Uni-directional release of ibuprofen from an asymmetric fibrous membrane enables effective peritendinous anti-adhesion.

Drug-loaded porous membranes have been deemed to be effective physicochemical barriers to separate postoperative adhesion-prone tissues in tendon healing. However, cell viability and subsequent tissue regeneration might be severely interfered with the unrestricted release and the locally excessive concentration of anti-inflammatory drugs. Herein, we report a double-layered membrane with sustained and uni-directional drug delivery features to prevent peritendinous adhesion without hampering the healing outcome. A vortex-assisted electrospinning system in combination with ibuprofen (IBU)-in-water emulsion was utilized to fabricate IBU-loaded poly-ʟ-lactic-acid (PLLA) fiber bundle membrane (PFB-IBU) as the anti-adhesion layer. The resultant highly porous structure, oleophilic and hydrophobic nature of PLLA fibers enabled in situ loading of IBU with a concentration gradient across the membrane thickness. Aligned collagen nanofibers were further deposited at the low IBU concentration side of the membrane for regulating cell growth and achieving uni-directional release of IBU. Drug release kinetics showed that the release amount of IBU from the high concentration side reached 79.32% at 14 d, while it was only 0.35% at the collagen side. Therefore, fibroblast proliferation at the high concentration side was successfully inhibited without affecting the oriented growth of tendon-derived stem cells at the other side. In vivo evaluation of the rat Achilles adhesion model confirmed the successful peritendinous anti-adhesion of our double-layered membrane, in that the macrophage recruitment, the inflammatory factor secretion and the deposition of pathological adhesion markers such as α-SMA and COL-III were all inhibited, which greatly improved the peritendinous fibrosis and restored the motor function of tendon.

NLRP3-Dependent Crosstalk between Pyroptotic Macrophage and Senescent Cell Orchestrates Trauma-Induced Heterotopic Ossification During Aberrant Wound Healing.

Heterotopic ossification (HO) represents an unwanted ossific wound healing response to the soft tissue injury which caused catastrophic limb dysfunction. Recent studies established the involvement of inflammation and cellular senescence in the tissue healing process, though their role in HO still remained to be clarified. Here, a novel crosstalk where the pyroptotic macrophages aroused tendon-derived stem cells (TDSCs) senescence is revealed to encourage osteogenic healing during trauma-induced HO formation. Macrophage pyroptosis blockade reduces the senescent cell burden and HO formation in NLRP3 knockout mice. Pyroptosis-driven IL-1ß and extracellular vesicles (EVs) secretion from macrophages are determined to motivate TDSCs senescence and resultant osteogenesis. Mechanistically, pyroptosis in macrophages enhances the exosomal release of high mobility group protein 1 (HMGB1), which directly bounds TLR9 in TDSCs to trigger morbid signaling. NF-κB signaling is confirmed to be the converging downstream pathway of TDSCs in response to HMGB1-containing EVs and IL-1ß. This study adds insights into aberrant regeneration-based theory for HO formation and boosts therapeutic strategy development.

Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult.

Heterotopic ossification (HO) is one of the most intractable disorders following musculoskeletal injury and is characterized by the ectopic presence of bone tissue in the soft tissue leading to severe loss of function in the extremities. Recent studies have indicated that immune cell infiltration and inflammation are involved in aberrant bone formation. In this study, we found increased monocyte/macrophage and mast cell accumulation during early HO progression. Macrophage depletion by clodronate liposomes and mast cell stabilization by cromolyn sodium significantly impeded HO formation. Therefore, we proposed that the dietary phytochemical quercetin could also suppress immune cell recruitment and related inflammatory responses to prevent HO. As expected, quercetin inhibited the monocyte-to-macrophage transition, macrophage polarization, and mast cell activation in vitro in a dose-dependent manner. Using a murine burn/tenotomy model, we also demonstrated that quercetin attenuated inflammatory responses and HO in vivo. Furthermore, elevated SIRT1 and decreased acetylated NFκB p65 expression were responsible for the mechanism of quercetin, and the beneficial effects of quercetin were reversed by the SIRT1 antagonist EX527 and mimicked by the SIRT agonist SRT1720. The findings in this study suggest that targeting monocyte/macrophage and mast cell activities may represent an attractive approach for therapeutic intervention of HO and that quercetin may serve as a promising therapeutic candidate for the treatment of trauma-induced HO by modulating SIRT1/NFκB signaling.

