Regulation of metabolic microenvironment with a nanocomposite hydrogel for improved bone fracture healing.

Bone nonunion poses an urgent clinical challenge that needs to be addressed. Recent studies have revealed that the metabolic microenvironment plays a vital role in fracture healing. Macrophages and bone marrow-derived mesenchymal stromal cells (BMSCs) are important targets for therapeutic interventions in bone fractures. Itaconate is a TCA cycle metabolite that has emerged as a potent macrophage immunomodulator that limits the inflammatory response. During osteogenic differentiation, BMSCs tend to undergo aerobic glycolysis and metabolize glucose to lactate. Copper ion (Cu2+) is an essential trace element that participates in glucose metabolism and may stimulate glycolysis in BMSCs and promote osteogenesis. In this study, we develop a 4-octyl itaconate (4-OI)@Cu@Gel nanocomposite hydrogel that can effectively deliver and release 4-OI and Cu2+ to modulate the metabolic microenvironment and improve the functions of cells involved in the fracture healing process. The findings reveal that burst release of 4-OI reduces the inflammatory response, promotes M2 macrophage polarization, and alleviates oxidative stress, while sustained release of Cu2+ stimulates BMSC glycolysis and osteogenic differentiation and enhances endothelial cell angiogenesis. Consequently, the 4-OI@Cu@Gel system achieves rapid fracture healing in mice. Thus, this study proposes a promising regenerative strategy to expedite bone fracture healing through metabolic reprogramming of macrophages and BMSCs.

Bone-targeting engineered small extracellular vesicles carrying anti-miR-6359-CGGGAGC prevent valproic acid-induced bone loss.

The clinical role and underlying mechanisms of valproic acid (VPA) on bone homeostasis remain controversial. Herein, we confirmed that VPA treatment was associated with decreased bone mass and bone mineral density (BMD) in both patients and mice. This effect was attributed to VPA-induced elevation in osteoclast formation and activity. Through RNA-sequencing, we observed a significant rise in precursor miR-6359 expression in VPA-treated osteoclast precursors in vitro, and further, a marked upregulation of mature miR-6359 (miR-6359) in vivo was demonstrated using quantitative real-time PCR (qRT-PCR) and miR-6359 fluorescent in situ hybridization (miR-6359-FISH). Specifically, the miR-6359 was predominantly increased in osteoclast precursors and macrophages but not in neutrophils, T lymphocytes, monocytes and bone marrow-derived mesenchymal stem cells (BMSCs) following VPA stimulation, which influenced osteoclast differentiation and bone-resorptive activity. Additionally, VPA-induced miR-6359 enrichment in osteoclast precursors enhanced reactive oxygen species (ROS) production by silencing the SIRT3 protein expression, followed by activation of the MAPK signaling pathway, which enhanced osteoclast formation and activity, thereby accelerating bone loss. Currently, there are no medications that can effectively treat VPA-induced bone loss. Therefore, we constructed engineered small extracellular vesicles (E-sEVs) targeting osteoclast precursors in bone and naturally carrying anti-miR-6359 by introducing of EXOmotif (CGGGAGC) in the 3'-end of the anti-miR-6359 sequence. We confirmed that the E-sEVs exhibited decent bone/osteoclast precursor targeting and exerted protective therapeutic effects on VPA-induced bone loss, but not on ovariectomy (OVX) and glucocorticoid-induced osteoporotic models, deepening our understanding of the underlying mechanism and treatment strategies for VPA-induced bone loss.

Melatonin Promotes BMSCs Osteoblastic Differentiation and Relieves Inflammation by Suppressing the NF-κB Pathways.

