Stiffness of photocrosslinkable gelatin hydrogel influences nucleus pulposus cell propertiesin vitro.

A key early sign of degenerative disc disease (DDD) is the loss of nucleus pulposus (NP) cells (NPCs). Accordingly, NPC transplantation is a treatment strategy for intervertebral disc (IVD) degeneration. However, in advanced DDD, due to structural damage of the IVD and scaffold mechanical properties, the transplanted cells are less viable and secrete less extracellular matrix, and thus, are unable to efficiently promote NP regeneration. In this study, we evaluated the encapsulation of NPCs in a photosensitive hydrogel made of collagen hydrolysate gelatin and methacrylate (GelMA) to improve NP regeneration. By adjusting the concentration of GelMA, we prepared hydrogels with different mechanical properties. After examining the mechanical properties, cell compatibility and tissue engineering indices of the GelMA-based hydrogels, we determined the optimal hydrogel concentration of the NPC-encapsulating GelMA hydrogel for NP regeneration as 5%. NPCs effectively combined with GelMA and proliferated. As the concentration of the GelMA hydrogel increased, the survival, proliferation and matrix deposition of the encapsulated NPCs gradually decreased, which is the opposite of NPCs grown on the surface of the hydrogel. The controllability of the GelMA hydrogels suggests that these NPC-encapsulating hydrogels are promising candidates to aid in NP tissue engineering and repairing endogenous NPCs.

Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae).

Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure-function relationships between enzymatic properties, toxicity and structural characterization of SEBIN.

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.

3D collagen porous scaffold carrying PLGA-PTX/SDF-1α recruits and promotes neural stem cell differentiation for spinal cord injury repair.

Spinal Cord Injury (SCI), one of the major factors of disability, can cause irreversible motor and sensory impairment. There are no effective therapeutic drugs and technologies available in domestic or foreign countries currently. Neural stem cells (NSCs), with the potential for multidirectional differentiation, are a potential treatment for SCI. However, it has been demonstrated that NSCs primarily differentiated into astrocytes rather than neurons due to the inflammatory microenvironment, and the current challenge remains to direct the differentiation of NSCs into neurons in the lesion site. It was reported that the microtubule-stabilizing agent paclitaxel (PTX) was able to promote the differentiation of NSCs into neurons rather than astrocytes after SCI. SDF-1α can recruit NSCs and thus guide the migration of stem cells. In this study, we developed a functional collagen scaffold by loading SDF-1α and nanoparticle-encapsulated PLGA-PTX into a 3D collagen porous scaffold, allowing for slow release of PTX. When the functional scaffolds were implanted into the injury site, it provided a neural regeneration conduit channel for the migration of NSCs and neuronal differentiation. Neural regeneration promoted the recovery of motor function and reduced glial scar formation after SCI. In conclusion, a 3D collagen porous scaffold combined with PLGA-PTX and SDF-1α is a promising therapeutic strategy for SCI repair.

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.

pH/Temperature Responsive Curcumin-Loaded Micelle Nanoparticles Promote Functional Repair after Spinal Cord Injury in Rats via Modulation of Inflammation.

BACKGROUND: The formation of an inhibitory inflammatory microenvironment after spinal cord injury (SCI) remains a great challenge for nerve regeneration. The poor local microenvironment exacerbates nerve cell death; therefore, the reconstruction of a favorable microenvironment through small-molecule drugs is a promising strategy for promoting nerve regeneration. METHODS: In the present study, we synthesized curcumin-loaded micelle nanoparticles (Cur-NPs) to increase curcumin bioavailability and analyzed the physical and chemical properties of Cur-NPs by characterization experiments. We established an in vivo SCI model in rats and examined the ability of hind limb motor recovery using Basso-Beattie-Bresnahan scoring and hind limb trajectory assays. We also analyzed neural regeneration after SCI using immunofluorescence staining. RESULTS: The nanoparticles achieved the intelligent responsive release of curcumin while improving curcumin bioavailability. Most importantly, the released curcumin attenuated local inflammation by modulating the polarization of macrophages from an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype. M2-type macrophages can promote cell differentiation, proliferation, matrix secretion, and reorganization by secreting or expressing pro-repair cytokines to reduce the inflammatory response. The enhanced inflammatory microenvironment supported neuronal regeneration, nerve remyelination, and reduced scar formation. These effects facilitated functional repair in rats, mainly in the form of improved hindlimb movements. CONCLUSION: Here, we synthesized pH/temperature dual-sensitive Cur-NPs. While improving the bioavailability of the drug, they were also able to achieve a smart responsive release in the inflammatory microenvironment that develops after SCI. The Cur-NPs promoted the regeneration and functional recovery of nerves after SCI through anti-inflammatory effects, providing a promising strategy for the repair of SCIs.

