Supramolecular Hydrogel with Ultra-Rapid Cell-Mediated Network Adaptation for Enhancing Cellular Metabolic Energetics and Tissue Regeneration.

Cellular energetics plays an important role in tissue regeneration, and the enhanced metabolic activity of delivered stem cells can accelerate tissue repair and regeneration. However, conventional hydrogels with limited network cell adaptability restrict cell-cell interactions and cell metabolic activities. In this work, it is shown that a cell-adaptable hydrogel with high network dynamics enhances the glucose uptake and fatty acid ß-oxidation of encapsulated human mesenchymal stem cells (hMSCs) compared with a hydrogel with low network dynamics. It is further shown that the hMSCs encapsulated in the high dynamic hydrogels exhibit increased tricarboxylic acid (TCA) cycle activity, oxidative phosphorylation (OXPHOS), and adenosine triphosphate (ATP) biosynthesis via an E-cadherin- and AMP-activated protein kinase (AMPK)-dependent mechanism. The in vivo evaluation further showed that the delivery of MSCs by the dynamic hydrogel enhanced in situ bone regeneration in an animal model. It is believed that the findings provide critical insights into the impact of stem cell-biomaterial interactions on cellular metabolic energetics and the underlying mechanisms.

Low-Dose Staphylococcal Enterotoxin C2 Mutant Maintains Bone Homeostasis via Regulating Crosstalk between Bone Formation and Host T-Cell Effector Immunity.

Studies in recent years have highlighted an elaborate crosstalk between T cells and bone cells, suggesting that T cells may be alternative therapeutic targets for the maintenance of bone homeostasis. Here, it is reported that systemic administration of low-dose staphylococcal enterotoxin C2 (SEC2) 2M-118, a form of mutant superantigen, dramatically alleviates ovariectomy (OVX)-induced bone loss via modulating T cells. Specially, SEC2 2M-118 treatment increases trabecular bone mass significantly via promoting bone formation in OVX mice. These beneficial effects are largely diminished in T-cell-deficient nude mice and can be rescued by T-cell reconstruction. Neutralizing assays determine interferon gamma (IFN-γ) as the key factor that mediates the beneficial effects of SEC2 2M-118 on bone. Mechanistic studies demonstrate that IFN-γ stimulates Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling, leading to enhanced production of nitric oxide, which further activates p38 mitogen-activated protein kinase (MAPK) and Runt-related transcription factor 2 (Runx2) signaling and promotes osteogenic differentiation. IFN-γ also directly inhibits osteoclast differentiation, but this effect is counteracted by proabsorptive factors tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß) secreted from IFN-γ-stimulated macrophages. Taken together, this work provides clues for developing innovative approaches which target T cells for the prevention and treatment of osteoporosis.

O-alg-THAM/gel hydrogels functionalized with engineered microspheres based on mesenchymal stem cell secretion recruit endogenous stem cells for cartilage repair.

Lacking self-repair abilities, injuries to articular cartilage can lead to cartilage degeneration and ultimately result in osteoarthritis. Tissue engineering based on functional bioactive scaffolds are emerging as promising approaches for articular cartilage regeneration and repair. Although the use of cell-laden scaffolds prior to implantation can regenerate and repair cartilage lesions to some extent, these approaches are still restricted by limited cell sources, excessive costs, risks of disease transmission and complex manufacturing practices. Acellular approaches through the recruitment of endogenous cells offer great promise for in situ articular cartilage regeneration. In this study, we propose an endogenous stem cell recruitment strategy for cartilage repair. Based on an injectable, adhesive and self-healable o-alg-THAM/gel hydrogel system as scaffolds and a biophysio-enhanced bioactive microspheres engineered based on hBMSCs secretion during chondrogenic differentiation as bioactive supplement, the as proposed functional material effectively and specifically recruit endogenous stem cells for cartilage repair, providing new insights into in situ articular cartilage regeneration.