Bone mesenchymal stem cells (BMSCs) play an important role in maintaining the dynamic balance of bone metabolism. Recent studies have reported that a decrease in the osteogenic function of MSCs is strongly associated with osteoporosis. Melatonin is a neuroendocrine hormone produced in the pineal gland and is essential in the physiological regulation. This study is aimed at exploring the effect of melatonin on MSCs osteoblastic differentiation and elucidate the underlying mechanisms. We isolated BMSCs from rat bone marrow and demonstrated that melatonin improved osteogenic differentiation of BMSCs by the alizarin red staining and ALP staining. We then showed that melatonin enhanced osteogenic gene expression in BMSCs, including ALP, Col 1, OCN, OPN, and RUNX2. We further revealed that melatonin inhibited the inflammatory response of BMSCs by suppressing the NF-κB signaling pathways. In light of this, we found that the NF-κB pathway-specific activator TNF-α activated the NF-κB pathway, inhibited osteogenic differentiation, and induced inflammatory response in BMSCs. Melatonin was found to reverse the inhibitory effect of TNF-α on osteogenic differentiation and inflammation in BMSCs. Taken together, these findings indicated that melatonin may have therapeutic potential to be used for the treatment of osteoporosis.

Recent advances in GelMA hydrogel transplantation for musculoskeletal disorders and related disease treatment.

Increasing data reveals that gelatin that has been methacrylated is involved in a variety of physiologic processes that are important for therapeutic interventions. Gelatin methacryloyl (GelMA) hydrogel is a highly attractive hydrogels-based bioink because of its good biocompatibility, low cost, and photo-cross-linking structure that is useful for cell survivability and cell monitoring. Methacrylated gelatin (GelMA) has established itself as a typical hydrogel composition with extensive biomedical applications. Recent advances in GelMA have focused on integrating them with bioactive and functional nanomaterials, with the goal of improving GelMA's physical, chemical, and biological properties. GelMA's ability to modify characteristics due to the synthesis technique also makes it a good choice for soft and hard tissues. GelMA has been established to become an independent or supplementary technology for musculoskeletal problems. Here, we systematically review mechanism-of-action, therapeutic uses, and challenges and future direction of GelMA in musculoskeletal disorders. We give an overview of GelMA nanocomposite for different applications in musculoskeletal disorders, such as osteoarthritis, intervertebral disc degeneration, bone regeneration, tendon disorders and so on.

Circulating MiRNA-21-enriched extracellular vesicles promote bone remodeling in traumatic brain injury patients.

Fracture combined with traumatic brain injury (TBI) is one of the most common and serious types of compound trauma in the clinic and is characterized by dysfunction of cellular communication in injured organs. Our prior studies found that TBI was capable of enhancing fracture healing in a paracrine manner. Exosomes (Exos), as small extracellular vesicles, are important paracrine vehicles for noncell therapy. However, whether circulating Exos derived from TBI patients (TBI-Exos) regulate the prohealing effects of fractures remains unclear. Thus, the present study aimed to explore the biological effects of TBI-Exos on fracture healing and reveal the potential molecular mechanism. TBI-Exos were isolated by ultracentrifugation, and the enriched miR-21-5 p was identified by qRT‒PCR analysis. The beneficial effects of TBI-Exos on osteoblastic differentiation and bone remodeling were determined by a series of in vitro assays. Bioinformatics analyses were conducted to identify the potential downstream mechanisms of the regulatory effect of TBI-Exos on osteoblasts. Furthermore, the role of the potential signaling pathway of TBI-Exos in mediating the osteoblastic activity of osteoblasts was assessed. Subsequently, a murine fracture model was established, and the effect of TBI-Exos on bone modeling was demonstrated in vivo. TBI-Exos can be internalized by osteoblasts, and in vitro, suppression of SMAD7 promoted osteogenic differentiation, whereas knockdown of miR-21-5 p in TBI-Exos strongly inhibited this bone-beneficial effect. Similarly, our results confirmed that preinjection of TBI-Exos led to enhanced bone formation, whereas knockdown of exosomal miR-21-5 p substantially impaired this bone-beneficial effect in vivo.

A Whole-Course-Repair System Based on Neurogenesis-Angiogenesis Crosstalk and Macrophage Reprogramming Promotes Diabetic Wound Healing.

