Disc regeneration by injectable fucoidan-methacrylated dextran hydrogels through mechanical transduction and macrophage immunomodulation.

Modulating a favorable inflammatory microenvironment that facilitates the recovery of degenerated discs is a key strategy in the treatment of intervertebral disc (IVD) degeneration (IDD). More interestingly, well-mechanized tissue-engineered scaffolds have been proven in recent years to be capable of sensing mechanical transduction to enhance the proliferation and activation of nucleus pulposus cells (NPC) and have demonstrated an increased potential in the treatment and recovery of degenerative discs. Additionally, existing surgical procedures may not be suitable for IDD treatment, warranting the requirement of new regenerative therapies for the restoration of disc structure and function. In this study, a light-sensitive injectable polysaccharide composite hydrogel with excellent mechanical properties was prepared using dextrose methacrylate (DexMA) and fucoidan with inflammation-modulating properties. Through numerous in vivo experiments, it was shown that the co-culture of this composite hydrogel with interleukin-1ß-stimulated NPCs was able to promote cell proliferation whilst preventing inflammation. Additionally, activation of the caveolin1-yes-associated protein (CAV1-YAP) mechanotransduction axis promoted extracellular matrix (ECM) metabolism and thus jointly promoted IVD regeneration. After injection into an IDD rat model, the composite hydrogel inhibited the local inflammatory response by inducing macrophage M2 polarization and gradually reducing the ECM degradation. In this study, we propose a fucoidan-DexMA composite hydrogel, which provides an attractive approach for IVD regeneration.

A multifunctional nanocomposite hydrogel with controllable release behavior enhances bone regeneration.

Autologous and allogeneic bone grafts remain the gold standard for repairing bone defects. However, donor shortages and postoperative infections contribute to unsatisfactory treatment outcomes. Tissue engineering technology that utilizes biologically active composites to accelerate the healing and reconstruction of segmental bone defects has led to new ideas for in situ bone repair. Multifunctional nanocomposite hydrogels were constructed by covalently binding silver (Ag+) core-embedded mesoporous silica nanoparticles (Ag@MSN) to bone morphogenetic protein-2 (BMP-2), which was encapsulated into silk fibroin methacryloyl (SilMA) and photo-crosslinked to form an Ag@MSN-BMP-2/SilMA hydrogel to preserve the biological activity of BMP-2 and slow its release. More importantly, multifunctional Ag+-containing nanocomposite hydrogels showed antibacterial properties. These hydrogels possessed synergistic osteogenic and antibacterial effects to promote bone defect repair. Ag@MSN-BMP-2/SilMA exhibited good biocompatibility in vitro and in vivo owing to its interconnected porosity and improved hydrophilicity. Furthermore, the multifunctional nanocomposite hydrogel showed controllable sustained-release activity that promoted bone regeneration in repairing rat skull defects by inducing osteogenic differentiation and neovascularization. Overall, Ag@MSN-BMP-2/SilMA hydrogels enrich bone regeneration strategies and show great potential for bone regeneration.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration.

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades. However, efficient harvest and handling of cell sheets remain challenging, including insufficient extracellular matrix content and poor mechanical strength. Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types. However, currently, there are no effective ways to apply mechanical loading to cell sheets. In this study, we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide) (PNIPAAm) to poly(dimethylsiloxane) (PDMS) surfaces. The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting. Subsequently, MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates. Upon maturation, the cell sheets were harvested by lowering the temperature. We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning. Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated. After implantation into the critical-sized calvarial defects of mice, the mechanically conditioned cell sheets significantly promoted new bone formation. Findings from this study reveal that thermo-responsive elastomer, together with mechanical conditioning, can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.

Quercetin-solid lipid nanoparticle-embedded hyaluronic acid functionalized hydrogel for immunomodulation to promote bone reconstruction.

