Osteoporotic bone defects, a severe complication of osteoporosis, are distinguished by a delayed bone healing process and poor repair quality. While bone marrow-derived mesenchymal stem cells (BMMSCs) are the primary origin of bone-forming osteoblasts, their mitochondrial function is impaired, leading to inadequate bone regeneration in osteoporotic patients. Melatonin is well-known for its antioxidant properties and regulation on bone metabolism. The present study postulated that melatonin has the potential to enhance the repair of osteoporotic bone defects by restoring the mitochondrial function of BMMSCs. In vitro administration of melatonin at varying concentrations (0.01, 1, and 100 mu M) demonstrated a significant dose-dependent improvement in the mitochondrial function of BMMSCs obtained from ovariectomized rats (OVX-BMMSCs), as indicated by an elevation in mitochondrial membrane potential, adenosine triphosphate synthesis and expression of mitochondrial respiratory chain factors. Melatonin reduced the level of mitochondrial superoxide by activating the silent information regulator type 1 (SIRT1) and its downstream antioxidant enzymes, particularly superoxide dismutase 2 (SOD2). The protective effects of melatonin were found to be nullified upon silencing of Sirt1 or Sod2, underscoring the crucial role of the SIRT1-SOD2 axis in the melatonin-induced enhancement of mitochondrial energy metabolism in OVX-BMMSCs. To achieve a sustained and localized release of melatonin, silk fibroin scaffolds loaded with melatonin (SF@MT) were fabricated. The study involved the surgical creation of bilateral femur defects in OVX rats, followed by the implantation of SF@MT scaffolds. The results indicated that the application of melatonin partially restored the mitochondrial energy metabolism and osteogenic differentiation of OVX-BMMSCs by reinstating mitochondrial redox homeostasis. These findings suggest that the localized administration of melatonin through bone implants holds potential as a therapeutic approach for addressing osteoporotic bone defects.

Volumetric muscle loss (VML) is a condition that results in the extensive loss of 20 % or more of skeletal muscle due to trauma or tumor ablation, leading to severe functional impairment and permanent disability. The current surgical interventions have limited functional regeneration of skeletal muscle due to the compromised self-repair mechanism. Melatonin has been reported to protect skeletal muscle from exercise-induced oxidative damage and holds great potential to treat muscle diseases. In this study, we hypothesize that melatonin can enhance myoblast differentiation and promote effective recovery of skeletal muscle following VML. In vitro administration of melatonin resulted in a significant enhancement of myogenesis in C2C12 myoblast cells, as evidenced by the upregulation of myogenic marker genes in a dose-dependent manner. Further experiments revealed that silent information of regulator type 3 (SIRT3) played a critical role in the melatonin-enhanced myoblast differentiation through enhancement of mitochondrial energy metabolism and activation of mitochondrial antioxidant enzymes such as superoxide dismutase 2 (SOD2). Silencing of Sirt3 completely abrogated the protective effect of melatonin on the mitochondrial function of myoblasts, evidenced by the increased reactive oxygen species, decreased adenosine triphosphate production, and down-regulated myoblast-specific marker gene expression. In order to attain a protracted and consistent release, liposome-encapsuled melatonin was integrated into gelatin methacryloyl hydrogel (GelMA-Lipo@MT). The implantation of GelMA-Lipo@MT into a tibialis anterior muscle defect in a VML model effectively stimulated the formation of myofibers and new blood vessels in situ, while concurrently inhibiting fibrotic collagen deposition. The findings of this study indicate that the incorporation of melatonin with GelMA hydrogel has facilitated the de novo vascularized skeletal muscle regeneration by augmenting mitochondrial energy metabolism. This represents a promising approach for the development of skeletal muscle tissue engineering, which could be utilized for the treatment of VML and other severe muscle injuries.

