| BackgroundMesenchymal stem cells(MSCs)are multipotent stem cells with multiple differentiation potential derived from various tissues,which has the characteristics of easy separation and amplification,multi-directional differentiation and low immunogenicity.Comparing with bone marrow mesenchymal stem cells(BMSCs),adipose derived mesenchymal stem cells(Ad-MSCs)have the advantages of abundant source,easy to obtain,easy to isolate and culture,low invasive,stable biological characteristics and so on.Therefore,it is considered to be an ideal seed cell in tissue engineering and regenerative medicine.The materials that is to forming tissue scaffolds need good biocompatibility,strong plasticity,high tensile strength and no immunogenicity.The major materials that manufacturing tissue engineering models including Collagen enzyme,polylactic acid,alginate and so on.However,every biological materials we mentioned have some limitations,in reality,it is difficult to control these biomaterials.Recently,the nano framework materials made up from self-assembly of nano peptide molecules that polypeptide is the basic structure unit have become a new research focus in the field of tissue engineering.This kind of nano peptide biological material has good biocompatibility and degradability,And the most prominent advantage is that it has biological activity,which endows the scaffold material with biological intelligence.With the rapid development of 3D printing technology,3D printing has been widely used in the field of Medicine.Which providing conditions to accurate and personalized medical.At the present,the high density printing of tissue engineering has been deeply explored and studied.Manufacturing the tissue engineering models by 3D printing technology with the using of so called "bio ink",making up from functionalized serf-assembling peptide hydrogel and Ad-MSCs.The Ad-MSCs with in the models were cultured in vitro,and the growth and differentiation of the cells were observed under different conditions.Studying on the feasibility of functional self-assembling nano peptide as a 3D printing material loaded with Ad-MSCs,exploring the new development direction of 3D printing.ObjectionThe purpose of this experiment is to observe the cells' growth status and multilineage within the tissue engineering models which manufactured by 3D printing technology with the using of so called "bio-ink",making up from functionalized serf-assembling peptide hydrogel and Ad-MSCs.Methods1.The primary Ad-MSCs was isolated and cultured,then it was subculture to P2,P3 for the following experiments.Flow cytometry was used to identify and analyze the cell immunophenotype;The cells were stained with phalloidin to observe the microfilament structures;The cells were differentiated into endothelial,osteogenic and adipogenic differentiation,then identified respectively.2.Dissolving three polypeptides with 10%sucrose-deionized-water-solution(RADA16-I,RGD,KLT).Mixing these three polypeptide-solutions according to the required concentration and volume ratio.Observing the mixed solution with a Atomic Force Microscopy(AFM).Put the mixed solution into Transwell,in order to form hydrogel.Scanning electron microscopy was used to observe the hydrogel.Three kinds of polypeptide-solutions were mixed with the cells to form hydrogel,and then cultured in vitro.3.Constructing the tissue engineering models by a 3D bio-printer with the bio-ink which consist of functionalized serf—assembling peptide hydroge and Ad-MSCs.Staining sliced bio-printed models with phalloidin.Observing the ability of multi-directional differentiation of Ad-MSCs in the models:Inducting osteogenic and adipogenic differentiation of Ad-MSCs in 3D models.Comparing with the contrast groups,after alizarin red and oil red O was stained on the models.Results1 The cells we isolated and cultured can be seen as a shuttle shape,and presented a vortex like growth trend.The cells we isolated and cultured expressed a cell-surface protein profile positive for CD 166,CD90 and CD29 and negative for CD31,CD34,CD45 and HLA-DR.Phalloidin staining showed the microfilament structures.After the induction of osteogenic and adipogenic differentiation,staining the cells with corresponding stains,we can see the characteristic colorings.2..Observing the self-assembing peptide hydrogel by an atomic force microscope(AFM),we can see that the nanofibers structures.The polypeptide hydrogel is colorless and transparent,and it is riched in water.The hydrogel was observed by scanning electron microscopy,and the structures of nano fiber was observed,the gaps can also be seen.The cells in the hydrogel which consist of polypeptide solutions and Ad-MSCs are arranged closely and in good condition.3.The tissueengineeringmodels were successfully constructed by the mixture solution.Ad-MSCs in the models had a good cellular growth behavior.After osteoinductive differentiation,Alizarin red S staining verified the formation of mineralized nodules.After adipogenic differentiation,Oil red O staining verified the formation of lipid droplets.Neither of the control groups colored.ConclusionAd-MSCs in the tissue engineering models which constructed by 3D bio-printer with the mixture solution had a good cellular growth behavior,and also had a strong ability of differentiation. |
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