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.

Extracellular vesicles from hydroxycamptothecin primed umbilical cord stem cells enhance anti-adhesion potential for treatment of tendon injury.

BACKGROUND: Peritendinous fibrosis represents a fibrotic healing process that usually occurs after tendon injury or surgery. This worldwide challenge hampers the functional rehabilitation and the mobility of extremities. However, effective treatment is still lacking at present. The aim of our study was to explore the effect of extracellular vesicles derived from hydroxycamptothecin primed human umbilical cord stem cells (HCPT-EVs) on post-traumatic tendon adhesion. METHODS: Extracellular vesicles derived from unprimed human umbilical cord mesenchymal stem cells (Unprimed EVs) or HCPT-EVs were isolated and characterized. A rat model of Achilles tendon injury was used to confirm the anti-adhesion effect of HCPT-EVs and compared with that of Unprimed EVs in vivo. In vitro, the inhibitory effects of HCPT-EVs on fibroblast proliferation, viability, and myofibroblast differentiation upon TGF-ß1 stimulation were compared with the effects of Unprimed EVs. For mechanistic analysis, the expression of endoplasmic reticulum stress (ERS)-associated proteins was examined among the effector cargos of HCPT-EVs and Unprimed EVs. The ERS antagonist salubrinal was used to determine the ERS dependence of the anti-adhesion effects of HCPT-EVs. RESULTS: There were no obvious differences between Unprimed EVs and HCPT-EVs in terms of morphology, particle size, characteristic protein expression, and cellular uptake. HCPT-EVs exhibited a fortified anti-adhesion effect after Achilles tendon injury compared with Unprimed EVs. Fibroblast proliferation and viability and myofibroblast differentiation were all inhibited by HCPT-EVs. These properties were superior for HCPT-EVs relative to Unprimed EVs. Mechanistically, HCPT-EVs contained more ERS-associated protein than Unprimed EVs and activated the ERS pathway in fibroblast to counteract myofibroblast differentiation. CONCLUSION: This study demonstrates that HCPT-EVs show high anti-adhesion potential for the treatment of tendon injury by provoking ERS in fibroblasts. HCPT-EVs represent a promising strategy for clinical use in treating adhesion-related diseases.

Beeswax-inspired superhydrophobic electrospun membranes for peritendinous anti-adhesion.

Posttraumatic peritendinous adhesion leads to limb disability. Physical barrier was widely used and thus focus was paid to fabricate the hydrophobic surfaces of electrospun membrane for anti-adhesion. However, current methods are limited and complicated. In this study, beeswax (Wax)/poly-L-lactic acid (PLA) anti-adhesion membranes were fabricated by blending electrospinning of Wax and PLA. The water contact angle was tested to investigate the hydrophobicity of the surfaces. Incorporation of Wax into PLA did not destroy the micro-pores between Wax/PLA fibers. After 7-day culture, proliferation of fibroblasts on Wax/PLA anti-adhesion membranes were significantly less than that on culture dish and PLA membranes. In rat Achilles adhesion model, least histological peritendinous adhesion formation was detected on the repaired sites in the group treated with Wax/PLA membranes than PLA membranes. Consequently, blending electrospinning of Wax into PLA is an easy method to fabricate hydrophobic surface of electrospun membrane with advanced peritendinous anti-adhesion outcome.

(-)-Epigallocatechin gallate-loaded polycaprolactone scaffolds fabricated using a 3D integrated moulding method alleviate immune stress and induce neurogenesis.