The repair of critical-sized bone defects poses a significant challenge due to the absence of periosteum, which plays a crucial role in coordinating the processes of osteogenesis and vascularization during bone healing. Herein, we hypothesized that melatonin-encapsuled silk Fibronin electrospun nanofibers (SF@MT) could provide intrinsic induction of both osteogenesis and angiogenesis, thereby promoting vascularized bone regeneration. The sustained release of melatonin from the SF@MT nanofibers resulted in favorable biocompatibility and superior osteogenic induction of bone marrow mesenchymal stem cells (BMMSCs). Interestingly, melatonin promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a BMMSC-dependent manner, potentially through the upregulation of vascular endothelial growth factor (VEGFA) expression in SF@MT-cultured BMMSCs. SF@MT nanofibers enhanced the BMMSC-mediated angiogenesis by activating the PI3K/Akt signaling pathway. In vivo experiments indicated that the implantation of SF@MT nanofibers into rat critical-sized calvarial defects significantly enhances the production of bone matrix and the development of new blood vessels, leading to an accelerated process of vascularized bone regeneration. Consequently, the utilization of melatonin-encapsulated silk Fibronin electrospun nanofibers shows great promise as a potential solution for artificial periosteum, with the potential to regulate the coupling of osteogenesis and angiogenesis in critical-sized bone defect repair.

Articular cartilage injury induced by collision or trauma is a common sports-related condition that may progress to pain, dysfunction, and secondary osteoarthritis (OA). Limited by self-renewal potential, car-tilage regeneration faces a quandary while bio-inspired novel strategies are urgently required. In this study, by a soft freezing method and surface modification technique, a multi-functional silk fibroin (SF) plus gelatin methacrylate (GelMA) scaffold laden with melatonin (MT) was prepared. SF-GelMA@MT scaf-fold demonstrated enhanced biomechanical characteristics and long-acting melatonin release. In vitro treatment with SF-GelMA@MT induced the synthesis of cartilage extracellular matrix (ECM) compo-nents. Mechanistically, sustained release of melatonin yielded robust chondroprotective effects via im-proving mitochondrial polarization and antioxidant properties. SF-GelMA@MT implantation boosted carti-lage renascence in a full-thickness cartilage defect model via mitochondria-associated sirtuins 1 (SIRT1)-superoxide dismutase 2 (SOD2) signaling pathway in vivo. In summary, this research proposed a well-designed bionic composite scaffold that promotes cartilage regeneration via mitochondrial function en-hancement, which is of tremendous potential for cartilage tissue engineering.(c) 2022 Published by Elsevier Ltd on behalf of The editorial office of Journal of Materials Science & Technology.

Over-accumulation of reactive oxygen species (ROS) causes mitochondrial dysfunction and impairs the osteogenic potential of bone marrow-derived mesenchymal stem cells (BMMSCs). Selenium (Se) protects BMMSCs from oxidative stress-induced damage; however, it is unknown whether Se supplementation can promote the repair of osteoporotic bone defects by rescuing the impaired osteogenic potential of osteoporotic BMMSCs (OP-BMMSCs). In vitro treatment with sodium selenite (Na2SeO3) successfully improved the osteogenic differentiation of OP-BMMSCs, as demonstrated by increased matrix mineralization and up-regulated osteogenic genes expression. More importantly, Na2SeO3 restored the impaired mitochondrial functions of OP-BMMSCs, significantly up-regulated glutathione peroxidase 1 (GPx1) expression and attenuated the intracellular ROS and mitochondrial superoxide. Silencing of Gpx1 completely abrogated the protective effects of Na2SeO3 on mitochondrial functions of OP-BMMSCs, suggesting the important role of GPx1 in protecting OP-BMMSCs from oxidative stress. We further fabricated Se-modified bone cement based on silk fibroin and calcium phosphate cement (SF/CPC). After 8 weeks of implantation, Se-modified bone cement significantly promoted bone defect repair, evidenced by the increased new bone tissue formation and enhanced GPx1 expression in ovariectomized rats. These findings revealed that Se supplementation rescued mitochondrial functions of OP-BMMSCs through activation of the GPx1-mediated antioxidant pathway, and more importantly, supplementation with Se in SF/CPC accelerated bone regeneration in ovariectomized rats, representing a novel strategy for treating osteoporotic bone fractures or defects.