Long Non-Coding RNA Malat1 Increases the Rescuing Effect of Quercetin on TNFα-Impaired Bone Marrow Stem Cell Osteogenesis and Ovariectomy-Induced Osteoporosis.

Osteoporosis, a common systematic bone homeostasis disorder related disease, still urgently needs innovative treatment methods. Several natural small molecules were found to be effective therapeutics in osteoporosis. In the present study, quercetin was screened out from a library of natural small molecular compounds by a dual luciferase reporter system. Quercetin was found to upregulate Wnt/ß-catenin while inhibiting NF-κB signaling activities, and thereby rescuing osteoporosis-induced tumor necrosis factor alpha (TNFα) impaired BMSCs osteogenesis. Furthermore, a putative functional lncRNA, Malat1, was shown to be a key mediator in quercetin regulated signaling activities and TNFα-impaired BMSCs osteogenesis, as mentioned above. In an ovariectomy (OVX)-induced osteoporosis mouse model, quercetin administration could significantly rescue OVX-induced bone loss and structure deterioration. Serum levels of Malat1 were also obviously rescued in the OVX model after quercetin treatment. In conclusion, our study demonstrated that quercetin could rescue TNFα-impaired BMSCs osteogenesis in vitro and osteoporosis-induced bone loss in vivo, in a Malat1-dependent manner, suggesting that quercetin may serve as a therapeutic candidate for osteoporosis treatment.

Alleviation of osteoarthritis by intra-articular transplantation of circulating mesenchymal stem cells.

This study aimed to evaluate the efficacy of intra-articular delivery of peripheral blood derived mesenchymal stromal cells (PB-MSCs) on the progression of trauma-induced osteoarthritis (OA) in mice. Adult male C57BL/6J mice subjected to destabilization of the medial meniscus surgeries (DMM) were randomly divided into four groups: sham surgery group; vehicle control group (treated with saline), PBMSC-treated group, or adipose tissue derived MSCs (AD-MSC)-treated group (n = 4 per group). PB-MSCs and AD-MSCs were harvested and cultured following previously established protocols, and pre-labeled with BrdU for 48 h before transplantation. PB-MSCs or AD-MSCs (5 × 105 cells/mouse; passage 3-5) were intra-articular injected into the right knee joints thrice post-surgery. The mice were euthanized at 8 weeks post-surgery and knee joint samples were collected for micro-CT and histological examinations. PB-MSCs administration significantly reduced subchondral bone volume comparing to the vehicle control group. Safranin O staining showed that PB-MSCs treatment ameliorated degeneration of articular cartilage, which was comparable to AD-MSCs treatment. The expression of catabolic marker MMP13 was significantly reduced in articular cartilage of the PB-MSCs treated group comparing to that of the vehicle control group. Co-expression of BrdU and Sox9 indicated that injected PB-MSCs differentiated in chondrocytes in situ, along with reduced levels of IL-6 within peripheral sera of PB-MSCs- and AD-MSCs-treated mice. Therefore, administration of PB-MSCs or AD-MSCs attenuated trauma-induced OA progression through inhibiting cartilage degradation and inflammation. PB-MSCs are ideal cell source for treating cartilage-associated diseases.

Malat1 attenuated the rescuing effects of docosahexaenoic acid on osteoarthritis treatment via repressing its chondroprotective and chondrogenesis activities.

Osteoarthritis (OA) is a degenerative disease associated with joint inflammation, articular cartilage degeneration and subchondral hypertrophy. Small molecules which both ameliorate chondrocyte OA phenotype and activate bone marrow-derived mesenchymal stem cells (BMSCs) chondrogenesis under inflammatory conditions have the therapeutical potential for OA treatment. In this study, we characterized a novel small molecule which could ameliorate OA progression via novel regulating mechanisms. Docosahexaenoic acid (DHA), a bioactive molecule, was screened from a small molecule library and showed anti-inflammatory and chondroprotective effects in OA chondrocytes, as well as ameliorated IL-1ß impaired BMSCs chondrogenesis in Wnt/ß-catenin and NF-κB signaling dependent manners. Furthermore, Malat1 was found to be the key mediator of DHA-mediating anti-inflammation chondroprotection and chondrogenesis. DHA also rescued cartilage loss and damage in a surgery-induced OA mice model. The elevation of serum Malat1 levels caused by OA was also downregulated by DHA treatment. Taken together, our findings demonstrated that DHA, with a dual-signaling repression property, exerted its anti-inflammation, chondroprotection and chondrogenesis function possibly via regulating Malat1 level, suggesting that it may be a possible drug candidate for OA patients with elevated MALAT1 expression levels.