Bone defects are a persistent challenge in clinical practice. Although repair therapies based on tissue-engineered materials, which are known to have a crucial role in defective bone regeneration, have gathered increased attention, the current treatments for massive bone defects have several limitations. In the present study, based on the immunomodulatory inflammatory microenvironment properties of quercetin, we encapsulated quercetin-solid lipid nanoparticles (SLNs) in a hydrogel. Temperature-responsive poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) modifications were coupled to the main chain of hyaluronic acid hydrogel, constructing a novel, injectable bone immunomodulatory hydrogel scaffold. Extensive in vitro and in vivo data showed that this bone immunomodulatory scaffold forms an anti-inflammatory microenvironment by decreasing M1 polarization, while elevating the M2 polarization. Synergistic effects on angiogenesis and anti-osteoclastic differentiation were observed. These findings further proved that administering quercetin SLNs encapsulated in a hydrogel can aid bone defect reconstruction in rats, providing new insights for large-scale bone defect repair.

The impact of Allgower-Donati suture pattern and postoperative sweet foods on wound suture breakage in experimental rats.

Background: Wound gnawing and/or scratching in rats often occurs in experimental models, causing suture breakage and wound dehiscence, and consequently affecting experimental results and wasting resources. This study aimed to investigate the impact of the combined postoperative use of the Allgower-Donati (A-D) suture pattern and sweet foods on suture breakage, inflammation, and healing in wounds. Materials and methods: Sprague Dawley (SD) rats (n = 48) were treated for linear wounds on the back by four procedures: simple suture, simple suture with postoperative sweet foods, A-D suture, and A-D suture with postoperative sweet foods. Additionally, CD68 immunofluorescence and CD31 immunohistochemistry were used to analyze wound inflammation and vascularization, respectively, on postoperative day 7. Sirius red staining was used to assess collagen deposition on postoperative day 14. Results: Gnawing and scratching of wound sutures were significantly reduced in treated rats (P < 0.01). Neovascularization and collagen deposition were significantly increased (P < 0.001), and inflammatory responses were significantly reduced (P < 0.001) in animals receiving AD sutures and postoperative sweet foods. CD31/CD68 analyses showed that A-D suture and postoperative sweet foods regulated wound angiogenesis and attenuated wound inflammation. Conclusions: Sweet food provision after A-D suture union surgery could reduce wound gnawing and/or scratching, suture breakage, incisional dehiscence, wound inflammation, and promote wound healing in rats.

Extracellular vesicles derived from bone marrow mesenchymal stem cells loaded on magnetic nanoparticles delay the progression of diabetic osteoporosis via delivery of miR-150-5p.

Extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) are emerged as carriers of therapeutic targets against bone disorders, yet its isolation and purification are limited with recent techniques. Magnetic nanoparticles (MNPs) can load EVs with a unique targeted drug delivery system. We constructed gold-coated magnetic nanoparticles (GMNPs) by decorating the surface of the Fe3O4@SiO2 core and a silica shell with poly(ethylene glycol) (PEG)-aldehyde (CHO) and examined the role of BMSC-EVs loaded on GMNPs in diabetic osteoporosis (DO). The osteoporosis-related differentially expressed miR-150-5p was singled out by microarray analysis. DO models were then established in Sprague-Dawley rats by streptozotocin injection, where poor expression of miR-150-5p was validated in the bone tissues. Next, GMNPE was prepared by combining GMNPs with anti-CD63, after which osteoblasts were co-cultured with the GMNPE-BMSC-EVs. The re-expression of miR-150-5p facilitated osteogenesis in osteoblasts. GMNPE could promote the enrichment of EVs in the bone tissues of DO rats. BMSC-EVs delivered miR-150-5p to osteoblasts, where miR-150-5p targeted MMP14 and consequently activated Wnt/ß-catenin pathway. This effect contributed to the enhancement of osteoblast proliferation and maturation. Furthermore, GMNPE enhanced the EV-based delivery of miR-150-5p to regulate the MMP14/Wnt/ß-catenin axis, resulting in promotion of osteogenesis. Overall, our findings suggest the potential of GMNP-BMSC-EVs to strengthen osteoblast proliferation and maturation in DO, showing promise as an appealing drug delivery strategy against DO. 1. GMNPs-BMSCs-EVs-miR-150-5p promotes the osteogenesis of DO rats. 2. miR-150-5p induces osteoblast proliferation and maturation by targeting MMP14. 3. Inhibition of MMP14 activates Wnt/ß-catenin and increases osteogenesis. 4. miR-150-5p activates the Wnt/ß-catenin pathway by downregulating MMP14.

