Wnt-3a protein promote neuronal differentiation of neural stem cells derived from adult mouse spinal cord

Abstract

Background: Wnt proteins as growth factor have multiple functions in neural development, and especially serve key roles in differentiation and development. Wnt-3a is an intercellular signaling molecule that is involved in a variety of morphogenetic events. The purpose of this study was to investigate the effects of Wnt-3a signal protein on proliferation and differentiation of neural stem cells derived from adult mouse spinal cord.

Methods: Adult mouse neural stem cells were cultured with serum free incubation. The recombined plasmid pSecTag2/Hygro B-Wnt3a for eukaryotic expression transfected adult neural stem cell, then the expression protein was detected by Western blot. The differentiation of adult neural stem cells was identified by the immunocytochemical technique.

Results: The inducing differentiated rates of neurons were improved greatly by Wnt-3a protein compared with control (p<0.05).

Conclusion: Wnt-3a has obvious influence on the neuronal differentiation of adult neural stem cell.     

Activation of the Canonical Wnt Signaling Pathway Induces Cementum Regeneration

Canonical Wnt signaling is important in tooth development but it is unclear whether it can induce cementogenesis and promote the regeneration of periodontal tissues lost because of disease. Therefore, the aim of this study is to investigate the influence of canonical Wnt signaling enhancers on human periodontal ligament cell (hPDLCs) cementogenic differentiation in vitro and cementum repair in a rat periodontal defect model. Canonical Wnt signaling was induced by (1) local injection of lithium chloride; (2) local injection of sclerostin antibody; and (3) local injection of a lentiviral construct overexpressing β‐catenin. The results showed that the local activation of canonical Wnt signaling resulted in significant new cellular cementum deposition and the formation of well‐organized periodontal ligament fibers, which was absent in the control group. In vitro experiments using hPDLCs showed that the Wnt signaling pathway activators significantly increased mineralization, alkaline phosphatase (ALP) activity, and gene and protein expression of the bone and cementum markers osteocalcin (OCN), osteopontin (OPN), cementum protein 1 (CEMP1), and cementum attachment protein (CAP). Our results show that the activation of the canonical Wnt signaling pathway can induce in vivo cementum regeneration and in vitro cementogenic differentiation of hPDLCs. © 2014 American Society for Bone and Mineral Research © 2015 American Society for Bone and Mineral Research     

Augmentation of smad‐dependent BMP signaling in neural crest cells causes craniosynostosis in mice

Craniosynostosis describes conditions in which one or more sutures of the infant skull are prematurely fused, resulting in facial deformity and delayed brain development. Approximately 20% of human craniosynostoses are thought to result from gene mutations altering growth factor signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis are incompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis have yet to be identified. Here, we show that enhanced bone morphogenetic protein (BMP) signaling through the BMP type IA receptor (BMPR1A) in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover, in vivo treatment with LDN‐193189, a selective chemical inhibitor of BMP type I receptor kinases, resulted in partial rescue of craniosynostosis. Enhanced signaling of the fibroblast growth factor (FGF) pathway, which has been implicated in craniosynostosis, was observed in both mutant and rescued mice, suggesting that augmentation of FGF signaling is not the sole cause of premature fusion found in this model. The finding that relatively modest augmentation of Smad‐dependent BMP signaling leads to premature cranial suture fusion suggests an important contribution of dysregulated BMP signaling to syndromic craniosynostoses and potential strategies for early intervention.     

Delayed Bone Regeneration Is Linked to Chronic Inflammation in Murine Muscular Dystrophy