Sesamin Promotes Osteoporotic Fracture Healing by Activating Chondrogenesis and Angiogenesis Pathways.

Osteoporotic fracture has been regarded as one of the most common bone disorders in the aging society. The natural herb-derived small molecules were revealed as potential treatment approaches for osteoporotic fracture healing. Sesamin is a member of lignan family, which possesses estrogenic activity and plays a significant role in modulating bone homeostasis. Our previous study reported the promoting effect of sesamin on postmenopausal osteoporosis treatment. However, the role of sesamin in osteoporotic fracture healing has not been well studied yet. In this study, we further investigated the putative treatment effect of sesamin on osteoporotic fracture healing. Our study indicated that sesamin could activate bone morphogenetic protein 2 (BMP2) signaling pathway and further promotes in vitro chondrogenesis and angiogenesis activities. This promoting effect was abolished by the treatment of ERα inhibitor. In the osteoporotic bone fracture model, we demonstrated that sesamin markedly improves the callus formation and increases the cartilaginous area at the early-stage, as well as narrowing the fracture gap, and expands callus volume at the late-stage fracture healing site of the OVX mice femur. Furthermore, the angiogenesis at the osteoporotic fracture site was also significantly improved by sesamin treatment. In conclusion, our research illustrated the therapeutic potential and underlying regulation mechanisms of sesamin on osteoporotic fracture healing. Our studies shed light on developing herb-derived bioactive compounds as novel drugs for the treatment of osteoporotic fracture healing, especially for postmenopausal women with low estrogen level.

MicroRNA-378 contributes to osteoarthritis by regulating chondrocyte autophagy and bone marrow mesenchymal stem cell chondrogenesis.

Osteoarthritis (OA) is the most common joint disease; thus, understanding the pathological mechanisms of OA initiation and progression is critical for OA treatment. MicroRNAs (miRNAs) have been shown to be involved in the progression of osteoarthritis, one candidate is microRNA-378 (miR-378), which is highly expressed in the synovium of OA patients during late-stage disease, but its function and the underlying mechanisms of how it contributes to disease progression remain poorly understood. In this study, miR-378 transgenic (TG) mice were used to study the role of miR-378 in OA development. miR-378 TG mice developed spontaneous OA and also exaggerated surgery-induced disease progression. Upon in vitro OA induction, miR-378 expression was upregulated and correlated with elevated inflammation and chondrocyte hypertrophy. Chondrocytes isolated from articular cartilage from miR-378 TG mice showed impaired chondrogenic differentiation. The bone marrow mesenchymal stem cells (BMSCs) collected from miR-378 TG mice also showed repressed chondrogenesis compared with the control group. The autophagy-related protein Atg2a, as well as chondrogenesis regulator Sox6, were identified as downstream targets of miR-378. Ectopic expression of Atg2a and Sox6 rescued miR-378-repressed chondrocyte autophagy and BMSC chondrogenesis, respectively. Anti-miR-378 lentivirus intra-articular injection in an established OA mouse model was shown to ameliorate OA progression, promote articular regeneration, and repress hypertrophy. Atg2a and Sox6 were again confirmed to be the target of miR-378 in vivo. In conclusion, miR-378 amplified OA development via repressing chondrocyte autophagy and by inhibiting BMSCs chondrogenesis, thus indicating miR-378 may be a potential therapeutic target for OA treatments.