Physiology-Inspired Multilayer Nanofibrous Membranes Modulating Endogenous Stem Cell Recruitment and Osteo-Differentiation for Staged Bone Regeneration.

Bone regeneration involves a cascade of sophisticated, multiple-staged cellular and molecular events, where early-phase stem cell recruitment mediated by chemokines and late-phase osteo-differentiation induced by pro-osteogenic factors play the crucial roles. Herein, enlightened by a bone physiological and regenerative mechanism, the multilayer nanofibrous membranes (PLLA@SDF-1α@MT01) consisting of PLLA/MT01 micro-sol electrospun nanofibers as intima and PLLA/PEG/SDF-1α electrospun nanofibers as adventitia are fabricated through micro-sol electrospinning and manual multi-layer stacking technologies. In vitro releasing profiles show that PLLA@SDF-1α@MT01 represents the rapid release of stromal cell-derived SDF-1α (SDF-1α) in the outer layers, while with long-term sustained release of MT01 in the inner layer. Owing to interconnected porosity like the natural bone extracellular matrix and improved hydrophilia, PLLA@SDF-1α@MT01 manifests good biocompatibility both in vitro and in vivo. Furthermore, PLLA@SDF-1α@MT01 can promote bone marrow mesenchymal stem cells (BMSCs) migration by amplifying the SDF-1α/CXCR4 axis and stimulating BMSCs osteo-differentiation via activating the MAPK pathway in vitro. PLLA@SDF-1α@MT01, with a programmed dual-delivery system, exhibits the synergetic promotion of bone regeneration and vascularization by emulating key characteristics of the staged bone repair in vivo. Overall, PLLA@SDF-1α@MT01 that mimics the endogenous cascades of bone regeneration can enrich the physiology-mimetic staged regenerative strategy and represent a promising tissue-engineered scaffold for the bone defect.

Selenium nanoparticles derived from Proteus mirabilis YC801 alleviate oxidative stress and inflammatory response to promote nerve repair in rats with spinal cord injury.

Microbial biotransformation and detoxification of biotoxic selenite into selenium nanoparticles (SeNPs) has emerged as an efficient technique for the utilization of selenium. SeNPs are characterized by high bioavailability and have several therapeutic effects owing to their antioxidant, anti-inflammatory and neuroprotective activities. However, their influence on microenvironment disturbances and neuroprotection after spinal cord injury (SCI) is yet to be elucidated. This study aimed to assess the influence of SeNPs on SCI and explore the underlying protective mechanisms. Overall, the proliferation and differentiation of neural stem cells were facilitated by SeNPs derived from Proteus mirabilis YC801 via the Wnt/ß-catenin signaling pathway. The SeNPs increased the number of neurons to a greater extent than astrocytes after differentiation and improved nerve regeneration. A therapeutic dose of SeNPs remarkably protected the integrity of the spinal cord to improve the motor function of the hind limbs after SCI and decreased the expression of several inflammatory factors such as tumor necrosis factor-α and interleukin-6 in vivo and enhanced the production of M2-type macrophages by regulating their polarization, indicating the suppressed inflammatory response. Besides, SeNPs reversed the SCI-mediated production of reactive oxygen species. In conclusion, SeNPs treatment holds the potential to improve the disturbed microenvironment and promote nerve regeneration, representing a promising therapeutic approach for SCI.