Duchenne muscular dystrophy (DMD) patients exhibit skeletal muscle weakness with continuous cycles of muscle fiber degeneration/regeneration, chronic inflammation, low bone mineral density, and increased risks of fracture. Fragility fractures and associated complications are considered as a consequence of the osteoporotic condition in these patients. Here, we aimed to establish the relationship between muscular dystrophy and fracture healing by assessing bone regeneration in mdx mice, a model of DMD with absence of osteoporosis. Our results illustrate that muscle defects in mdx mice impact the process of bone regeneration at various levels. In mdx fracture calluses, both cartilage and bone deposition were delayed followed by a delay in cartilage and bone remodeling. Vascularization of mdx fracture calluses was also decreased during the early stages of repair. Dystrophic muscles are known to contain elevated numbers of macrophages contributing to muscle degeneration. Accordingly, we observed increased macrophage recruitment in the mdx fracture calluses and abnormal macrophage accumulation throughout the process of bone regeneration. These changes in the inflammatory environment subsequently had an impact on the recruitment of osteoclasts and the remodeling phase of repair. Further damage to the mdx muscles, using a novel model of muscle trauma, amplified both the chronic inflammatory response and the delay in bone regeneration. In addition, PLX3397 treatment of mdx mice, a cFMS (colony stimulating factor receptor 1) inhibitor in monocytes, partially rescued the bone repair defect through increasing cartilage deposition and decreasing the number of macrophages. In conclusion, chronic inflammation in mdx mice contributes to the fracture healing delay and is associated with a decrease in angiogenesis and a transient delay in osteoclast recruitment. By revealing the role of dystrophic muscle in regulating the inflammatory response during bone repair, our results emphasize the implication of muscle in the normal bone repair process and may lead to improved treatment of fragility fractures in DMD patients. © 2014 American Society for Bone and Mineral Research.     

Bone Regeneration by Lactoferrin Released from a Gelatin Hydrogel

The objective of this study is to evaluate the potential of lactoferrin (LF), an iron-binding glycoprotein, to induce bone regeneration. A biodegradable gelatin hydrogel was prepared to allow LF release in vivo in a sustained fashion. When subcutaneously implanted into the back of mice, the gelatin hydrogel incorporating LF showed a longer LF retention period at the site of implantation than that of LF solution injection. An in vitro culture experiment with 3T3E1 cells (mouse-derived osteoblasts) revealed that the cells were proliferated to a significantly greater extent by the repeated addition of LF compared with a single addition of LF at the same dose. Following the implantation of gelatin hydrogels incorporating LF into a skull bone defect of rats, a significantly stronger bone regeneration at the defect was observed than in LF-free- or low-LF-treated rats. It is concluded that the sustained release from the gelatin hydrogels enables LF to enhance the in vivo activity of bone regeneration.     

Asymmetrically porous PLGA/Pluronic F127 membrane for effective guided bone regeneration

Porous guided bone regeneration (GBR) membranes with selective permeability, hydrophilicity and adhesiveness to bone were prepared with PLGA and Pluronic F127 using an immersion precipitation method. The porous PLGA/Pluronic F127 membranes were fabricated by immersing the PLGA/Pluronic F127 mixture solution (in tetraglycol) in a mold into water. The PLGA/Pluronic F127 mixture was precipitated in water by the diffusion of water into PLGA/Pluronic F127 mixture solution. It was observed that the membrane has an asymmetric column-shape porous structure. The top surface of the membrane (water contact side) had nano-size pores (approx. 50 nm) which can effectively prevent from fibrous connective tissue invasion but permeate nutrients, while the bottom surface (mold contact size) had micro-size pores (approx. 40 μm) which can improve adhesiveness with bone. From the investigations of mechanical property, water absorbability, model nutrient permeability and preliminary in vivo bone regeneration, the hydrophilized porous PLGA/F127 (5 wt%) membrane seems to be a good candidate as a GBR membrane for the effective permeation of nutrients and osteoconductivity, as well as good mechanical strength to maintain a secluded space for bone regeneration.     

A trial to prepare biodegradable collagen–hydroxyapatite composites for bone repair