Bisphosphonate-based hydrogel mediates biomimetic negative feedback regulation of osteoclastic activity to promote bone regeneration.

The intricate dynamic feedback mechanisms involved in bone homeostasis provide valuable inspiration for the design of smart biomaterial scaffolds to enhance in situ bone regeneration. In this work, we assembled a biomimetic hyaluronic acid nanocomposite hydrogel (HA-BP hydrogel) by coordination bonds with bisphosphonates (BPs), which are antiosteoclastic drugs. The HA-BP hydrogel exhibited expedited release of the loaded BP in response to an acidic environment. Our in vitro studies showed that the HA-BP hydrogel inhibits mature osteoclastic differentiation of macrophage-like RAW264.7 cells via the released BP. Furthermore, the HA-BP hydrogel can support the initial differentiation of primary macrophages to preosteoclasts, which are considered essential during bone regeneration, whereas further differentiation to mature osteoclasts is effectively inhibited by the HA-BP hydrogel via the released BP. The in vivo evaluation showed that the HA-BP hydrogel can enhance the in situ regeneration of bone. Our work demonstrates a promising strategy to design biomimetic biomaterial scaffolds capable of regulating bone homeostasis to promote bone regeneration.

Cranial Bone Transport Promotes Angiogenesis, Neurogenesis, and Modulates Meningeal Lymphatic Function in Middle Cerebral Artery Occlusion Rats.

BACKGROUND: Ischemic stroke is a leading cause of death and disability worldwide. However, the time window for quickly dissolving clots and restoring cerebral blood flow, using tissue-type plasminogen activator treatment is rather limited, resulting in many patients experiencing long-term functional impairments if not death. This study aims to determine the roles of cranial bone transport (CBT), a novel, effective, and simple surgical technique, in the recovery of ischemic stroke using middle cerebral artery occlusion (MCAO) rat model. METHODS: CBT was performed by slowly sliding a bone segment in skull with a special frame and a speed of 0.25 mm/12 hours for 10 days following MCAO. Morris water maze, rotarod test, and catwalk gait analysis were used to study the neurological behaviors, and infarct area and cerebral flow were evaluated during CBT process. Immunofluorescence staining of CD31 and Nestin/Sox2 (sex determining region Y box 2) was performed to study the angiogenesis and neurogenesis. OVA-A647 (ovalbumin-Alexa Fluor 647) was intracisterna magna injected to evaluate the meningeal lymphatic drainage function. RESULTS: CBT treatment has significantly reduced the ischemic lesions areas and improved the neurological deficits in MCAO rats compared with the rats in the control groups. CBT treatment significantly promoted angiogenesis and neurogenesis in the brain of MCAO rats. The drainage function of meningeal lymphatic vessels in MCAO rats was significantly impaired compared with normal rats. Ablation of meningeal lymphatic drainage led to increased neuroinflammation and aggravated neurological deficits and ischemic injury in MCAO rats. CBT treatment significantly improved the meningeal lymphatic drainage function and alleviated T-cell infiltration in MCAO rats. CONCLUSIONS: This study provided evidence for the possible mechanisms on how CBT attenuates ischemic stroke injury and facilitates rapid neuronal function recovery, suggesting that CBT may be an alternative treatment strategy for managing ischemic stroke.

DANCR Mediates the Rescuing Effects of Sesamin on Postmenopausal Osteoporosis Treatment via Orchestrating Osteogenesis and Osteoclastogenesis.