Mechanically enhanced composite hydrogel scaffold for in situ bone repairs.

High-efficiency repair of critical bone defects is a pressing problem in clinical practice. However, most biological replacement materials do not simultaneously satisfy the dual requirements of mechanical strength and cell compatibility. In this study, chitosan methacryloyl (CSMA) and ß-tricalcium phosphate (ß-TCP) were subjected to photo-crosslinking to form the CSMA/ß-TCP composite hydrogel, which has strong mechanical properties contributing to bone regeneration. In addition, its scaffold can alter the morphology of bone marrow mesenchymal stem cells (BMSCs), promote their proliferation, enhance the expression of alkaline phosphatase (ALP), and augment the nodular deposition of calcium. Meanwhile, the expressions of osteogenic proteins (ALP, osteocalcin, and osteopontin) were upregulated and the regulatory mechanism of the Hippo signaling pathway was verified. Moreover, animal experiments have confirmed that CSMA/ß-TCP has adequate biocompatibility and bone regeneration. These results demonstrate the immense potential of the CSMA/ß-TCP composite hydrogel in bone regeneration therapy.

Electrospun nanofibrous membrane for biomedical application.

Electrospinning is a simple, cost-effective, flexible, and feasible continuous micro-nano polymer fiber preparation technology that has attracted extensive scientific and industrial interest over the past few decades, owing to its versatility and ability to manufacture highly tunable nanofiber networks. Nanofiber membrane materials prepared using electrospinning have excellent properties suitable for biomedical applications, such as a high specific surface area, strong plasticity, and the ability to manipulate their nanofiber components to obtain the desired properties and functions. With the increasing popularity of nanomaterials in this century, electrospun nanofiber membranes are gradually becoming widely used in various medical fields. Here, the research progress of electrospun nanofiber membrane materials is reviewed, including the basic electrospinning process and the development of the materials as well as their biomedical applications. The main purpose of this review is to discuss the latest research progress on electrospun nanofiber membrane materials and the various new electrospinning technologies that have emerged in recent years for various applications in the medical field. The application of electrospun nanofiber membrane materials in recent years in tissue engineering, wound dressing, cancer diagnosis and treatment, medical protective equipment, and other fields is the main topic of discussion in this review. Finally, the development of electrospun nanofiber membrane materials in the biomedical field is systematically summarized and prospects are discussed. In general, electrospinning has profound prospects in biomedical applications, as it is a practical and flexible technology used for the fabrication of microfibers and nanofibers.

CD271 antibody-functionalized microspheres capable of selective recruitment of reparative endogenous stem cells for in situ bone regeneration.

In the strategy of in situ bone regeneration, it used to be difficult to specifically recruit bone marrow mesenchymal stem cells (BM-MSCs) by a single marker. Recently, CD271 has been considered to be one of the most specific markers to isolate BM-MSCs; however, the effectiveness of CD271 antibodies in recruiting BM-MSCs has not been explored yet. In this study, we developed novel CD271 antibody-functionalized chitosan (CS) microspheres with the aid of polydopamine (PDA) coating to recruit endogenous BM-MSCs for in situ bone regeneration. The CS microspheres were sequentially modified with PDA and CD271 antibody through dopamine self-polymerization and bioconjugation, respectively. In vitro studies showed that the CD271 antibody-functionalized microspheres selectively captured significantly more BM-MSCs from a fluorescently labeled heterotypic cell population than non-functionalized controls. In addition, the PDA coating was critical for supporting stable adhesion and proliferation of the captured BM-MSCs. Effective early recruitment of CD271+ stem cells by the functionalized microspheres at bone defect site of SD rat was observed by the CD271/DAPI immunofluorescence staining, which led to significantly enhanced new bone formation in rat femoral condyle defect over long term. Together, findings from this study have demonstrated, for the first time, that the CD271 antibody-functionalized CS microspheres are promising for in situ bone regeneration.