Introduction: Accelerated imbalance between bone formation and bone resorption is associated with bone loss in postmenopausal osteoporosis. Studies have shown that this loss is accompanied by an increase in bone marrow adiposity. Melatonin was shown to improve impaired bone formation capacity of bone marrow-derived mesen-chymal stem cells from ovariectomized rats (OVX-BMMSCs).Objectives: To investigate whether the anti-osteoporosis effect of melatonin involves regulation of the equilibrium between osteogenic and adipogenic differentiation of osteoporotic BMMSCs.Methods: To induce osteoporosis, female Sprague-Dawley rats received ovariectomy (OVX). Primary BMMSCs were isolated from tibiae and femurs of OVX and sham-op rats and were induced towards osteogenic or adipo-genic differentiation. Matrix mineralization was determined by Alizarin Red S (ARS) and lipid formation was evaluated by Oil Red O. OVX rats were injected with melatonin through the tail vein. Bone microarchitecture was determined using micro computed tomography and marrow adiposity were examined by histology staining.Results: OVX-BMMSCs exhibited a compromised osteogenic potential and an enhanced lineage differentiation towards adipocytes. In vitro melatonin improved osteogenic differentiation of OVX-BMMSCs and promoted matrix mineralization by enhancing the expression of transcription factor RUNX2 in a dose-dependent manner. More-over, melatonin significantly inhibited lipid formation and suppressed OVX-BMMSCs adipogenesis by down -regulating peroxisome proliferator-activated receptor gamma (PPAR gamma). Intravenous injection of melatonin prevented bone mass reduction and bone architecture destruction in ovariectomized rats. Importantly, there was a signifi-cant inhibition of adipose tissue formation in the bone marrow. Mechanistic investigations revealed that SIRT1 was involved in melatonin-mediated determination of stem cell fate. Inhibition of SIRT1 abolished the protective effects of melatonin on bone formation by inducing BMMSCs towards adipocyte differentiation.Conclusions: Melatonin reversed the differentiation switch of OVX-BMMSCs from osteogenesis to adipogenesis by activating the SIRT1 signaling pathway. Restoration of stem cell lineage commitment by melatonin prevented marrow adipose tissue over-accumulation and protected from bone loss in postmenopausal osteoporosis.The translational potential of this article: Determination of stem cell fate towards osteoblasts or adipocytes plays a pivotal role in regulating bone metabolism. This study demonstrates the protective effect of melatonin on bone mass in estrogen-deficient rats by suppressing adipose tissue accumulation in the bone marrow. Melatonin may serve as a promising candidate for the treatment of osteoporosis in clinics.

Recent evidence indicates that the mitochondrial functions of chondrocytes are impaired in the pathogenesis of osteoarthritis (OA). Melatonin can attenuate cartilage degradation through its antioxidant functions. This study aims to investigate whether melatonin could rescue the impaired mitochondrial functions of OA chondrocytes and protect cartilage metabolism. OA chondrocytes showed a compromised matrix synthesis capacity associated with mitochondrial dysfunction and aberrant oxidative stress. In vitro treatments with melatonin promoted the expression of cartilage extracellular matrix (ECM) components, improved adenosine triphosphate production, and attenuated mitochondrial oxidative stress. Mechanistically, either silencing of SOD2 or inhibition of SIRT1 abolished the protective effects of melatonin on mitochondrial functions and ECM synthesis. To achieve a sustained release effect, a melatonin-laden drug delivery system (DDS) was developed and intra-articular injection with DDS successfully improved cartilage matrix degeneration in a posttraumatic rat OA model. These findings demonstrate that melatonin-mediated recharge of mitochondria to rescue the mitochondrial functions of chondrocytes represents a promising therapeutic strategy to protect cartilage from OA.