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.

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.

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.

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.

Graded-Three-Dimensional Cell-Encapsulating Hydrogel as a Potential Biologic Scaffold for Disc Tissue Engineering.

BACKGROUND: Intervertebral disk (IVD) degeneration, which can cause lower back pain, is a major predisposing factor for disability and can be managed through multiple approaches. However, there is no satisfactory strategy currently available to reconstruct and recover the natural properties of IVDs after degeneration. As tissue engineering develops, scaffolds with embedded cell cultures have proved critical for the successful regeneration of IVDs. METHODS: In this study, an integrated scaffold for IVD replacement was developed. Through scanning electron microscopy and other mechanical measurements, we characterized the physical properties of different hydrogels. In addition, we simulated the physiological structure of natural IVDs. Nucleus pulposus (NP) cells and annulus fibrosus-derived stem cells (AFSCs) were seeded in gelatin methacrylate (GelMA) hydrogel at different concentrations to evaluate cell viability and matrix expression. RESULTS: It was found that different concentrations of GelMA hydrogel can provide a suitable environment for cell survival. However, hydrogels with different mechanical properties influence cell adhesion and extracellular matrix component type I collagen, type II collagen, and aggrecan expression. CONCLUSION: This tissue-engineered IVD implant had a similar structure and function as the native IVD, with the inner area mimicking the NP tissue and the outer area mimicking the stratified annulus fibrosus tissue. The new integrated scaffold demonstrated a good simulation of disc structure. The preparation of efficient and regeneration-promoting tissue-engineered scaffolds is an important issue that needs to be explored in the future. It is hoped that this work will provide new ideas and methods for the further construction of functional tissue replacement discs.

[Experimental study of resveratrol-solid lipid nanoparticles in promotion of osteogenic differentiation of bone marrow mesenchymal stem cells].

Objective: To investigate the effect of solid lipid nanoparticles (SLNs) on enhancing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro by resveratrol (Res), and provide a method for the treatment of bone homeostasis disorders. Methods: Res-SLNs were prepared by high-temperature emulsification and low-temperature solidification method, and then the 2nd-3rd generation BMSCs from Sprague Dawley rat were co-cultured with different concentrations (0, 0.1, 1, 5, 10, 20 µmol/L) of Res and Res-SLNs. The effects of Res and Res-SLNs on the cell viability of BMSCs were detected by cell counting kit 8 (CCK-8) and live/dead cell staining; the effects of Res and Res-SLNs on the osteogenic differentiation of BMSCs were detected by alkaline phosphatase (ALP) staining and alizarin red S (ARS) staining after osteogenic differentiation induction, and the optimal concentration of Res-SLNs for gene detection was determined. Anti-osteocalcin (OCN) immunofluorescence staining and real-time fluorescent quantitative PCR (RT-qPCR) were used to detect the effect of Res and Res-SLNs on osteoblast-related genes (ALP and OCN) of BMSCs. Results: Live/dead cell staining showed that there was no significant difference in the number of dead cells between Res and Res-SLNs groups; CCK-8 detection showed that the activity of BMSCs in Res group was significantly reduced at the concentration of 20 µmol/L (P<0.05), while Res-SLNs activity was not affected by Res concentration (P>0.05). After osteogenic differentiation, the staining intensity of ALP and ARS in both groups was dose-dependent. The percentage of ALP positive staining area and the percentage of mineralized nodule area in Res group and Res-SLNs group reached the maximum at the concentrations of 10 µmol/L and 1 µmol/L, respectively (P<0.05), and then decreased gradually; the most effective concentration of Res-SLNs was 1 µmol/L. The expression of OCN and the relative expression of ALP and OCN mRNA in Res-SLNs group were significantly higher than those in Res group (P<0.05). Conclusion: Encapsulation of SLNs can improve the effect of Res on promoting osteogenesis, and achieve the best effect of osteogenic differentiation of BMSCs at a lower concentration, which is expected to be used in the treatment of bone homeostasis imbalance diseases.