Comparison of clinical efficacy of suprapatellar and infrapatellar intramedullary nailing in treating tibial shaft fractures.

OBJECTIVES: To compare clinical efficacies of suprapatellar and infrapatellar intramedullary nailing approaches in treating tibial shaft fractures. METHODS: Patients (n=110) admitted with tibial shaft fractures in our hospital from January 2017 to June 2020, who underwent procedures with internal fixation intramedullary nails, were retrospectively divided into suprapatellar and infrapatellar approach groups (n = 55 each) based on the surgical method used for fracture repair. The clinical and functional outcomes of the knee were assessed six months after the surgery. RESULTS: Six months after the operation, the pooled value for excellent and good efficacy rates in the suprapatellar approach group, as indicated by Hospital for Special Surgery (HSS) Knee scoring system, was 90.91%, which was significantly higher than that in the infrapatellar approach group (76.36%). The degree of pain (visual analogue scale (VAS) score) of the patients in the suprapatellar approach group was over 2-fold lower than in the infrapatellar approach group (P < 0.001).The Lysholm knee score, range of motion (ROM), SF-36p, and SF-36M scores in the suprapatellar approach group were significantly higher than those in the infrapatellar approach group (P < 0.001). CONCLUSION: Suprapatellar approach had significantly higher clinical efficiency than infrapatellar approach, and can significantly reduce the degree of pain, promote the recovery of patients with knee joint involvement, improve the physical and psychological well-being, reduce the number of cases of postoperative delayed healing.

Gelatin methacrylate hydrogel scaffold carrying resveratrol-loaded solid lipid nanoparticles for enhancement of osteogenic differentiation of BMSCs and effective bone regeneration.

Critical-sized bone defects caused by traumatic fractures, tumour resection and congenital malformation are unlikely to heal spontaneously. Bone tissue engineering is a promising strategy aimed at developing in vitro replacements for bone transplantation and overcoming the limitations of natural bone grafts. In this study, we developed an innovative bone engineering scaffold based on gelatin methacrylate (GelMA) hydrogel, obtained via a two-step procedure: first, solid lipid nanoparticles (SLNs) were loaded with resveratrol (Res), a drug that can promote osteogenic differentiation and bone formation; these particles were then encapsulated at different concentrations (0.01%, 0.02%, 0.04% and 0.08%) in GelMA to obtain the final Res-SLNs/GelMA scaffolds. The effects of these scaffolds on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone regeneration in rat cranial defects were evaluated using various characterization assays. Our in vitro and in vivo investigations demonstrated that the different Res-SLNs/GelMA scaffolds improved the osteogenic differentiation of BMSCs, with the ideally slow and steady release of Res; the optimal scaffold was 0.02 Res-SLNs/GelMA. Therefore, the 0.02 Res-SLNs/GelMA hydrogel is an appropriate release system for Res with good biocompatibility, osteoconduction and osteoinduction, thereby showing potential for application in bone tissue engineering.

Efficacy of repeated intravenous tranexamic acid in reducing perioperative bleeding of acetabular fractures.

BACKGROUND: Application of tranexamic acid (TXA) in the treatment of acetabular fractures could reduce intraoperative and postoperative blood loss. OBJECTIVE: To investigate the effect of single and repeated intravenous infusion of TXA on blood loss of acetabular fractures. METHODS: 120 patients with acetabular fractures admitted to our hospital from January 2017 to September 2020 were retrospectively divided into three groups: Patients accepted 1g TXA at preoperative 30 minutes were defined as single TXA group (n = 40); Patients accepted 1g TXA at preoperative 30 minutes and 1g TXA at 3 hours after the start of surgery were defined as repeated TXA group (n = 40); Patients accepted normal saline at preoperative 30 minutes were defined as control group (n = 40). RESULTS: The total blood loss in single TXA group and repeated TXA group were significantly lower than control group, and the total blood loss in the repeated TXA group was significantly lower than single TXA group (P < 0.05). The hidden blood loss from surgery to postoperative 1 day in repeated TXA group was significantly lower than single TXA group and the control group(P < 0.05). No significant differences were observed in the operative time, postoperative transfusion rate and thrombosis rate among the three groups (P >  0.05). CONCLUSION: Repeated TXA is more recommended during acetabular fracture surgery since it can reduce the total blood loss without increasing the operative time, postoperative transfusion rate and thrombosis rate compared with single TXA.