Accumulation of senescent bone marrow-derived mesenchymal stem cells (BMMSCs) has led to an age-related bone loss. However, the role of stem cell senescence in estrogen deficiency-induced osteoporosis remains elusive. Though melatonin plays a vital role in bone metabolism regulation, the underlying mechanisms of melatonin-mediated antiosteoporosis are partially elucidated. Therefore, this study purposed to explore (1) whether estrogen deficiency causes cellular senescence of BMMSCs, and if so, (2) the potential of melatonin in preventing bone loss via senescence signaling inhibition. BMMSCs derived from ovariectomized (OVX) rats (OVX BMMSCs) showed an impaired osteogenic capacity, albeit having comparable levels of senescence biomarkers than the sham cells. When exposed to low levels of hydrogen peroxide (H2O2), OVX BMMSCs rapidly exhibited senescence-associated phenotypes such as the increased activity of senescence-associated β-galactosidase (SA-β-gal) and upregulation of cell cycle inhibitors. Notably, the in vitro treatment with melatonin hindered H2O2-induced senescence in OVX BMMSCs and restored their osteogenic capacity. Treatment with either SIRT1 inhibitor (sirtinol) or melatonin receptor antagonists (luzindole and 4-P-PDOT) eliminated melatonin protective effects, thus indicating its potential in preventing stem cell senescence via SIRT1 activation through the melatonin membrane receptors. Following in vivo intravenous administration with melatonin, it successfully protected the bone microstructure and preserved the antisenescence property of BMMSCs in OVX rats. Collectively, our findings demonstrated that melatonin protected against estrogen deficiency-related bone loss by improving the resistance of BMMSCs to cellular senescence. Therefore, melatonin-mediated antisenescence effect on stem cells provides vital information to facilitate the development of a novel and effective strategy for treating postmenopausal OP.

Reactive oxygen species (ROS) are implicated in induction of inflammatory response and cartilage degradation in osteoarthritis (OA). Melatonin has been shown to improve the chondrogenic differentiation and promote cartilage matrix synthesis in mesenchymal stem cells. However, the underlying mechanisms of melatonin-regulated antioxidant activity in OA cartilage are not known. The aim of this study was to explore the effect of melatonin on nuclear factor-erythroid 2-related factor 2 (NRF2), a key antioxidant transcription factor, and its target antioxidant genes in early-stage OA cartilage. Primary chondrocytes were isolated from rats with surgically induced OA. In vitro treatment of melatonin significantly increased cartilage matrix synthesis and upregulated antioxidant enzymes, mainly heme oxygenase 1 (HO-1), while decreasing matrix degradation enzymes and intracellular ROS. In vivo intraarticular injection of melatonin effectively ameliorated cartilage degeneration in an experimental rat OA model. Inhibition of melatonin membrane receptors by Luzindole or 4-P-PDOT reversed the beneficial effects of melatonin on cartilage matrix synthesis, implying that melatonin receptor-mediated pathway is involved in its anti-arthritic effects. Interestingly, melatonin showed no significant effect on the mRNA level of Nrf2 but significantly increased its protein level. Silencing of Nrf2 or HO-1 expression abolished the protective effects of melatonin, as shown by increased ROS levels and matrix degradation enzyme expression. Microarray assays revealed that miR-146a, a predicted target for Nrf2, was significantly upregulated in OA chondrocytes but was markedly reduced by melatonin treatment. Overexpression of miR-146a diminished the protective effects of melatonin by inhibiting NRF2 expression and aggravating OA-induced cartilage degradation. These findings demonstrate that melatonin supports the anabolic metabolism of cartilage matrix in OA chondrocytes by enhancing the protein levels of NRF2 via suppressing miR-146a. Melatonin-mediated activation of the NRF2/HO-1 axis prevents cartilage degeneration and represents a promising therapeutic target for treatment of early-stage OA. © 2022 American Society for Bone and Mineral Research (ASBMR).

Loss of extracellular matrix (ECM) of cartilage due to oxidative stress injury is one of the main characteristics of osteoarthritis (OA). As a bioactive molecule derived from the traditional Chinese Burdock, arctiin exerts robust antioxidant properties to modulate redox balance. However, the potential therapeutic effects of arctiin on OA and the underlying mechanisms involved are still unknown. Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) tool, Burdock-extracted small molecule arctiin was identified as a potential anti-arthritic component. In vitro, treatment using arctiin rescued the interleukin (IL)-1β-induced activation of proteinases and promoted the cartilage ECM synthesis in human chondrocytes. In vivo, intraperitoneal injection of arctiin ameliorated cartilage erosion and encountered subchondral bone sclerosis in the post-traumatic OA mice. Transcriptome sequencing uncovered that arctiin-enhanced cartilage matrix deposition was associated with restricted oxidative stress. Mechanistically, inhibition of nuclear factor erythroid 2-related factor 2 (NRF2) abolished arctiin-mediated anti-oxidative and anti-arthritic functions. To further broaden the application prospects, a gellan gum (GG)-based bioactive gel (GG-CD@ARC) encapsulated with arctiin was made to achieve long-term and sustained drug release. Intra-articular injection of GG-CD@ARC counteracted cartilage degeneration in the severe (12 weeks) OA mice model. These findings indicate that arctiin may be a promising anti-arthritic agent. Furthermore, GG-modified bioactive glue loaded with arctiin provides a unique strategy for treating moderate to severe OA.