Diabetic wound (DW) therapy is currently a big challenge in medicine and strategies to enhance neurogenesis and angiogenesis have appeared to be a promising direction. However, the current treatments have failed to coordinate neurogenesis and angiogenesis simultaneously, leading to an increased disability rate caused by DWs. Herein, a whole-course-repair system is introduced by a hydrogel to concurrently achieve a mutually supportive cycle of neurogenesis-angiogenesis under a favorable immune-microenvironment. This hydrogel can first be one-step packaged in a syringe for later in situ local injections to cover wounds long-termly for accelerated wound healing via the synergistic effect of magnesium ions (Mg2+ ) and engineered small extracellular vesicles (sEVs). The self-healing and bio-adhesive properties of the hydrogel make it an ideal physical barrier for DWs. At the inflammation stage, the formulation can recruit bone marrow-derived mesenchymal stem cells to the wound sites and stimulate them toward neurogenic differentiation, while providing a favorable immune microenvironment via macrophage reprogramming. At the proliferation stage of wound repair, robust angiogenesis occurs by the synergistic effect of the newly differentiated neural cells and the released Mg2+ , allowing a regenerative neurogenesis-angiogenesis cycle to take place at the wound site. This whole-course-repair system provides a novel platform for combined DW therapy.

Hallmarks of peripheral nerve function in bone regeneration.

Skeletal tissue is highly innervated. Although different types of nerves have been recently identified in the bone, the crosstalk between bone and nerves remains unclear. In this review, we outline the role of the peripheral nervous system (PNS) in bone regeneration following injury. We first introduce the conserved role of nerves in tissue regeneration in species ranging from amphibians to mammals. We then present the distribution of the PNS in the skeletal system under physiological conditions, fractures, or regeneration. Furthermore, we summarize the ways in which the PNS communicates with bone-lineage cells, the vasculature, and immune cells in the bone microenvironment. Based on this comprehensive and timely review, we conclude that the PNS regulates bone regeneration through neuropeptides or neurotransmitters and cells in the peripheral nerves. An in-depth understanding of the roles of peripheral nerves in bone regeneration will inform the development of new strategies based on bone-nerve crosstalk in promoting bone repair and regeneration.

The role of the immune microenvironment in bone, cartilage, and soft tissue regeneration: from mechanism to therapeutic opportunity.

Bone, cartilage, and soft tissue regeneration is a complex spatiotemporal process recruiting a variety of cell types, whose activity and interplay must be precisely mediated for effective healing post-injury. Although extensive strides have been made in the understanding of the immune microenvironment processes governing bone, cartilage, and soft tissue regeneration, effective clinical translation of these mechanisms remains a challenge. Regulation of the immune microenvironment is increasingly becoming a favorable target for bone, cartilage, and soft tissue regeneration; therefore, an in-depth understanding of the communication between immune cells and functional tissue cells would be valuable. Herein, we review the regulatory role of the immune microenvironment in the promotion and maintenance of stem cell states in the context of bone, cartilage, and soft tissue repair and regeneration. We discuss the roles of various immune cell subsets in bone, cartilage, and soft tissue repair and regeneration processes and introduce novel strategies, for example, biomaterial-targeting of immune cell activity, aimed at regulating healing. Understanding the mechanisms of the crosstalk between the immune microenvironment and regeneration pathways may shed light on new therapeutic opportunities for enhancing bone, cartilage, and soft tissue regeneration through regulation of the immune microenvironment.

The clinical efficacy and safety of double reverse traction repositor and traction table in the reduction of unstable intertrochanteric fractures in elderly patients: a retrospective comparative study.