[Comparison of effectiveness of two cannulated screw configurations in the treatment of femoral neck fractures in young adults].

OBJECTIVE: To investigate and analyze the effectiveness of internal fixation with the two different cannulated screw implanting methods of rhomboid and inverted triangle in the treatment of femoral neck fracture in young adults. METHODS: The clinical data of 38 young adults with femoral neck fracture who met the selection criteria between January 2018 and August 2019 were retrospectively analyzed. According to the different methods of cannulated screw implanting, the patients were divided into two groups, 19 cases in each group. The trial group was treated with closed reduction and cannulated screw rhombic distribution internal fixation, while the control group was treated with closed reduction and cannulated screw inverted triangular distribution internal fixation. There was no significant differences in patients' gender, age, cause of injury, Garden classification of fracture, and time from injury to operation between the two groups ( P>0.05). The fracture healing time, the incidence of nonunion, femoral neck shortening, and femoral head necrosis were recorded and compared between the two groups; the effectiveness was evaluated by Harris score and visual analogue scale (VAS) score at last follow-up. RESULTS: The incisions of the two groups healed by first intention. All patients were followed up 12-24 months with an average of 15.5 months. There were 1 case of fracture nonunion and 2 cases of shortening of femoral neck in the trial group; while there were 2 cases of fracture nonunion, 1 case of necrosis of femoral head, and 6 cases of femoral neck shortening in the control group; the difference in the incidence of complications (15.8% vs. 47.4%) between the two groups was significant ( χ 2=4.385, P=0.036). The remaining 18 cases in the trial group and 17 cases in the control group all achieved osteonal union, and the healing time was (14.8±1.6) weeks and (15.9±1.3) weeks, respectively, showing no significant difference between the two groups ( t=1.265, P=0.214). At last follow-up, Harris score and VAS score of the trial group were 88.9±4.3 and 1.1±0.7, respectively, while those of the control group were 86.9±5.9 and 1.3±0.9, respectively, showing no significant difference ( t=0.603, P=0.550; t=1.152, P=0.257). Hip function was evaluated in accordance with Harris score, the results were excellent in 12 cases, good in 6 cases, and fair in 1 case in the trial group, the excellent and good rate was 94.74%; the results were excellent in 10 cases, good in 7 cases, and fair in 2 cases in the control group, the excellent and good rate was 89.47%; there was no significant difference in the excellent and good rate between the two groups ( χ 2=0.368, P=0.544). CONCLUSION: The short-term effectiveness of the two kinds of cannulated screw implanting methods is clear, rhomboid fixation of 4 screws has strong stability with stress distribution, which can effectively reduce the incidence of femoral neck shortening, fracture nonunion, femoral head necrosis, and other complications.

Substrate Topography Regulates Differentiation of Annulus Fibrosus-Derived Stem Cells via CAV1-YAP-Mediated Mechanotransduction.