As one of the leading causes of bone fracture in postmenopausal women and in older men, osteoporosis worldwide is attracting more attention in recent decades. Osteoporosis is a common disease mainly resulting from an imbalance of bone formation and bone resorption. Pharmaceutically active compounds that both activate osteogenesis, while repressing osteoclastogenesis hold the potential of being therapeutic medications for osteoporosis treatment. In the present study, sesamin, a bioactive ingredient derived from the seed of Sesamum Indicum, was screened out from a bioactive compound library and shown to exhibit dual-regulating functions on these two processes. Sesamin was demonstrated to promote osteogenesis by upregulating Wnt/ß-catenin, while repressing osteoclastogenesis via downregulating NF-κB signaling . Furthermore, DANCR was found to be the key regulator in sesamin-mediated bone formation and resorption . In an ovariectomy (OVX)-induced osteoporotic mouse model, sesamin could rescue OVX-induced bone loss and impairment. The increased serum level of DANCR caused by OVX was also downregulated upon sesamin treatment. In conclusion, our results demonstrate that sesamin plays a dual-functional role in both osteogenesis activation and osteoclastogenesis de-activation in a DANCR-dependent manner, suggesting that it may be a possible medication candidate for osteoporotic patients with elevated DNACR expression levels.

Asiatic acid protects articular cartilage through promoting chondrogenesis and inhibiting inflammation and hypertrophy in osteoarthritis.

Natural small molecules have become attractive in osteoarthritis (OA) treatment. This study aims to investigate the effect of asiatic acid (AA) on OA development in vitro and in vivo. Chondrocytes were pretreated with AA at optimized concentrations and subsequently treated with interleukin-1 beta (IL-1ß). Inflammatory mediator nitric oxide (NO) was measured by Griess method. The mRNA expression level of inflammatory markers nitric oxide synthase (iNOS) and cyclooxygenase 2 (Cox2), as well as chondrogenic or hypertrophic markers including SRY-box transcription factor 9 (Sox9), Aggrecan, Collagen 2a1 (Col II), and Matrix metalloproteinase-13 (Mmp13) were measured by using real-time PCR analysis. The nuclear factor-kappa B (NF-κB) signaling activity was determined by dual luciferase assay and Western blot analysis. Surgery-induced OA animal model was constructed, and AA was administrated to study its effect on OA pathogenesis. AA induced a dose-dependent inhibitory effect up to -67.4% on NO production. AA could repress iNOS and Cox2 protein expression levels (-77.2% and -73.4%, respectively) in IL-1ß induced chondrocytes. AA increased the formation of cartilage extracellular matrix components including glycosaminoglycans (GAGs) and collagen type II. AA also mRNA expression of chondrogenesis marker including Aggrecan, Sox9, Col II and Fibronectin (402.87%, 151.04%, 314.15% and 187.76%, respectively) as well as hypertrophic marker Mmp13 (-67.8%). AA repressed the chondrocyte inflammation by directly inhibiting NF-κB signaling activity, which was revealed by the inhibition effect of AA on IκBα phosphorylation (-105.4%) and NF-κB/p65 translocation (-60.9%) induced by IL-1ß. Furthermore, In vivo OA study indicated the protective effect of AA on OA progression by preventing articular cartilage from degeneration and destruction. AA treatment could significantly reduce OA score (16.125 vs 5.25) and repress mRNA expression level of Mmp13 and Col X (23.5, vs 2.375 and 18.125 vs 94.5). Taken together, our findings suggest that AA could effectively rescue IL-1ß induced chondrocytes and protected cartilage in OA progression, which shed light on a potential novel therapeutic strategy of OA treatment.

Vasoactive Intestinal Peptide Promotes Fracture Healing in Sympathectomized Mice.