Background: Currently, we found that double reverse traction repositor (DRTR) is a treatment with operation convenience and fast in our clinical work. However, the clinical efficacy and safety of DRTR in the reduction of unstable intertrochanteric fractures in elderly patients remain unknown. Therefore, the study aimed to compare the clinical efficacy and safety of DRTR and traction table (TT) in the reduction of unstable intertrochanteric fractures in elderly patients. Methods: From October 2018 to December 2020, the elderly patients with unstable intertrochanteric fractures were reviewed. 22 patients treated with TT and 20 patients treated with DRTR met the inclusion criteria of this study, and baseline clinical characteristics were recorded. The reduction time, operation time, incision length and intraoperative blood loss were reviewed. The safety outcome was assessed by postoperative complications, and the efficacy outcomes were evaluated by the fracture healing time based on the radiographs conducted at each follow-up (1, 3, 6, 12 months after surgery) and hip function (hip flexion, Harris Hip Score) at the final follow-up (12 months after surgery). Results: There were no significant differences in terms of demographics and fracture characteristics of cases enrolled. In DRTR group, the average intraoperative reduction time [(34.8±7.6) min] and the average operation time [(87.1±12.2) min] were superior to those [(56.6±9.3); (123.1±15.0) min] in TT group (P<0.0001). However, there were no statistical significance in terms of the average incision lengths [(6.4±0.9) vs. (6.8±1.1) cm; P=0.1619], , the average intraoperative blood loss [(152.6±22.9) vs. (146.8±20.7) mL; P=0.3941], the average fracture healing times [(13.8±1.5) vs. (14.4±1.8) weeks; P=0.2350] and the average Harris hip score a year after operation [(84.4±6.6) vs. (82.7±7.2); P=0.4496] between the two groups. One patient in TT group experienced lower extremity intermuscular venous thrombosis postoperatively. No other operation-related complications were observed postoperatively nor during follow-up. Conclusions: Minimally invasive reduction with DRTR in unstable intertrochanteric fractures could effectively shorten the intraoperative reduction time and operation time in this study. Therefore, minimally invasive reduction with DRTR might be a good choice for intertrochanteric reduction of unstable intertrochanteric fractures.

Advances in the therapeutic application and pharmacological properties of kinsenoside against inflammation and oxidative stress-induced disorders.

Extensive research has implicated inflammation and oxidative stress in the development of multiple diseases, such as diabetes, hepatitis, and arthritis. Kinsenoside (KD), a bioactive glycoside component extracted from the medicinal plant Anoectochilus roxburghii, has been shown to exhibit potent anti-inflammatory and anti-oxidative abilities. In this review, we summarize multiple effects of KD, including hepatoprotection, pro-osteogenesis, anti-hyperglycemia, vascular protection, immune regulation, vision protection, and infection inhibition, which are partly responsible for suppressing inflammation signaling and oxidative stress. The protective action of KD against dysfunctional lipid metabolism is also associated with limiting inflammatory signals, due to the crosstalk between inflammation and lipid metabolism. Ferroptosis, a process involved in both inflammation and oxidative damage, is potentially regulated by KD. In addition, we discuss the physicochemical properties and pharmacokinetic profiles of KD. Advances in cultivation and artificial synthesis techniques are promising evidence that the shortage in raw materials required for KD production can be overcome. In addition, novel drug delivery systems can improve the in vivo rapid clearance and poor bioavailability of KD. In this integrated review, we aim to offer novel insights into the molecular mechanisms underlying the therapeutic role of KD and lay solid foundations for the utilization of KD in clinical practice.

Metformin alleviates bone loss in ovariectomized mice through inhibition of autophagy of osteoclast precursors mediated by E2F1.

BACKGROUND: Postmenopausal bone loss, mainly caused by excessive bone resorption mediated by osteoclasts, has become a global public health burden. Metformin, a hypoglycemic drug, has been reported to have beneficial effects on maintaining bone health. However, the role and underlying mechanism of metformin in ovariectomized (OVX)-induced bone loss is still vague. RESULTS: In this study, we demonstrated for the first time that metformin administration alleviated bone loss in postmenopausal women and ovariectomized mice, based on reduced bone resorption markers, increased bone mineral density (BMD) and improvement of bone microstructure. Then, osteoclast precursors administered metformin in vitro and in vivo were collected to examine the differentiation potential and autophagical level. The mechanism was investigated by infection with lentivirus-mediated BNIP3 or E2F1 overexpression. We observed a dramatical inhibition of autophagosome synthesis and osteoclast formation and activity. Treatment with RAPA, an autophagy activator, abrogated the metformin-mediated autophagy downregulation and inhibition of osteoclastogenesis. Additionally, overexpression of E2F1 demonstrated that reduction of OVX-upregulated autophagy mediated by metformin was E2F1 dependent. Mechanistically, metformin-mediated downregulation of E2F1 in ovariectomized mice could downregulate BECN1 and BNIP3 levels, which subsequently perturbed the binding of BECN1 to BCL2. Furthermore, the disconnect between BECN1 and BCL2 was shown by BNIP3 overexpression. CONCLUSION: In summary, we demonstrated the effect and underlying mechanism of metformin on OVX-induced bone loss, which could be, at least in part, ascribed to its role in downregulating autophagy during osteoclastogenesis via E2F1-dependent BECN1 and BCL2 downregulation, suggesting that metformin or E2F1 inhibitor is a potential agent against postmenopausal bone loss. Video abstract.

