The Study On The Construction Of In Vitro Models Of Craniomaxillofacial Bone With 3D Bio-printing Technology

Part ? The exploration of 3D bioprinting technology to simulate the microenvironment of bone marrowObjective The three-dimensional(3D)marrow microenvironment plays an essential role in regulating human cord blood-derived CD34+Cells(hCB-CD34+)migration,proliferation and differentiation.Extensive in vitro and in vivo studies have aimed to recapitulate the main components of the bone marrow(BM)niche.Nonetheless,the models are limited by a lack of heterogeneity and compound structure.Here,we fabricated coaxial extruded core-shell tubular scaffolds and extrusion-based bioprinted cell-laden mesh scaffolds to mimic the functional niche in vitro.Methods A multicellular mesh scaffold and two different core-shell tubular scaffolds were developed with human bone marrow-derived mesenchymal stromal cells(BMSCs)in comparison with a conventional 2D co-culture system.A clear cell-cell connection was established in all three bioprinted constructs.Cell distribution and morphology were observed in different systems with scanning electron microscopy(SEM).Collected hCB-CD34+cells were characterized by various stem cell-specific and lineage-specific phenotypic parameters.Results The results showed that compared with hCB-CD34+cells co-cultured with BMSCs in Petri dishes,the self-renewal potential of hCB-CD34+cells was stronger in the tubular sacffolds after 14 days.Besides,cells in these core-shell constructs tended to obtain stronger differentiation potential of lymphoid and megakaryocytes,while cells encapsulated in mesh scaffolds obtained stronger differentiation tendency into erythroid cells.Conclusions 3D bioprinting technology could partially simulate the niche of human hematopoietic stem cells.The three models have their potential in sternness maintenance and multilineage differentiation.This study can provide initial effective guidance in the directed differentiation research and related screening of drug models for haematological diseases.Part ? Construction of an in vitro osteogenetic model of composite bio-ink for 3D bioprintingObjective The optimization of the of biomaterials is an important method to strengthen the 3D model of bone tissue regeneration in vitro.In this study,a new type of composite bio-ink was prepared by synthesizing polydopamine-coated nano-hydroxyapatite particles(DHA).By mixing DHA with GelMA or GelMA with collagen fibres,we used the different formulations to bioprint adipose-derived mesenchymal stem cells(ADSC)laden scaffolds with a digital light processing(DLP)bioprinter.The ability of scaffolds to induce osteogenic differentiation in vitro was detected.Methods Compared with the pure GelMA hydrogel,we set up groups as follows:GelMA+DHA with low/high concentration,GelMA+nano hydroxyapatite particles(nHA),GelMA+DHA with low concentration+collagen.Bio-ink formulations based on 5%GelMA and 0.5%photoinitiator LAP were used to print cell-free scaffolds and the characteristics of the materials were explored,including water absorption,swelling,structural stability,protein release,enzyme digestion,and mechanical properties.After NIH 3T3 cells-laden scaffolds were bioprinted,the biocompatibility of each groups and the ability to promote cell proliferation were evaluated.Osteogenesis was induced in vitro in the ADSCs-laden scaffolds.Cell viability and alkaline phosphatase(ALP)activity,an osteogenic induction marker,were detected during the culture process.The expression of osteogenic markers like type I collagen and RUNX2 was detected by immunofluorescence assay.The degree of tissue mineralization was evaluated by histochemical staining.Scanning electron microscopy(SEM)was used to observe the interaction between the cells and between the cells and materials.Results Compared with pure hydrogel bio-ink,composite bio-ink obtained stronger mechanical properties and helped maintain a stable structure for a long time to support cell proliferation.The hydrogel added with DHA enhanced the cell interaction in the porous network of the scaffolds,and enhanced the expression of osteogenic signals such as type I collagen and RUNX2.In addition,histochemical staining also suggested that the degree of mineralization in the DHA added group was significantly increased.The microstructure under the SEM also demonstrated that the DHA composite material promoted cell adhesion and proliferation.Conclusion This study suggested that GelMA with DHA as a composite bio-ink can promote the ability of DLP printed scaffolds loaded with ADSCs to induce bone formation in vitro.The high biocompatibility,enhanced mechanical properties and the ability to promote bone differentiation of this composite material make it possible to become a promising solution to the problem of bone tissue repair.