Regeneration of annulus fibrosus (AF) through tissue engineering techniques shows promise as a treatment for patients with degenerative disc disease (DDD). Yet, it remains challenging because of the intrinsic heterogeneity of AF tissue and shortage of in-depth knowledge of its structure-function correlation. In the current study, we fabricated fibrous poly(ether carbonate urethane)urea (PECUU) scaffolds with various fiber sizes to mimic the microstructural feature of native AF and aimed to regulate the differentiation of AF-derived stem cells (AFSCs) by controlling the topographical cues of the scaffold. We found that the morphology of AFSCs varied significantly on scaffolds with various fiber sizes. Meanwhile, the expression of the phenotypic marker genes of outer AF was up-regulated on scaffolds with large fibers. Meanwhile, enhanced expression of the phenotypic marker genes of inner AF was seen on scaffolds with small fibers. Such topography-dependent gene expression in AFSCs approximated the biochemical profile of AF tissue in various zones. Moreover, cell spreading and nucleus translocation of Yes-associated protein (YAP) were facilitated with increased fiber size. Formation and maturation of focal adhesions of AFSCs were also promoted. We also found that Caveolin-1 (CAV1) positively modulated the mechano-responses of YAP in response to substrate topography. In conclusion, depending on the activation of the CAV1-YAP mechanotransduction axis, tuning the fiber size of scaffolds can effectively induce changes in cell shape, adhesions, and extracellular matrix expression. This work may therefore provide new insights in the design of novel materials toward AF tissue regeneration.

Neobavaisoflavone protects osteoblasts from dexamethasone-induced oxidative stress by upregulating the CRNDE-mediated Nrf2/HO-1 signaling pathway.

Neobavaisoflavone (NBIF) is a flavonoid, which has a variety of pharmacological activities. However, the mechanism of NBIF in the treatment of osteoporosis still needs further exploration. The differentiation of osteoblast MC-3T3-E1 cells after treatment was observed by Alizarin red staining. Cell counting kit-8 and flow cytometry were used to detect viability, apoptosis, and reactive oxygen species (ROS) levels of treated MC-3T3-E1 cells, respectively. Malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were tested by ELISA kits. The expressions of lncRNA MALAT1, MEG3, CRNDE, Runx2, osteocalcin (OCN), osteopontin (OPN), collagen I (col-I), nuclear Nrf2, cytoplasm Nrf2, heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1) in treated MC-3T3-E1 cells were examined by Quantitative real-time PCR or Western blot. Dexamethasone (Dex) inhibited the viability of MC-3T3-E1 cells, while the appropriate amount of NBIF had no significantly effect on cell viability. Dex downregulated CRNDE expression, whereas NBIF upregulated CRNDE. Overexpressed CRNDE and NBIF reversed the inhibitory effects of Dex on cell viability, differentiation and levels of SOD, GSH-Px, Runx2, OCN, OPN, col-I, nuclear Nrf2, HO-1 and NQO1 while reversing the promoting effect of Dex on apoptosis and the levels of ROS, MDA, LDH and cytoplasm Nrf2 in MC-3T3-E1 cells, respectively, but shCRNDE further reversed the effects of NBIF in MC-3T3-E1 cells. NBIF protected osteoblasts from Dex-induced oxidative stress by upregulating the CRNDE-mediated Nrf2/HO-1 signaling pathway.

Regulation of differentiation of annulus fibrosus-derived stem cells using heterogeneous electrospun fibrous scaffolds.