Vasoactive intestinal peptide (VIP) as a neuromodulator and neurotransmitter played a significant role in modulating bone homeostasis. Our previous study reported an essential role of VIP in in vitro BMSCs osteogenesis and in vivo bone defect repair. VIP was also revealed to have a promoting effect on embryonic skeletal element development. However, the role of VIP in fracture healing is not known yet. We hypothesized that the disorder of sympathetic nervous system impairs bone structure and fracture healing, whereas VIP may rescue the sympathetic inhibition effects and promote fracture healing. We employed a 6-hydroxydopamine (6-OHDA) induced sympathectomy mice model (sympathectomized mice), in which successful sympathetic inhibition was confirmed by a decreased level of norephedrine (NE) in the spleen. In the sympathectomized mice, the femoral micro-architecture, bone density and mechanical properties were all impaired compared to the vehicle control mice. The femoral fracture was created in the vehicle or sympathectomized mice. Vehicle mice were locally injected with PBS as a negative control, and the sympathectomized mice were treated with injection of PBS or VIP. VIP expression at the fracture site was significantly decreased in sympathectomized mice. The fracture healing was repressed upon 6-OHDA treatment and rescued by VIP treatment. Micro-CT examination showed that the femoral bone micro-architecture at the fracture sites and mechanical properties were all impaired. Simultaneously, the expression level of osteogenic markers OCN and OPN were reduced in sympathectomized mice compared with vehicle group. While the VIP treatment rescued the repression effects of 6-OHDA on bone remodeling and significantly promoted bone quality and mechanical properties as well as increased osteogenesis marker expression in the sympathectomized mice. VIP administration promoted bone fracture healing by inhibiting bone resorption, making it a putative new alternative treatment strategy for fracture healing.

Local administration of allogeneic or autologous bone marrow-derived mesenchymal stromal cells enhances bone formation similarly in distraction osteogenesis.

BACKGROUND AIMS: Distraction osteogenesis (DO) is a surgical technique to promote bone regeneration that requires a long time for bone healing. Bone marrow-derived mesenchymal stromal cells (MSCs) have been applied to accelerate bone formation in DO. Allogeneic MSCs are attractive, as they could be ready to use in clinics. Whether allogeneic MSCs would have an effect similar to autologous MSCs with regard to promoting bone formation in DO is still unknown. This study compares the effect of autologous MSCs versus allogeneic MSCs on bone formation in a rat DO model. METHODS: Rat bone marrow-derived MSCs were isolated, characterized and expanded in vitro. Adult rats were subjected to right tibia transverse osteotomy. On the third day of distraction, each rat received one injection of phosphate-buffered saline (PBS), autologous MSCs or allogeneic MSCs at the distraction site. Tibiae were harvested after 28 days of consolidation for micro-computed tomography examination, mechanical test and histological analysis. RESULTS: Results showed that treatment with both allogeneic and autologous MSCs promoted bone formation, with significantly higher bone mass, mechanical properties and mineral apposition rate as well as expression of angiogenic and bone formation markers at the regeneration sites compared with the PBS-treated group. No statistical difference in bone formation was found between the allogeneic and autologous MSC treatment groups. CONCLUSIONS: This study indicates that allogeneic and autologous MSCs have a similar effect on promoting bone consolidation in DO. MSCs from an allogeneic source could be used off-the-shelf with DO to achieve early bone healing.

Rejuvenated ageing mesenchymal stem cells by stepwise preconditioning ameliorates surgery-induced osteoarthritis in rabbits.

AIMS: Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model. METHODS: Mesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU), quantitative polymerase chain reaction (q-PCR), ß-Gal, Rosette forming, and histological staining were compared in the manipulated human mesenchymal stem cells (hM-MSCs) and their controls. The anterior cruciate ligament transection (ACLT) rabbit models were locally injected with two millions, four millions, or eight millions of hM-MSCs or phosphate-buffered saline (PBS). Osteoarthritis Research Society International (OARSI) scoring was performed to measure the pathological changes in the affected joints after staining. Micro-CT analysis was conducted to determine the microstructural changes in subchondral bone. RESULTS: Stepwise preconditioning approach significantly enhanced the proliferation and chondrogenic potential of ageing hMSCs at early passage. Interestingly, remarkably lower immunogenicity and senescence was also found in hM-MSCs. Data from animal studies showed cartilage damage was retarded and subchondral bone remodelling was prevented by the treatment of preconditioned MSCs. The therapeutic effect depended on the number of cells applied to animals, with the best effect observed when treated with eight millions of hM-MSCs. CONCLUSION: This study demonstrated a reliable and feasible stepwise preconditioning strategy to improve the safety and efficacy of ageing MSCs for the prevention of OA development. Cite this article: Bone Joint Res 2021;10(1):10-21.