Osteoarthritis (OA) is a common articular degenerative disease characterized by loss of cartilage matrix and subchondral bone sclerosis. Kartogenin (KGN) has been reported to improve chondrogenic differentiation of mesenchymal stem cells. However, the therapeutic effect of KGN on OA-induced cartilage degeneration was still unclear. This study aimed to explore the protective effects and underlying mechanisms of KGN on articular cartilage degradation using mice with post-traumatic OA. To mimic the in vivo arthritic environment, in vitro cultured chondrocytes were exposed to interleukin-1β (IL-1β). We found that KGN barely affected the cell proliferation of chondrocytes; however, KGN significantly enhanced the synthesis of cartilage matrix components such as type II collagen and aggrecan in a dose-dependent manner. Meanwhile, KGN markedly suppressed the expression of matrix degradation enzymes such as MMP13 and ADAMTS5. In vivo experiments showed that intra-articular administration of KGN ameliorated cartilage degeneration and inhibited subchondral bone sclerosis in an experimental OA mouse model. Molecular biology experiments revealed that KGN modulated intracellular reactive oxygen species in IL-1β-stimulated chondrocytes by up-regulating nuclear factor erythroid 2-related factor 2 (NRF2), while barely affecting its mRNA expression. Microarray analysis further revealed that IL-1β significantly up-regulated miR-146a that played a critical role in regulating the protein levels of NRF2. KGN treatment showed a strong inhibitory effect on the expression of miR-146a in IL-1β-stimulated chondrocytes. Over-expression of miR-146a abolished the anti-arthritic effects of KGN not only by down-regulating the protein levels of NRF2 but also by up-regulating the expression of matrix degradation enzymes. Our findings demonstrate, for the first time, that KGN exerts anti-arthritic effects via activation of the miR-146a-NRF2 axis and KGN is a promising heterocyclic molecule to prevent OA-induced cartilage degeneration.

Abstract Background Postoperative delirium (POD) is widely reported as a common postoperative complication following total joint arthroplasty (TJA) of the hip and knee in elderly patients, leading to many adverse effects. We sought to investigate predictors of delirium after TJA. Methods PubMed, EMBASE, Cochrane Library and Web of Science were searched up to 2020 for studies examining POD following TJA in elderly patients. Pooled odds ratio (OR) and mean difference (MD) of those who experienced delirium compared to those who did not were calculated for each variable. The Newcastle-Ottawa Scale (NOS) was used for the study quality evaluation. Results Fifteen studies with 31 potential factors were included. In the primary analysis, 9 factors were associated with POD, comprising advanced age (MD 3.81; 95% confidence interval (CI) 1.80–5.83), dementia (OR 24.85; 95% CI 7.26–85.02), hypertension (OR 2.26; 95% CI 1.31–3.89), diabetes (OR 2.02; 95% CI 1.15–3.55), stroke (OR 14.61; 95% CI 5.26–40.55), psychiatric illness (OR 2.72; 95% CI 1.45–5.08), use of sedative-hypnotics (OR 6.42; 95% CI 2.53–16.27), lower preoperative levels of hemoglobin (MD − 0.56; 95% CI − 0.89−− 0.22), and lower preoperative mini-mental state examination score (MD − 0.40; 95% CI − 0.69−− 0.12). Twelve studies were included in the systematic review, of which 24 factors were additionally correlated with POD using single studies. Conclusions Strategies and interventions should be implemented for the elderly patients receiving TJA surgeries with potential predictors identified in this meta-analysis.