BACKGROUND: Tissue engineering of the annulus fibrosus (AF) shows promise as a treatment for patients with degenerative disc disease (DDD). However, it remains challenging due to the intrinsic heterogeneity of AF tissue. Fabrication of scaffolds recapitulating the specific cellular, componential, and microstructural features of AF, therefore, is critical to successful AF tissue regeneration. METHODS: Poly-L-lactic acid (PLLA) fibrous scaffolds with various fiber diameters and orientation were prepared to mimic the microstructural characteristics of AF tissue using electrospinning technique. AF-derived stem cells (AFSCs) were cultured on the PLLA fibrous scaffolds for 7 days. RESULTS: The morphology of AFSCs significantly varied when cultured on the scaffolds with various fiber diameters and orientation. AFSCs were nearly round on scaffolds with small fibers. However, they became spindle-shaped on scaffolds with large fibers. Meanwhile, upregulated expression of collagen-I gene happened in cells cultured on scaffolds with large fibers, while enhanced expression of collagen-II and aggrecan genes was seen on scaffolds with small fibers. The production of related proteins also showed similar trends. Further, culturing AFSCs on a heterogeneous scaffold by overlaying membranes with different fiber sizes led to the formation of a hierarchical structure approximating native AF tissue. CONCLUSION: Findings from this study demonstrate that fibrous scaffolds with different fiber sizes effectively promoted the differentiation of AFSCs into specific cells similar to the types of cells at various AF zones. It also provides a valuable reference for regulation of cell differentiation and fabrication of engineered tissues with complex hierarchical structures using the physical cues of scaffolds. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Effective AF repair is an essential need for treating degenerative disc disease. Tissue engineering is a promising approach to achieving tissue regeneration and restoring normal functions of tissues. By mimicking the key structural features of native AF tissue, including fiber size and alignment, this study deciphered the effect of scaffold materials on the cell differentiation and extracellular matrix deposition, which provides a solid basis for designing new strategies toward more effective AF repair and regeneration.

Mechanical Stimulation and Diameter of Fiber Scaffolds Affect the Differentiation of Rabbit Annulus Fibrous Stem Cells.

BACKGROUND: Degeneration of the annulus fibrosus (AF), an important structure of the intervertebral disc, is one of the main causes of degenerative disc disease. Fabrication of scaffolds replicating the stratified microstructure of the AF is critical for the successful regeneration of AF. METHODS: In this study, we cultured rabbit AF-derived stem cells (AFSCs) using fabricated electrospun fibrous poly-L-lactic acid scaffolds with different diameters. We applied cyclic tensile strain (CTS) on the scaffolds to regulate the differentiation of AFSCs into specific cell types that resided at the inner, middle, and outer zones of the AF. RESULTS: We found that the morphologies of AFSCs on the smaller-fiber-diameter scaffolds were nearly round, whereas spindle-like cells morphologies were observed on large-diameter scaffolds. CTS enhanced these phenomena and made the cells slender. The expression levels of collagen-I in cells increased as a function of the fiber diameter, whereas collagen-II and aggrecan exhibited opposite trends. Moreover, the application of CTS upregulated the gene expressions of collagen-I, collagen-II, and aggrecan. CONCLUSION: Overlaying the scaffolds with different CTS-stimulated cells could eventually lead to engineered AF tissues with hierarchical structures that approximated the native AF tissue. Thus, the proposed methodologies could be potentially applied for AF regeneration.

miR-346-3p promotes osteoclastogenesis via inhibiting TRAF3 gene.

MicroRNAs (miRNAs) modulate gene expression and regulate many physiological and pathological conditions. However, their modulation and effect in osteoclastogenesis remain unknown. In this study, we investigated the role of miR-346-3p in regulating the osteoclast differentiation from RAW264.7 cells. We used the miRNA microarray assay, miR-346-3p mimic transfection, tartrate resistant acid phosphatase (TRAP) staining, bone resorption assay, qRT-PCR, and western blot. Our results showed that the expression of miR-346-3p was significantly upregulated during osteoclast differentiation. Further, by transfecting cells with miR-346-3p mimic, we observed an increased number of TRAP-positive multinucleated cells, increased pit area caused by bone resorption, and enhanced expression of osteoclast-specific genes and proteins. Conversely, miR-346-3p inhibition attenuated the osteoclast differentiation and function. Software-mediated prediction and validation using luciferase reporter assay showed that TRAF3, a negative regulator of osteoclast differentiation, was inhibited by miR-346-3p overexpression. Our results showed that miR-346-3p directly targeted TRAF3 mRNA via binding to its 3'-UTR and inhibited the expression of TRAF3 protein. Taken together, our results revealed that miR-346-3p promotes the regulation of osteoclastogenesis by suppressing the TRAF3 gene. In conclusion, miR-346-3p could be a novel therapeutic target for bone loss-related pathogenesis.