Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization

BACKGROUND The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine.Stem cells can self-organise into microsized organ units,partially modelling tissue function and regeneration.Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases.However,the lack of vasculature limits the utility of dental pulp organoids.AIM To improve survival and aid in recovery after stem cell transplantation,we demonstrated the three-dimensional(3D)self-assembly of adult stem cell-human dental pulp stem cells(hDPSCs)and endothelial cells(ECs)into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium(CM).METHODS During culture,primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM.The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids.The biological characteristics of the organoids were analysed,and the regulatory pathways associated with angiogenesis were studied.RESULTS The combination of these two agents resulted in prevascularized human dental pulp organoids(Vorganoids)that more closely resembled dental pulp tissue in terms of morphology and function.Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis.The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids.CONCLUSION In this innovative study,we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration,facilitating the development of clinical treatment strategies.

Prevascularized spongy-like hydrogels maintain their angiogenic potential after prolonged hypothermic storage

The chronic shortage of organs and tissues for transplantation represents a dramatic burden on healthcare systems worldwide.Tissue engineering offers a potential solution to address these shortages,but several challenges remain,with prevascularization being a critical factor for in vivo survival and integration of tissue engineering products.Concurrently,a different challenge hindering the clinical implementation of such products,regards their efficient preservation from the fabrication site to the bedside.Hypothermia has emerged as a potential solution for this issue due to its milder effects on biologic systems in comparison with other cold preservation methodologies.Its impact on prevascularization,however,has not been well studied.In this work,3D prevascularized constructs were fabricated using adipose-derived stromal vascular fraction cells and preserved at 4◦C using Hypothermosol or basal culture media(α-MEM).Hypothermosol efficiently preserved the structural and cellular integrity of prevascular networks as compared to constructs before preservation.In contrast,the use ofα-MEM led to a clear reduction in prevascular structures,with concurrent induction of high levels of apoptosis and autophagy at the cellular level.In vivo evaluation using a chorioallantoic membrane model demonstrated that,in opposition toα-MEM,Hypothermosol preservation retained the angiogenic potential of constructs before preservation by recruiting a similar number of blood vessels from the host and presenting similar integration with host tissue.These results emphasize the need of studying the impact of preservation techniques on key properties of tissue engineering constructs such as prevascularization,in order to validate and streamline their clinical application.

A novel bioartificial pancreas fabricated via islets microencapsulation in anti-adhesive core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized in vivo

Islets transplantation is a promising treatment for type 1 diabetes mellitus. However, severe host immune rejection and poor oxygen/nutrients supply due to the lack of surrounding capillary network often lead to transplantation failure. Herein, a novel bioartificial pancreas is constructed via islets microencapsulation in core-shell microgels and macroencapsulation in a hydrogel scaffold prevascularized in vivo. Specifically, a hydrogel scaffold containing methacrylated gelatin (GelMA), methacrylated heparin (HepMA) and vascular endothelial growth factor (VEGF) is fabricated, which can delivery VEGF in a sustained style and thus induce subcutaneous angiogenesis. In addition, islets-laden core-shell microgels using methacrylated hyaluronic acid (HAMA) as microgel core and poly(ethylene glycol) diacrylate (PEGDA)/carboxybetaine methacrylate (CBMA) as shell layer are prepared, which provide a favorable microenvironment for islets and simultaneously the inhibition of host immune rejection via anti-adhesion of proteins and immunocytes. As a result of the synergistic effect between anti-adhesive core-shell microgels and prevascularized hydrogel scaffold, the bioartificial pancreas can reverse the blood glucose levels of diabetic mice from hyperglycemia to normoglycemia for at least 90 days. We believe this bioartificial pancreas and relevant fabrication method provide a new strategy to treat type 1 diabetes, and also has broad potential applications in other cell therapies.

Heterotopic bone formation in the musculus latissimus dorsi of sheep usingβ-tricalcium phosphate scaffolds:evaluation of different seeding techniques

Osseous reconstruction of large bone defects remains a challenge in oral and maxillofacial surgery.In addition to autogenous bone grafts,which despite potential donor-site mobility still represent the gold standard in reconstructive surgery,many studies have investigated less invasive alternatives such as in vitro cultivation techniques.This study compared different types of seeding techniques on pureβ-tricalcium phosphate scaffolds in terms of bone formation and ceramic resorption in vivo.Cylindrical scaffolds loaded with autologous cancellous bone,venous blood,bone marrow aspirate concentrate or extracorporeal in vitro cultivated bone marrow stromal cells were cultured in sheep on a perforator vessel of the musculus latissimus dorsi over a 6-month period.Histological and histomorphometric analyses revealed that scaffolds loaded with cancellous bone were superior at promoting heterotopic bone formation and ceramic degradation,with autogenous bone and bone marrow aspirate concentrate inducing in vivo formation of vital bone tissue.These results confirm that autologous bone constitutes the preferred source of osteoinductive and osteogenic material that can reliably induce heterotopic bone formation in vivo.

Direct-writing Process and in vivo Evaluation of Prevascularized Composite Constructs for Muscle Tissue Engineering Application

Tissue engineered skeletal muscle is expected to treat muscle defects caused by trauma and disease.However,designing and manufacturing thick and complex tissue engineered skeletal muscle requires vascularization to ensure its internal cell viability and nutrient supply in vitro.In this article,we developed a set of Direct-Writing(DW)bio-printing procedure to manufacture a prevascularized composite construct with Human Umbilical Vein Endothelial Cell(HUVEC)and C2C12 cells for muscle tissue engineering application.We put the cells into the construct during the DW process to obtain the prevascularization and intend to promote its vascularization in vivo later.The constructs with cells or without cells were implanted respectively into nude mice back for 3 weeks,after which the mice healthily live for all the time and all the implants are tightly bonded to the host.From immunohistochemical analysis,CD31-positive blood vessels existed in the implanted samples with cells are more substantial than those without cells,but the implanted samples with HUVEC and C2C12 cells have much more number of small blood vessels distributing evenly.Moreover,the implants with cells,especially that with HUVEC and C2C12 cells,are able to get better fusion with the host skin and subcutaneous tissues.Histological analysis demonstrates that our DW-based constructs have the potential to be getting to vascularize the tissue engineered muscle.