Postmenopausal osteoporosis (OP) is one of the most common bone diseases that affects millions of aging women. Reduced osteogenesis and increased oxidative stress have been implicated in bone marrow mesenchymal stem cells (BMMSCs) derived from OP patients. Melatonin has shown positive effects on osteoblast differentiation and bone formation; however, it was unknown whether melatonin could restore OP-impaired osteogenic potential of BMMSCs and what the underlying mechanisms entailed. The objective of this study is to investigate (1) whether melatonin can restore the impaired osteogenic potential of OP BMMSCs by preserving their antioxidant functions, and if so, (2) whether intravenous administration of melatonin can prevent OP-induced bone loss in ovariectomized (OVX) rats. Ovariectomies were performed in female rats and BMMSCs were isolated from the osteoporotic rats 3 months later. In vitro treatment with melatonin successfully improved the osteogenic differentiation of OP BMMSCs, as evidenced by increased levels of matrix mineralization and osteoblast-specific genes. In melatonin-treated OP BMMSCs, intracellular oxidative stress was significantly attenuated, while levels of intracellular antioxidant enzymes were noticeably up-regulated - particularly superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1). Silent information regulator type 1 (SIRT1) was involved in the melatonin-mediated recovery of osteogenesis and antioxidant functions. Meanwhile, in vivo injections of melatonin via the tail vein successfully ameliorated the bone micro-architecture in ovariectomized rat femurs. Further experiments confirmed that BMMSCs derived from melatonin-treated OVX rats exerted well-preserved antioxidant properties and osteogenic potential. Our findings demonstrate that the administration of melatonin is a promising strategy for treating patients with postmenopausal OP by preserving the antioxidant properties and osteogenic potential of their BMMSCs.

Immobilization can lead to intervertebral disc degeneration. The biomechanical characteristics of such discs have not so far been investigated at the micro- or nanoscale, the level at which cells sense and respond to the surrounding environment. This study aimed to characterize changes in the elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale and correlate this with micro-biomechanical properties of the nucleus pulposus after immobilization, in addition to observation of tissue histology and its gene expressions. An immobilization system was used on the rat tail with an external fixation device. The elastic modulus was measured using both nano and micro probes for atomic force microscopy after 4 and 8 weeks of immobilization. Histology of the tissue was observed following hematoxylin and eosin staining. Gene expression in the annulus fibrosus tissue was quantified using real-time reverse transcription-polymerase chain reaction. The elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale increased from 74.07 ± 17.06 MPa in the control to 90.06 ± 25.51 MPa after 8 weeks (P = 0.007), and from 33.51 ± 9.33 kPa to 43.18 ± 12.08 kPa at the microscale (P = 0.002). After immobilization for 8 weeks, a greater number of cells were observed by histology to be aggregated within the nucleus pulposus. Collagen II (P = 0.007) and aggrecan (P = 0.003) gene expression were downregulated whereas collagen I (P = 0.002), MMP-3 (P < 0.001), MMP-13 (P < 0.001) and ADAMTs-4 (P < 0.001) were upregulated. Immobilization not only influenced individual collagen fibrils at the nanoscale, but also altered the micro-biomechanics and cell response in the nucleus pulposus. These results suggest that significant changes occur in intervertebral discs at both scales after immobilization, a situation about which clinicians should be aware when immobilization has to be used clinically.

Osteoarthritis (OA) is characterized by the progressive destruction of articular cartilage, which is involved in the imbalance between extracellular matrix (ECM) synthesis and degradation. MicroRNA-140-5p (miR-140) is specifically expressed in cartilage and plays an important role in OA-induced matrix degradation. The aim of this study was to investigate (1) whether intra-articular injection of melatonin could ameliorate surgically induced OA in mice and (2) whether melatonin could regulate matrix-degrading enzymes at the posttranscriptional level by targeting miR-140. In an in vitro OA environment induced by interleukin-1 beta (IL-1β), melatonin treatment improved cell proliferation of human chondrocytes, promoted the expression of cartilage ECM proteins (e.g., type II collagen and aggrecan), and inhibited the levels of IL-1β-induced proteinases, such as matrix metalloproteinase 9 (MMP9), MMP13, ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4), and ADAMTS5. Both the microarray and polymerase chain reaction (PCR) experiments revealed that miR-140 was a melatonin-responsive microRNA and melatonin upregulated miR-140 expression, which was suppressed by IL-1β stimulation. In vivo experiments demonstrated that intra-articular injection of melatonin prevented disruptions of cartilage matrix homeostasis and successfully alleviated the progression of surgery-induced OA in mice. Transfection of miR-140 antagomir completely counteracted the antiarthritic effects of melatonin by promoting matrix destruction. Our findings demonstrate that melatonin protects the articular cartilage from OA-induced degradation by targeting miR-140, and intra-articular administration of melatonin may benefit patients suffering from OA.