MicroRNA-378 Suppressed Osteogenesis of MSCs and Impaired Bone Formation via Inactivating Wnt/ß-Catenin Signaling.

MicroRNAs (miRNAs) have been reported to serve as silencers to repress gene expression at post-transcriptional levels. Multiple miRNAs have been demonstrated to play important roles in osteogenesis. MicroRNA (miR)-378, a conserved miRNA, was reported to mediate bone metabolism and influence bone development, but the detailed function and underlying mechanism remain obscure. In this study, the miR-378 transgenic (TG) mouse was developed to study the role of miR-378 in osteogenic differentiation as well as bone formation. The abnormal bone tissues and impaired bone quality were displayed in the miR-378 TG mice, and a delayed healing effect was observed during bone fracture of the miR-378 TG mice. The osteogenic differentiation of mesenchymal stem cells (MSCs) derived from this TG mouse was also inhibited. We also found that miR-378 mimics suppressed, whereas anti-miR-378 promoted osteogenesis of human MSCs. Two Wnt family members, Wnt6 and Wnt10a, were identified as bona fide targets of miR-378, and their expression was decreased by this miRNA, which eventually induced the inactivation of Wnt/ß-catenin signaling. Finally, the short hairpin (sh)-miR-378-modified MSCs were locally injected into the fracture sites in an established mouse fracture model. The results indicated that miR-378 inhibitor therapy could promote bone formation and stimulate the healing process in vivo. In conclusion, miR-378 suppressed osteogenesis and bone formation via inactivating Wnt/ß-catenin signaling, suggesting that miR-378 may be a potential therapeutic target for bone diseases.

Administration of allogeneic mesenchymal stem cells in lengthening phase accelerates early bone consolidation in rat distraction osteogenesis model.

BACKGROUND: Distraction osteogenesis (DO) is a surgical technique to promote bone regeneration which may require long duration for bone consolidation. Bone marrow-derived mesenchymal stem cells (MSCs) have been applied to accelerate bone formation in DO. However, the optimal time point for cell therapy in DO remains unknown. This study sought to determine the optimal time point of cell administration to achieve early bone consolidation in DO. We hypothesized that the ratio of circulating MSCs to peripheral mononuclear cells and the level of cytokines in serum might be indicators for cell administration in DO. METHODS: Unilateral tibial osteotomy with an external fixator was performed in adult Sprague Dawley rats. Three days after osteotomy, the tibia was lengthened at 0.5 mm/12 h for 5 days. At first, 5 rats were used to analyze the blood components at 6 different time points (3 days before lengthening, on the day lengthening began, or 3, 6, 10, or 14 days after lengthening began) by sorting circulating MSCs and measuring serum levels of stromal cell-derived factor 1 (SDF-1) and interleukin 1ß. Then, 40 rats were used for cell therapy study. A single dose of 5 × 105 allogeneic MSCs was locally injected at the lengthening site on day 3, 6, or 10 after lengthening began, or 3 doses of MSCs were injected at the three time points. Sequential X-ray radiographs were taken weekly. Endpoint examinations included micro-computed tomography analysis, mechanical testing, histomorphometry, and histology. RESULTS: The number of circulating MSCs and serum level of SDF-1 were significantly increased during lengthening, and then decreased afterwards. Single injection of MSCs during lengthening phase (on day 3, but not day 6 or 10) significantly increased bone volume fraction, mechanical maximum loading, and bone mineralization of the regenerate. Triple injections of MSCs at three time points also significantly increased bone volume and maximum loading of the regenerates. CONCLUSION: This study demonstrated that bone consolidation could be accelerated by a single injection of MSCs during lengthening when the ratio of peripheral MSCs to mononuclear cells and the serum SDF-1 presented at peak levels concurrently, suggesting that day 3 after lengthening began may be the optimal time point for cell therapy to promote early bone consolidation.

Vasoactive Intestinal Peptide Stimulates Bone Marrow-Mesenchymal Stem Cells Osteogenesis Differentiation by Activating Wnt/ß-Catenin Signaling Pathway and Promotes Rat Skull Defect Repair.

