Dual crosslinking silk fibroin/pectin-based bioink development and the application on neural stem/progenitor cells spheroid laden 3D bioprinting.

The human nervous system is an incredibly intricate physiological network, and neural cells lack the ability to repair and regenerate after a brain injury. 3-dimensional (3D) bioprinting technology offers a promising strategy for constructing biomimetic organ constructs and in vitro brain/disease models. The bioink serves as a pivotal component that emulates the microenvironment of biomimetic construct and exerts a profound influence on cellular behaviors. In this study, a series of mechanically adjustable and dual crosslinking bioinks were developed using photocrosslinkable methacrylated silk fibroin (SilMA) in combination with the ionic crosslinking material, pectin, or pectin methacryloyl (PecMA) with silk fibroin (SF) supplementation. SilMA/pectin exhibited superior properties, with SilMA providing biocompatibility and adjustable mechanical properties, while the addition of pectin enhanced printability. The porous structure supported neural cell growth, and 15 % SilMA/0.5 % pectin bioinks displayed excellent printability and shape fidelity. Neural stem/progenitor cells (NSPCs)-loaded bioinks were used to construct a 3D brain model, demonstrating sustained vitality and high neuronal differentiation without the need for growth factors. The SilMA/pectin bioinks demonstrated adjustable mechanical properties, favorable biocompatibility, and an environment highly conducive to neural induction, offering an alternative approach for neural tissue engineering applications or in vitro brain models.

Dental Pulp Stem Cell Transplantation with 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside Accelerates Alveolar Bone Regeneration in Rats.

INTRODUCTION: Although the therapeutic potential of human dental pulp stem cells (hDPSCs) has been studied for bone regeneration, the therapeutic efficiency needs further consideration and examinations for clinical applications. Thus, the aims of this study were to evaluate the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG) on the osteogenic differentiation of hDPSCs and to examine the therapeutic efficiency of the THSG-enhanced osseous potential of hDPSCs in alveolar bony defects of rats. METHODS: Expressions of osteogenic messenger RNAs (including ALP, RUNX2, BGLAP, and AMBN) were examined by quantitative real-time polymerase chain reaction. Alizarin red S staining was conducted to analyze THSG-induced mineralization of hDPSCs. To investigate the regenerative effects of THSG-treated hDPSCs on dental alveolar bone, bony defects were created in male Sprague-Dawley rats. Defects were treated with Matrigel (Corning Inc, Corning, NY), hDPSCs, or hDPSCs + THSG. After 2 weeks, defect healing was evaluated by micro-computed tomographic and histologic analyses. RESULTS: In the cell model, THSG induced osteogenesis-associated genes (ALP, RUNX2, and BGLAP) and an enamel-related gene (AMBN), resulting in mineralization as detected by alizarin red S staining after 2 weeks of treatment. In the animal model, THSG increased all parameters of bone formation (the relative bone volume, trabecular thickness, trabecular number, and trabecular separation) in alveolar bony defects of rats. THSG not only improved the quality of newly formed bone but also the quantity of new bone. CONCLUSIONS: These results showed important findings in revealing the THSG-enhanced osteogenic differentiation of hDPSCs and THSG-facilitated bone regeneration, which may provide an alternative option for cell-based regenerative therapy.

Maintenance of the spheroid organization and properties of glandular progenitor cells by fabricated chitosan based biomaterials.

Dysfunctional salivary gland (SG) is an unsolved clinical challenge, which is presented as xerostomia. Cell therapy is a promising treatment for restoring SG function. Salispheres are spheroid cellular organizations derived from SG stem cells. Benefitting from these cellular organizations, SG stem cells can be expanded to regenerate SG. During in vitro culture, the spontaneous reorganization of salispheres may change the features of residing SG stem cells. Therefore, it is imperative to explore ways to maintain the spheroid structure of salispheres during cell expansion in vitro. Herein, we explored biomaterial approaches using chitosan. Chitosan based biomaterials were fabricated in different forms to offer distinct interactive surfaces for cultured salispheres. The number and size of the salispheres increase in the chitosan-containing systems without increasing the incidence of spheroid cavitation. The effect of chitosan increases with high chitosan concentrations, which is optimum when chitosan is fabricated in a soluble form. The chitosan effect contributes to the regulation of the intercellular interactions and polarization within the spheroid structures. By retarding the process of salisphere cavitation, chitosan preserves the features of salivary gland progenitor cells in the cultured salispheres. The results suggest that the chitosan-containing system could effectively maintain the primitive structures and properties of salispheres during in vitro expansion, which demonstrates the potential application of salispheres for cell therapy of dysfunctional SG.

Chitosan biomaterials induce branching morphogenesis in a model of tissue-engineered glandular organs in serum-free conditions.

Glandular organs feature ramified structures that are important for regulating physiological transport. The aim of this study was to develop a biomaterial-assisted, serum-free culture system to generate branching structures in explants of glandular organs. The fetal mammary gland (MG) was selected as the model organ to study the formation of glandular structure. Among the many biomaterials tested, chitosan demonstrated a superior effect in promoting branch formation in MGs. The morphogenetic effect toward MG branching was chitosan specific and not observed with other analogs with similar chemical compositions or structures. The molecular weight and specific linkages in the chitosan polymer were important parameters in mediating the morphogenetic effect. MG explants from different anatomical locations effectively promoted structure formation. Blocking endogenous fibroblast growth factor 10 (FGF10) inhibited the morphogenetic effect of chitosan, indicating that the chitosan effect was FGF10 dependent. This work demonstrates the feasibility of creating a serum-free system that is competent in facilitating tissue morphogenesis in MG. MG tissue structure can be efficiently generated in a biocompatible system, which was assisted by biomaterials.

Biomaterial mediated epithelial-mesenchymal interaction of salivary tissue under serum free condition.

Many organs develop from epithelial-mesenchymal interactions such that in order to regenerate these organs, it might be a preferable strategy to recapitulate this process. However, in the current culture system designed for tissue interaction, the supplement of serum is required. The aim of this study is to explore the possibility of reproducing epithelial-mesenchymal interaction and ensuing morphogenesis in a serum-free condition. In accordance with the previous studies, by using a standard model of murine fetal submandibular gland (SMG), the tissue interaction and the morphogenesis were largely dependent on serum. Nonetheless, when tissue recombinants were cultivated on polyvinylidene fluoride (PVDF), but not on other biomaterials, the serum-deprived effect could be rescued. On PVDF, SMG tissue recombinant was able to increase epithelial size, de novo synthesize basement membrane, and develop new branches without serum. Although the gene expression levels of selected morphogens were not significantly altered, the precise localization of morphogenetic-decisive extracellular matrix such as type III collagen and the superior adsorbing capacity of essential diffusible factors like fibroblast growth factor 7 (FGF7) might account for PVDF effect. Accordingly, the result demonstrates that it is possible to establish a serum-free system that is competent in facilitating epithelial-mesenchymal interaction of salivary tissue. With PVDF, the crosstalk between SMG epithelia and mesenchyme could be sustained without serum.