Autologous chondrocyte implantation (ACI) is a promising approach to repair cartilage defects; however, the cartilage trauma-induced inflammatory environment compromises its clinical outcomes. Cell-derived decellularized extracellular matrix (DECM) has been used as a culture substrate for mesenchymal stem cells (MSCs) to improve the cell proliferation and lineage-specific differentiation. In this study, DECM deposited by synovium-derived MSCs was used as an in vitro expansion system for rabbit articular chondrocytes and the response of DECM-expanded chondrocytes to pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was evaluated. Compared with those grown on tissue culture polystyrene (TCPS), the proliferation rate was significantly improved in DECM-expanded chondrocytes. TCPS- and DECM-expanded chondrocytes were isolated and induced to redifferentiation in a high-density pellet culture. DECM-expanded chondrocytes exerted a stronger resistance to 1 ng/mL of IL-1β than TCPS-expanded cells, but the production of cartilage matrix in both groups was inhibited by 5 ng/mL of IL-1β. When exposed to 1 or 5 ng/mL of TNF-α, DECM-expanded chondrocytes showed higher levels of cartilage matrix synthesis than TCPS-expanded cells. In addition, the gene expression of IL-1β- or TNF-α-induced matrix degrading enzymes (MMP3, MMP9, MMP13, and ADAMTS5) was significantly lower in DECM-expanded chondrocytes than TCPS-expanded cells. Furthermore, we found that SIRT1 inhibition by nicotinamide completely counteracted the protective effect of DECM on chondrocytes in the presence of IL-1β or TNF-α, indicating that the SIRT1 signaling pathway was involved in the DECM-mediated enhancement of anti-inflammatory properties of chondrocytes. Taken together, this work suggests that stem cell-derived DECM is a superior culture substrate for in vitro chondrocyte expansion by improving proliferation and enhancing the anti-inflammatory properties of chondrocytes. DECM-expanded chondrocytes with enhanced anti-inflammatory properties hold great potential in clinically ACI-based cartilage repair.

Osteoarthritis is a chronic degenerative joint disease involving both articular cartilage and subchondral bone. Kartogenin (KGN) was recently identified to improve in vivo cartilage repair; however, its effect on bone formation is unknown. The aim of this study was to investigate the effect of KGN on antioxidant properties and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). Human BM-MSCs were treated with KGN at concentrations ranging from 10 −8 M to 10 −6 M. Our results indicated that KGN improved cell proliferation and attenuated intracellular reactive oxygen species. The levels of antioxidant enzymes and osteogenic differentiation of BM-MSCs were enhanced by KGN in a dose-dependent manner. Furthermore, KGN-treated BM-MSCs showed upregulation of silent information regulator type 1 (SIRT1) and increased phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK), indicating that KGN activated the AMPK-SIRT1 signaling pathway in BM-MSCs. Inhibition of SIRT1 by nicotinamide reversed the antioxidant effect of KGN on BM-MSCs and suppressed osteogenic differentiation. In conclusion, our results demonstrated that KGN improved intracellular antioxidant properties and promoted osteogenic differentiation of BM-MSCs by activating the AMPK-SIRT1 signaling pathway. Thus, KGN may have the potential for not only articular cartilage repair but also the clinical application of MSCs in bone tissue engineering.

Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) is a central event in bone formation. However, oxidative stress has a deleterious impact on BM-MSC osteogenesis. In this study, we hypothesized that oxidative stress influenced BM-MSC osteogenesis differently in the early or late stages, in which silent information regulator type 1 (SIRT1) played a critical role. A continuous exposure to sublethal concentrations of hydrogen peroxide (H2 O2 ), ranging from 25 to 100 µM for 21 days, resulted in the complete inhibition of BM-MSC osteogenesis. We found that a 7-day treatment with H2 O2 inhibited the lineage commitment of BM-MSCs toward osteoblasts, as evidenced by a significant reduction of alkaline phosphatase activity (a typical marker for early osteogenesis). However, moderate oxidative stress did not affect late-differentiated BM-MSCs, as there were comparable levels of matrix mineralization (a typical marker for late osteogenesis). In addition, we observed a spontaneous up-regulation of SIRT1 and intracellular antioxidant enzymes such as superoxide dismutase 2, catalase, and glutathione peroxidase 1, which accounted for the enhanced resistance to oxidative stress upon osteogenic differentiation. Activation of SIRT1 by resveratrol rescued the effect of H2 O2 on early-differentiated BM-MSCs and inhibition of SIRT1 by nicotinamide intensified the effect of H2 O2 on late-differentiated BM-MSCs, indicating that the SIRT1-mediated pathway was actively involved in MSC osteogenesis and antioxidant mechanisms. Our findings uncovered the relationship between SIRT1 and resistance to H2 O2 -induced oxidative stress during BM-MSC osteogenesis, which could provide a new strategy for protecting MSCs from extracellular oxidative stress.

Mesenchymal stem cells (MSCs) are promising cell sources for regenerative medicine. Growing evidence has indicated that mechanical stimuli are crucial for their lineage-specific differentiation. However, the effect of mechanical loading on redox balance and the intracellular antioxidant system in MSCs was unknown. In this study, human bone marrow-derived MSCs (BM-MSCs) were subjected to cyclic stretch at the magnitude of 2.5%, 5%, and 10%. Cell proliferation, intracellular reactive oxygen species (ROS), expression of antioxidant enzymes, and osteogenic differentiation were evaluated. RNA was extracted and subjected to DNA microarray analysis. Sirtinol and compound C were used to investigate the underlying mechanisms involved silent information regulator type 1 (SIRT1) and AMP-activated protein kinase (AMPK). Our results showed that mechanical stretch at appropriate magnitudes increased cell proliferation, up-regulated extracellular matrix organization, and down-regulated matrix disassembly. After 3 days of stretch, intracellular ROS in BM-MSCs were decreased but the levels of antioxidant enzymes, especially superoxide dismutase 1 (SOD1), were up-regulated. Osteogenesis was improved by 5% stretch rather than 10% stretch, as evidenced by increased matrix mineralization and osteogenic marker gene expression. The expression of SIRT1 and phosphorylation of AMPK were enhanced by mechanical stretch; however, inhibition of SIRT1 or AMPK abrogated the stretch-induced antioxidant effect on BM-MSCs and inhibited the stretch-mediated osteogenic differentiation. Our findings reveal that mechanical stretch induced antioxidant responses, attenuated intracellular ROS, and improved osteogenesis of BM-MSCs. The stretch-induced antioxidant effect was through activation of the AMPK-SIRT1 signaling pathway. Our findings demonstrated that appropriate mechanical stimulation can improve MSC antioxidant functions and benefit bone regeneration.

Decellularized extracellular matrix (ECM) derived from stem cells has been shown as a promising biomaterial for bone regeneration because of the promotion effect on osteogenesis in mesenchymal stem cells (MSCs). However, bone regeneration is also influenced by bone resorption and little is known about the effect of cell-derived ECM on osteoclast differentiation. In this study, ECM was deposited by MSCs and, after decellularization, the effect of ECM on osteoclastogenesis of bone marrow monocytes (BMMs) was investigated in comparison to standard tissue culture polystyrene. Our results showed that cell derived ECM improved BMM proliferation but potently inhibited osteoclast differentiation, evidenced by down-regulation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, areas of actin rings, and osteoclast-specific gene expression. ECM-mediated attenuation of intracellular reactive oxygen species (ROS) was suggested to play a rival role in the inhibition of osteoclastogenesis, because exogenous hydrogen peroxide supplementation partially rescued the ECM-inhibited osteoclastogenesis. Furthermore, rather than collagen type I, fibronectin in the ECM contributed to ECM-mediated anti-osteoclastogenesis. In conclusion, stem cell-derived decellularized ECM significantly suppressed osteoclastogenesis via the attenuation of intracellular ROS. The anti-osteoclastogenic property of cell derived ECM may benefit its clinical use for modulating bone remodeling and promoting bone tissue engineering. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.