This paper is a trial to prepare collagen-hydroxyapatite composites in vitro by an alternate immersion method. Collagen sponges of different biodegradabilities were prepared through chemical cross-linking of Type I collagen with glutaraldehyde (GA) at concentrations of 0.2, 1.0, and 2.0 wt%. The sponges were immersed at 37°C in Tris-HCl-buffered solution containing 200 mM CaCl₂ (pH 7.4) for 2 h and then in an aqueous solution of 120 mM Na₂HPO₄ (pH 9.3) for a 2 h further (one immersion cycle). The alternate immersion cycle was repeated for different times to obtain collagen-hydroxyapatite composites. The characterization of the resulting composites was performed by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The weight of composites increased with an increase in immersion cycles and the rate of increase became greater with higher GA cross-linking levels for collagen sponge preparation. The pH of the phosphate solution decreased with the immersion cycle, which suggests H⁺ generation accompanied hydroxyapatite formation. Irrespective of the GA concentration and immersion cycle, every composite showed IR absorption bands attributable to phosphate and hydroxyl groups at 950-1100 or 550-650 and 3000-3500 cm^-1 and broad peaks specific to hydroxyapatite on the XRD charts. SEM study revealed small white clusters of hydroxyapatite interspersed uniformly on/in the collagen framework without any preferential orientation. The composite prepared from 0.2 wt% GA cross-linked collagen sponge which showed favourable characteristics was applied to a rat skull defect to evaluate its osteoconductivity as well as biodegradability. The formation of new bone tissue was histologically observed at the defect 12 weeks after application in marked contrast to the collagen sponge alone. The composite degraded without any inflammation reaction. It is concluded that the collagen-hydroxyapatite composite prepared by the present method is a biodegradable biomaterial of osteoconductivity applicable to bone repair.     

Studies on nerve cell affinity of biodegradable modified chitosan films

Chitosan, a natural polysaccharide that has excellent biocompatibility and biodegradability,can be used as nerve conduit material. The purpose of this work was to study the ability of chitosan and some chitosan-derived materials to facilitate nerve cell attachment, differentiation and growth. The biomaterials studied were chitosan, poly-L-lysine-blended chitosan (CP), collagen-blended chitosan (CC) and albumin-blended chitosan (CA), with collagen control material. Culture of PC12 cells and fetal mouse cerebral cortex (FMCC) cells on these biomaterials was used to evaluate their nerve cell affinity. The composite materials, including CP, CC and CA, had significantly improved nerve cell affinity compared to chitosan, as established by increasing attachment, differentiation and growth of PC12 cells. FMCC cells could also grow better on composite materials than on chitosan. CP exhibited the best nerve cell affinity among these three types of composite material. CP is an even better material in promoting neurite outgrowth than collagen, a substrate that is widely used in tissue engineering, suggesting that CP is a promising candidate material for nerve regeneration.     

Solid Free-Form Fabrication-Based PCL/HA Scaffolds Fabricated with a Multi-head Deposition System for Bone Tissue Engineering

In this study, we fabricated polycaprolactone/hydroxyapatite (PCL/HA) scaffolds with a multi-head deposition system, a solid free-form fabrication technology that was developed in our previous study. The bone regeneration potential of the scaffolds was compared with that of PCL scaffolds fabricated with the same system. The fabricated scaffolds had a pore size of 400 μm and a porosity of 66.7%. The PCL/HA scaffolds had higher mechanical strength and modulus than the PCL scaffolds. To compare the osteogenic potential, the two types of scaffolds were seeded with rat osteoblasts and cultured in vitro or implanted subcutaneously into athymic mice. The cells cultured on PCL/HA scaffolds expressed higher levels of osteopontin and osteonectin, both of which are osteogenic proteins. The PCL/HA scaffolds resulted in larger bone area and calcium deposition in the implants compared to the PCL scaffolds.     

Photopolymerized poly(ethylene glycol)/poly(L-lysine) hydrogels for the delivery of neural progenitor cells

Neural progenitor cells (NPCs) have shown promise in a number of models of disease and injury, but for these cells to be safe and effective, they must be directed to differentiate appropriately following transplantation. We have developed a photopolymerized hydrogel composed of macromers of poly(ethylene glycol) (PEG) bound to poly(L-lysine) (PLL) that supports NPC survival and directs differentiation. Green fluorescent protein (GFP) positive NPCs were encapsulated in these gels and demonstrated survival up to 17 days. When encapsulated in the gels at a photoinitiator concentration of 5.0 mg/ml, few NPCs (0.5 ± 0.25%) demonstrated apoptosis. Furthermore, 55 ± 6% of the NPCs cultured within the gels in epidermal growth factor (EGF) containing media differentiated into a mature neuronal cell type (neurofilament 200 positive) while the remainder 44 ± 8% were undifferentiated (nestin positive). A small percentage, 1 ± 0.4%, expressed the astrocytic marker glial acidic fibrilary protein. Photopolymerized PEG/PLL gels promote the survival and direct the differentiation of NPCs, making this system a promising delivery vehicle for NPCs in the treatment of injuries and diseases of the central nervous system.