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.

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.

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.

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.

Schistosoma japonicum cystatin suppresses osteoclastogenesis via manipulating the NF­κB signaling pathway.

Abnormal osteoclastic activation and secretion of cysteine proteinases result in excessive bone resorption, which is one of the primary factors in the development of bone metabolic disorders, such as rheumatoid arthritis and osteoporosis. Mammalian cystatins have been demonstrated to restrain osteoclastic bone resorption and to alleviate severe osteolytic destruction via blocking the activity of cysteine proteinases. However, the specific effects of parasite cystatins on the formation and function of osteoclasts remain unclear. The purpose of the current study was to explore the effects of cystatins from Schistosoma japonicum (Sj­Cys) on macrophage colony­stimulating factor (M­CSF) and receptor activator of NF­κB ligand (RANKL)­induced osteoclast differentiation, as well as the underlying molecular mechanisms. Recombinant Sj­Cys (rSj­Cys) dose­dependently restrained osteoclast formation, with a half­maximal inhibitory concentration (IC50) value of 0.3 µM, and suppressed osteoclastic bone resorptive capability in vitro. The findings were based on tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays, respectively. However, the cell viability assay showed that the repression of rSj­Cys on osteoclast formation did not depend on effects on cell viability or apoptosis. Based on the results of reverse transcription­quantitative PCR and western blot analysis, it was found that rSj­Cys downregulated the expression levels of osteoclastogenesis­related genes and proteins, by interfering with M­CSF and RANKL­induced NF­κB signaling and downstream transcription factors during early­phase osteoclastogenesis. Overall, the results of the present study revealed that rSj­Cys exerted an inhibitory role in osteoclast differentiation and could be a prospective biotherapeutic candidate for the treatment and prevention of bone metabolic disorders.

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.

Electrospun fibers: an innovative delivery method for the treatment of bone diseases.

INTRODUCTION: The treatment performances of current surgical therapeutic materials for injuries caused by high-energy trauma, such as prolonged bone defects, nerve-fiber disruptions, and repeated spasms or adhesions of vascular tendons after repair, are poor. Drug-loaded electrospun fibers have become a novel polymeric material for treating orthopedic diseases owing to their three-dimensional structures, thus providing excellent controlled drug-release responses and high affinity with local tissues. Herein, we reviewed the morphology of electrospun nanofibers, methods for loading drugs on the fibers, and modification methods to improve drug permeability and bioavailability. We highlight innovative applications of drug-loaded electrospun fibers in different treatments, including bone and cartilage defects, tendon and soft-tissue adhesion, vascular remodeling, skin grafting, and nervous-system injuries. AREAS COVERED: With the rapid development of electrospinning technologies and advancement of tissue engineering, drug-loaded electrospun fibers are becoming increasingly important in controlled drug release, wound closure, and tissue regeneration and repair. EXPERT OPINION: Drug-loaded electrospun fibers exhibit a broad range of application prospects and great potential in treating orthopedic diseases. Accordingly, a plethora of novel treatments utilizing the different morphological features of electrospun fibers, the distinctive pharmacokinetics, pharmacodynamics characteristics of different drugs, and the diverse onset characteristics of different diseases, is proposed.

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.