Traction-bed-assisted reduction and double-plate fixation for treatment of comminuted femoral intertrochanteric fractures with coronal split.

Background: A coronal comminuted femoral intertrochanteric fracture is a special type of fracture that easily leads to internal fixation failure, and the current internal fixation techniques remain controversial. This study aims to evaluate the effect of traction-bed-assisted reduction and double-plate internal fixation in the treatment of comminuted and coronally split intertrochanteric femoral fracture. Method: Retrospective analyses of the clinical data of 83 patients diagnosed with, and treated for, comminuted and coronally split intertrochanteric femoral fracture from December 2017 to November 2019 were conducted. Among the total number of 83 patients, 40 patients received traction-bed-assisted reduction and PFNA fixation (the control group), whereas 43 patients received traction-bed-assisted reduction and double-plate internal fixation (the experimental group). The major indicators for the research analysis such as the general information of patients, perioperative data, and follow-up data of both groups were collected, sorted out, and meticulously analyzed. Results: The time taken for traction-bed-assisted reduction and double-plate intern fixation in the experimental group was significantly shorter than that in the control group (P < .05). The post-operative Harris Hip Score (HHS) at 3 months and at the final follow-up after the surgery was significantly better in the experimental group compared with that in the control group, both of which were statistically significant (P < .05). However, there were statistically no significant differences between the two groups in terms of preoperative hemoglobin (Hb) level, amount of intraoperative total blood loss, immediate post-operative Hb level, incidence of wound infection within 14 days post-operatively, time taken to step up on the ground after surgery, HHS 2 weeks after surgery, time taken for fracture healing, and the incidence of complications (P > .05). Conclusion: The use of a traction bed to achieve adequate reduction, followed by internal fixation using double plates, comparatively takes less time for both reduction and operation in the treatment of comminuted and coronally split intertrochanteric femoral fractures, which also restores proper hip joint movements relatively early and hence provides better hip joint functions in the long run.

The Regulatory Role of Ferroptosis in Bone Homeostasis.

Ferroptosis is an iron-dependent form of programmed cell death and an important type of biological catabolism. Through the action of divalent iron or ester oxygenase, ferroptosis can induce lipid peroxidation and cell death, regulating a variety of physiological processes. The role of ferroptosis in the modulation of bone homeostasis is a significant topic of interest. Herein, we review and discuss recent studies exploring the mechanisms and functions of ferroptosis in different bone-related cells, including mesenchymal stem cells, osteoblasts, osteoclasts, and osteocytes. The association between ferroptosis and disorders of bone homeostasis is also explored in this review. Overall, we aim to provide a detailed overview of ferroptosis, summarizing recent understanding on its role in regulation of bone physiology and bone disease pathogenesis.

MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2.