Bone defect regeneration is a complex process that involves the coordination of a variety of different type of cells. As bone tissues are innervated and rich in nerve fibers, the neuropeptides released from various never fibers could regulate bone development, metabolism, and remodeling. Among all the neuropeptides, vasoactive intestinal peptide (VIP) could modulate the functions of both osteoblasts and osteoclasts, and may play a vital role in bone marrow mesenchymal stem cell (BMSC) osteogenesis during bone repair. In this study, we investigated the role of VIP in bone formation and the mechanisms of VIP in mediating BMSC osteogenic differentiation, and its possibility in clinical application of bone defect reconstruction. Our in vitro study results indicated that VIP promoted BMSC osteogenic differentiation by activating Wnt/ß-catenin signaling pathway in BMSCs. VIP could also stimulate tube formation of EA.hy926 endothelial cell and increase vascular endothelial growth factor (VEGF) expression in BMSCs. Furthermore, in the rat skull defect model, VIP-conjugated functionalized hydrogel significantly enhanced cranial bone defect repair compared with the control group, with increased bone formation and angiogenesis. Taken together, as a member of neuropeptides, VIP could promote the BMSCs osteogenesis and angiogenesis differentiation in vitro and stimulate bone repair in vivo by activating Wnt/ß-catenin signaling pathway. The knowledge obtained from this study emphasized the close association between innervation and bone repair process, and VIP may be a potential therapeutic agent for augmenting bone repair.

Conformational manipulation of scale-up prepared single-chain polymeric nanogels for multiscale regulation of cells.

Folded single chain polymeric nano-objects are the molecular level soft material with ultra-small size. Here, we report an easy and scalable method for preparing single-chain nanogels (SCNGs) with improved efficiency. We further investigate the impact of the dynamic molecular conformational change of SCNGs on cellular interactions from molecular to bulk scale. First, the supramolecular unfoldable SCNGs efficiently deliver siRNAs into stem cells as a molecular drug carrier in a conformation-dependent manner. Furthermore, the conformation changes of SCNGs enable dynamic and precise manipulation of ligand tether structure on 2D biomaterial interfaces to regulate the ligand-receptor ligation and mechanosensing of cells. Lastly, the dynamic SCNGs as the building blocks provide effective energy dissipation to bulk biomaterials such as hydrogels, thereby protecting the encapsulated stem cells from deleterious mechanical shocks in 3D matrix. Such a bottom-up molecular tailoring strategy will inspire further applications of single-chain nano-objects in the biomedical area.

MiR-378a suppresses tenogenic differentiation and tendon repair by targeting at TGF-ß2.

BACKGROUND: Tendons are a crucial component of the musculoskeletal system and responsible for transmission forces derived from muscle to bone. Patients with tendon injuries are often observed with decreased collagen production and matrix degeneration, and healing of tendon injuries remains a challenge as a result of limited understanding of tendon biology. Recent studies highlight the contribution of miR-378a on the regulation gene expression during tendon differentiation. METHODS: We examined the tendon microstructure and tendon repair with using miR-378a knock-in transgenic mice, and the tendon-derived stem cells were also isolated from transgenic mice to study their tenogenic differentiation ability. Meanwhile, the expression levels of tenogenic markers were also examined in mouse tendon-derived stem cells transfected with miR-378a mimics during tenogenic differentiation. With using online prediction software and luciferase reporter assay, the binding target of miR-378a was also studied. RESULTS: Our results indicated miR-378a impairs tenogenic differentiation and tendon repair by inhibition collagen and extracellular matrix production both in vitro and in vivo. We also demonstrated that miR-378a exert its inhibitory role during tenogenic differentiation through binding at TGFß2 by luciferase reporter assay and western blot. CONCLUSIONS: Our investigation suggests that miR-378a could be considered as a new potential biomarker for tendon injury diagnosis or drug target for a possible therapeutic approach in future clinical practice.