OBJECTIVES: In peripheral neuropathy, the underlying mechanisms of nerve and muscle degeneration include chronic inflammation and oxidative stress in fibrotic tissues. (-)-Epigallocatechin gallate (EGCG) is a major, active component in green tea and may scavenge free radical oxygen and attenuate inflammation. Conservative treatments such as steroid injection only deal with early, asymptomatic, peripheral neuropathy. In contrast, neurolysis and nerve conduit implantation work effectively for treating advanced stages. MATERIALS AND METHODS: An EGCG-loaded polycaprolactone (PCL) porous scaffold was fabricated using an integrated moulding method. We evaluated proliferative, oxidative and inflammatory activity of rat Schwann cells (RSCs) and rat skeletal muscle cells (RSMCs) cultured on different scaffolds in vitro. In a rat radiation injury model, we assessed the morphological, electrophysiological and functional performance of regenerated sciatic nerves and gastrocnemius muscles, as well as oxidative stress and inflammation state. RESULTS: RSCs and RSMCs exhibited higher proliferative, anti-oxidant and anti-inflammatory states in an EGCG/PCL scaffold. In vivo studies showed improved nerve and muscle recovery in the EGCG/PCL group, with increased nerve myelination and muscle fibre proliferation and reduced macrophage infiltration, lipid peroxidation, inflammation and oxidative stress indicators. CONCLUSIONS: The EGCG-modified PCL porous nerve scaffold alleviates cellular oxidative stress and repairs peripheral nerve and muscle structure in rats. It attenuates oxidative stress and inflammation in vivo and may provide further insights into peripheral nerve repair in the future.

Hydroxycamptothecin Inhibits Peritendinous Adhesion via the Endoplasmic Reticulum Stress-Dependent Apoptosis.

Traumatic peritendinous fibrosis is a worldwide clinical problem resulting in severe limb disability. Hydroxycamptothecin (HCPT) is an anti-neoplastic drug widely exploited in clinical practice. It has shown potential of anti-fibrosis in recent years. We previously demonstrated that HCPT inhibited the characterization of fibrosis in vitro. However, it is still unclear whether it ameliorates peritendinous adhesion in an in vivo animal tendon injury model. The underlying mechanism is also worth investigating. The present study aims to determine whether HCPT inhibits tendon adhesion and to explore the underlying mechanisms. In a rat tendon injury model, we observed that topical application of HCPT significantly attenuated peritendinous adhesion as revealed by the results of macroscopic observation, biomechanical, histological, immunohistochemical evaluation, western blot, and quantitative PCR (q-PCR) analyses. Furthermore, western blot and q-PCR analyses revealed that this phenomenon is correlated with HCPT activation of endoplasmic reticulum (ER) stress. In addition, in vitro studies show that HCPT significantly inhibits fibroblast proliferation and induces apoptosis by reducing the expression of extracellular matrix (ECM) proteins COL3A1 and α-smooth muscle actin (α-SMA). Finally, we employed small interfering RNA (siRNA) to target inositol requiring kinase 1 (IRE1) and activated transcription factor 6 (ATF-6) to verify that the effect of inhibitory fibrosis of HCPT disappears after knockdown of ATF-6 and IRE1, thereby suggesting that an anti-fibrotic effect of HCPT is mediated by the ER-dependent apoptotic pathway. In conclusion, our results indicate that HCPT inhibits peritendinous fibrosis through the ER-dependent apoptotic pathway and might serve as a potential solution to prevent traumatic peritendinous adhesion.

Follistatin-like protein 1 suppressed pro-inflammatory cytokines expression during neuroinflammation induced by lipopolysaccharide.

Follistain-like protein 1 (FSTL1), has been recently demonstrated to be involved in the embryo development of nervous system and glioblastoma. However, the role of FSTL1 in neuroinflammation remains unexplored. In this study, the expression of FSTL1 in astrocytes was verified and its role was studied in neuroinflammation induced by in vivo intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) or LPS treatment to astrocytes in vitro. FSTL1 was significantly induced after ICV LPS injection or LPS treatment. FSTL1 suppressed upregulation of pro-inflammatory cytokines in astrocytes after LPS treatment. Moreover, FSTL1 downregulated expression of pro-inflammatory cytokines through suppressing MAPK/p-ERK1/2 pathway in astrocytes. Our results suggest that FSTL1 may play an anti-inflammatory role in neuroinflammation mediated by astrocytes.