and late release. Therefore, we have recommended that an early arthrolysis would be preferable due to its multiple advantages, and the conventionally observed interval of > 1 year after the injury, could be shortened. LEVEL OF EVIDENCE: Level III; Retrospective Cohort Design; Therapeutic Study.

KLF2/PPARγ axis contributes to trauma-induced heterotopic ossification by regulating mitochondrial dysfunction.

Trauma-induced heterotopic ossification (HO) is a complex disorder after musculoskeletal injury and characterized by aberrant extraskeletal bone formation. Recent studies shed light on critical role of dysregulated osteogenic differentiation in aberrant bone formation. Krupel-like factor 2 (KLF2) and peroxisome proliferator-activated receptor gamma (PPARγ) are master adapter proteins that link cellular responses to osteogenesis; however, their roles and relationships in HO remain elusive. Using a murine burn/tenotomy model in vivo, we identified elevated KLF2 and reduced PPARγ levels in tendon stem/progenitor cells (TSPCs) during trauma-induced HO formation. Both KLF2 inhibition and PPARγ promotion reduced mature HO, whereas the effects of PPARγ promotion were abolished by KLF2 overexpression. Additionally, mitochondrial dysfunction and reactive oxygen species (ROS) production also increased after burn/tenotomy, and improvements in mitochondrial function (ROS scavenger) could alleviate HO formation, but were abolished by KLF2 activation and PPARγ suppression by affecting redox balance. Furthermore, in vitro, we found increased KLF2 and decreased PPARγ levels in osteogenically induced TSPCs. Both KLF2 inhibition and PPARγ promotion relieved osteogenesis by improving mitochondrial function and maintaining redox balance, and effects of PPARγ promotion were abolished by KLF2 overexpression. Our findings suggest that KLF2/PPARγ axis exerts regulatory effects on trauma-induced HO through modulation of mitochondrial dysfunction and ROS production in TSPCs by affecting redox balance. Targeting KLF2/PPARγ axis and mitochondrial dysfunction can represent attractive approaches to therapeutic intervention in trauma-induced HO.

Endogenous hydrogen sulfide accelerated trauma-induced heterotopic ossification through the Ca2+/ERK pathway-enhanced aberrant osteogenic activity.

Unveiling of the mechanism involved in the occurrence and development of trauma-induced heterotopic ossification (tHO) is highly demanding due to current ineffective clinical treatment for it. Previous studies proposed that hydrogen sulfide (H2S) was vital for fate determination of stem cells, suggesting a potential role in the regulation of tHO development. In the current study, We found that expression of metabolic enzyme within sulfur conversion pathway was enhanced after tendon injury, leading to H2S accumulation within the tHO region. Increased production of endogenous H2S was shown to promote aberrant osteogenic activity of tendon-derived stem cells (TDSCs), which accelerated tHO formation. The inhibition of metabolic enzyme of H2S production or directly absorption of H2S could abolished osteogenic induction of TDSCs and the formation of tHO. Mechanistically, through RNA sequencing combined with rescue experiments, we demonstrated that activation of Ca2+/ERK pathway was the downstream molecular event of H2S-induced osteogenic commitment of TDSCs and tHO. For treatment strategy exploration, zine oxide nanoparticles (ZnO) as an effective H2S elimination material was validated to ideally halt the tHO formation in this study. Furthermore, in terms of chirality of nanoparticles, D-ZnO or L-ZnO nanoparticles showed superiority over R-ZnO nanoparticles in both clearing of H2S and inhibition of tHO. Our study not only revealed the mechanism of tHO through the endogenous gas signaling event from a new perspective, but also presented a applicable platform for elimination of the inordinate gas production, thus aiding the development of clinical treatment for tHO.

Preclinical assessment of IL-1ß primed human umbilical cord mesenchymal stem cells for tendon functional repair through TGF-ß/IL-10 signaling.

Background: Inadequate repair capacity and disturbed immune compartments are the main pathological causes of tendinopathy. Transplantation of mesenchymal stem cells (MSCs) become an effective clinic option to alleviate tendinopathy. Interleukin-1ß (IL-1ß) could confer on MSCs enhanced immunoregulatory capability to remodel the repair microenvironment favoring tissue repair. Therefore, IL-1ß activated UC-MSCs (1ßUC-MSCs) may exert favorable efficacy in promoting tendon repair in a preclinical tendinopathy rat model. Methods: Tendon-derived stem cells (TDSCs) were isolated and characterized. In vitro, the levels of immunoregulatory-related cytokines such as IL-1ß, IL-6, IL-10, and TGF-ß secreted by 1ßUC-MSCs and unprimed UC-MSCs was measured. And tendon-specific markers expressed by TDSCs cultured with primed cultured medium (CM) or unprimed CM were detected. In vivo, Achilles tendinopathy was induced by 30 µL collagenase I injection in Sprague Dawley rats. One week later, the rats were randomly injected with UC-MSCs primed with IL-1ß (106 cells per tendon), UC-MSCs, or PBS. After rats were sacrificed, histological evaluation, electron microscopy, biomechanical tests, gait performance were conducted to evaluate the structural and functional recovery of Achilles tendons. The inflammation and metabolic state of the extracellular matrix, and the potential mechanism were assessed by immunohistochemical staining and Western blot. Results: UC-MSCs were activated by IL-1ß to secrete higher levels of IL-10 and TGF-ß while the secretion levels of IL-6 and IL-1ß were not changed significantly, promoting a higher expression level of COL I and TNMD in TDSCs under proinflammatory environment. In vivo, the transplanted 1ßUC-MSCs could survive up to 5 weeks after injection with tenogenic differentiation and improved tendon healing histologically semi-quantified by modified Bonar scores. This structural regeneration was further confirmed by observation of ultrastructural morphology, and led to good functional recovery including improved biomechanical properties and gait performance. During this process, the inflammatory response and metabolism of the extracellular matrix was improved through TGF-ß/IL-10 pathway. Conclusion: This study demonstrated that the transplantation of UC-MSCs activated by IL-1ß exhibited satisfactory ability for promoting tendon functional repair in a tendinopathy rat model. During this process, the balance of inflammatory response and extracellular matrix metabolism was remodeled, and the TGF-ß/Smad2/3 and IL-10 signaling pathways were activated simultaneously. We cautiously conclude that the IL-1ß primed UC-MSCs could be a promising strategy for enhancing the ability of MSCs to treat tendinopathy.

lncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/ß-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification.

BACKGROUND: Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms. RESULTS: On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/ß-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/ß-catenin axis. CONCLUSIONS: Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

Macrophage-Derived TGF-ß and VEGF Promote the Progression of Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is a pathological bone formation process caused by musculoskeletal trauma. HO is characterized by aberrant endochondral ossification and angiogenesis. Our previous studies have indicated that macrophage inflammation is involved in traumatic HO formation. In this study, we found that macrophage infiltration and TGF-ß signaling activation are presented in human HO. Depletion of macrophages effectively suppressed traumatic HO formation in a HO mice model, and macrophage depletion significantly inhibited the activation of TGF-ß/Smad2/3 signaling. In addition, the TGF-ß blockade created by a neutralizing antibody impeded ectopic bone formation in vivo. Notably, endochondral ossification and angiogenesis are attenuated following macrophage depletion or TGF-ß inhibition. Furthermore, our observations on macrophage polarization revealed that M2 macrophages, rather than M1 macrophages, play a critical role in supporting HO development by enhancing the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Our findings on ectopic bone formation in HO patients and the mice model indicate that M2 macrophages are an important contributor for HO development, and that inhibition of M2 polarization or TGF-ß activity may be a potential method of therapy for traumatic HO.

Non-electric bioelectrical analog strategy by a biophysical-driven nano-micro spatial anisotropic scaffold for regulating stem cell niche and tissue regeneration in a neuronal therapy.

The slow regenerating rate and misdirected axonal growth are primary concerns that disturb the curative outcome of peripheral nerve repair. Biophysical intervention through nerve scaffolds can provide efficient, tunable and sustainable guidance for nerve regrowth. Herein, we fabricate the reduced graphene oxide (rGO)/polycaprolactone (PCL) scaffold characterized with anisotropic microfibers and oriented nanogrooves by electrospinning technique. Adipose-derived stem cells (ADSCs) are seeded on the scaffolds in vitro and the viability, neural differentiation efficiency and neurotrophic potential are investigated. RGO/PCL conduits reprogram the phenotype of seeded cells and efficiently repair 15 mm sciatic nerve defect in rats. In summary, biophysical cues on nerve scaffolds are key determinants to stem cell phenotype, and ADSC-seeded rGO/PCL oriented scaffolds are promising, controllable and sustainable approaches to enable peripheral nerve regeneration.

Surgical resection outcomes of post-traumatic elbow heterotopic ossification: multicenter case series at a minimum 5-year follow-up.

BACKGROUND: Heterotopic ossification (HO), a common complication after elbow trauma, causes severe limb disability, Surgical resection is usually performed for post-traumatic elbow HO (PTEHO) to regain mobility. Though it was heavily reported, there has been no long-term (minimum 5-year) follow-up. PATIENTS AND METHODS: 173 patients who underwent PTEHO resection were followed up for minimum 5 years in 4 hospitals between January/2015 and August/2016. Demographics, disease characteristics, preoperative and minimum 5-year assessments were collected. After controlling for potential variables when dividing long-term ROM into <120° and ≥120°, risk factors for ROM recovery to modern functional arc were identified through multivariable regression analysis. RESULTS: Clinically important improvements in ROM of 39°â†’124° were obtained at final follow-up, and 74.6% achieved modern functional arc (≥120°). Mayo Elbow Performance Index (MEPI) had clinically important increases of 69→93 points at final follow-up, and 96.5% reported excellent-to-good. Pain (Numerical Rating Scale, 1.9→0.6 points) and ulnar nerve symptoms were improved. Total complication rate was 15.6%, including new-onset ulnar nerve symptoms (5.8%), HO recurrence with clinical symptoms (6.9%), elbow instability (1.7%), and joint infection (1.2%). Previously reported high body mass index (BMI, p=0.002) and long disease duration (p=0.033) were equally identified as risk factors for not achieving modern functional arc, meanwhile tobacco use (p=0.024) and ankylosed HO (p<0.001) were found to be new risk factors. CONCLUSION: Surgical resection yields satisfactory outcomes for PTEHO at long-term of minimum 5 years. High BMI, tobacco use, long disease duration, and ankylosed HO would negatively affect ROM recovery to modern functional arc (≥120°). LEVEL OF EVIDENCE: Level IV, therapeutic study.