MicroRNAs (miRNAs) broadly regulate normal biological functions of bone and the progression of fracture healing and osteoporosis. Recently, it has been reported that miR-1224-5p in fracture plasma is a potential therapy for osteogenesis. To investigate the roles of miR-1224-5p and the Rap1 signaling pathway in fracture healing and osteoporosis development and progression, we used BMMs, BMSCs, and skull osteoblast precursor cells for in vitro osteogenesis and osteoclastogenesis studies. Osteoblastogenesis and osteoclastogenesis were detected by ALP, ARS, and TRAP staining and bone slice resorption pit assays. The miR-1224-5p target gene was assessed by siRNA-mediated target gene knockdown and luciferase reporter assays. To explore the Rap1 pathway, we performed high-throughput sequencing, western blotting, RT-PCR, chromatin immunoprecipitation assays and immunohistochemical staining. In vivo, bone healing was judged by the cortical femoral defect, cranial bone defect and femoral fracture models. Progression of osteoporosis was evaluated by an ovariectomy model and an aged osteoporosis model. We discovered that the expression of miR-1224-5p was positively correlated with fracture healing progression. Moreover, in vitro, overexpression of miR-1224-5p slowed Rankl-induced osteoclast differentiation and promoted osteoblast differentiation via the Rap1-signaling pathway by targeting ADCY2. In addition, in vivo overexpression of miR-1224-5p significantly promoted fracture healing and ameliorated the progression of osteoporosis caused by estrogen deficiency or aging. Furthermore, knockdown of miRNA-1224-5p inhibited bone regeneration in mice and accelerated the progression of osteoporosis in elderly mice. Taken together, these results identify miR-1224-5p as a key bone osteogenic regulator, which may be a potential therapeutic target for osteoporosis and fracture nonunion.

Enhanced tissue regeneration through immunomodulation of angiogenesis and osteogenesis with a multifaceted nanohybrid modified bioactive scaffold.

Major traumatic tissue defects are common clinical problems often complicated by infection and local vascular dysfunction, processes which hinder the healing process. Although local application of growth factors or stem cells through various tissue engineering techniques are promising methods for the repair of tissue defects, limitations in their clinical application exist. Herein, we synthesized multifaceted nanohybrids composed of Quaternized chitosan (QCS), Graphene oxide (GO), and Polydopamine (PDA; QCS-GO-PDA). Covalent grafting of QCS and GO at a mass ratio of 5:1 (5QCS-1GO) displayed excellent biocompatibility and enhanced osteogenic ability, while addition of PDA (5QCS-1GO-PDA) reduced the level of reactive oxygen species (ROS). 5QCS-1GO-PDA was able to achieve wound tissue regeneration by reducing the inflammatory response and enhancing angiogenesis. Furthermore, Polylactic acid/hydroxyapatite (PLA/HA) composite scaffolds were printed using Selective Laser Sintering (SLS) and the hybrid nanomaterial (5QCS-1GO-PDA) was used to coat the PLA/HA scaffold (5QCS-1GO-PDA@PLA/HA) to be used for rapid bone regeneration. 5QCS-1GO-PDA not only improved angiogenesis and osteogenic differentiation, but also induced M2-type polarization of macrophages and promoted bone regeneration via the BMP2/BMPRs/Smads/Runx2 signaling pathway. The bidirectional enhanced healing ability of the multifaceted nanohybrids 5QCS-1GO-PDA provides a promising method of effectively treating tissue defects.

SHIP1 Activator AQX-1125 Regulates Osteogenesis and Osteoclastogenesis Through PI3K/Akt and NF-κb Signaling.

With the worldwide aging population, the prevalence of osteoporosis is on the rise, particularly the number of postmenopausal women with the condition. However, the various adverse side effects associated with the currently available treatment options underscore the need to develop novel therapies. In this study, we investigated the use of AQX-1125, a novel clinical-stage activator of inositol phosphatase-1 (SHIP1), in ovariectomized (OVX) mice, identifying a protective role. We then found that the effect was likely due to increased osteogenesis and mineralization and decreased osteoclastogenesis caused by AQX-1125 in a time- and dose-dependent manner. The effect against OVX-induced bone loss was identified to be SHIP1-dependent as pretreatment of BMSCs and BMMs with SHIP1 RNAi could greatly diminish the osteoprotective effects. Furthermore, SHIP1 RNAi administration in vivo induced significant bone loss and decreased bone mass. Mechanistically, AQX-1125 upregulated the expression level and activity of SHIP1, followed upregulating the phosphorylation levels of PI3K and Akt to promote osteoblast-related gene expressions, including Alp, cbfa1, Col1a1, and osteocalcin (OCN). NF-κB signaling was also inhibited through suppression of the phosphorylation of IκBα and P65 induced by RANKL, resulting in diminished osteoclastogenesis. Taken together, our results demonstrate that AQX-1125 may be a promising candidate for preventing and treating bone loss.