Research On The Functional Hydrogel-based Biofabrication Of Stem Cell-embedded Microspheres And Induction Of Biological Anisotropy

Objective:Our study reports the optimization of electrospray human bone marrow stromal cells(hBMSCs)embedded Alginate-Gelatin(Alg-Gel)microspheres for the purpose of their assembly in 3D printed poly(s-caprolactone)(PCL)scaffold for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct.Then,uses human tendon stem/progenitor cells(hTSPCs)and Collagen type I(COL I)mix with the Iron Oxide nanoparticles(IOPs)in the magnetic field to induce the COL I-IOPs-hTSPCs anisotropy for promoting cell proliferation and tenogenic differentiation for the purpose of their application in tissue engineering.Methods:The fabrication of the Alg-Gel microspheres using an electrospray technique was optimized in term of polydispersity,yield of microspheres and circularity,varying fabrication conditions.PCL scaffolds were designed and printed by melt-extrusion.Then four groups were set:Alg-hBMSCs microspheres cultured in 2D well plate group(Alg-hBMSCs+2D),Alg-Gel-hBMSCs microspheres cultured in 2D well plate group(Alg-Gel-hBMSCs+2D),Alg-Gel-hBMSCs microspheres embedded in 3D PCL scaffold cultured in 2D well plate group(Alg-Gel-hBMSCs+PCL+2D)and Alg-Gel-hBMSCs microspheres cultured in 3D bioreactor group(Alg-Gel-hBMSCs+3D).Cell viability,proliferation and chondrogenic differentiation were evaluated and mechanical test was performed.Then,the hTSPCs embedded in COL I group(COL I-hTSPCs),hTSPCs embedded in COL I with random IOPs group(COL I-R/IOPs-hTSPCs)and hTSPCs embedded in COL I with aligned IOPs group(COL I-A/IOPs-hTSPCs)were set.COL I-A/IOPs-hTSPCs hydrogel was fabricated and induced anisotropy in the magnetic field before gelated.After cross-linking and gelling,the stable and anisotropic structure was formed.Contraction of hydrogel,cell viability,proliferation,alignment of IOPs and hTSPCs,cell morphology and the formation of cell strings were evaluated and the expression of tenogenic related genes and lineage/cross-linking genes were analyzed.Results:Non-aggregated,low polydispersity and almost spherical microspheres of average diameter of 200-300 μm were produced with Alginate 1.5 w:v%,Gelatin(type B)concentration of 0.5 w:v%and CaCl2 coagulating bath concentration of 3.0 w:v%,using 30G needle size and 8kV and 0.6bar voltage and air pressure respectively.Alginate with Gelatin hydrogel improved viability and promoted hBMSCs proliferation better than Alginate microspheres.Interestingly,hBMSCs embedded in microspheres assembled in 3D printed PCL scaffold and cultured in 3D bioreactor were more proliferative in comparison to the previous two groups(p<0.05).Similarly,the GAG content,GAG/DNA ratio as well as COL Ⅱ and ACAN gene expression were increased in the last two groups(p<0.05).For the anisotropic biomaterial-cell exploration,the detached hydrogel was contracted heavily even more than 80%during the first 24 hours while the non-detached samples showed the homogeneous content in all the three group.The cell proliferation in the anisotropic COL I-A/IOPs-hTSPCs group was significantly better than the other two groups from day 3(p<0.05)and on day 7 as well(p<0.05).The hTSPCs in anisotropic group had a more stretched morphology with a higher cell aspect ratio on day 7(p<0.05),indicated the potential of tenogenic differentiation.Also,the cells in anisotropic group aligned with the same direction as the A/IOPs during the culture,while the other two groups showed the random cells without the homogenic arrangement,which showed the anisotropy promote the proliferation of the hTSPCs by its alignment.Besides,the anisotropy induced the hTSPCs to contact with each other lengthways forming the cell strings and both the cell strings number and cell number in one string were significant more than the other groups(p<0.05).During the experiment,the alignment of A/IOPs decreased while the hTSPCs in anisotropic group increasing and was even higher than the A/IOPs on day 7.Lastly,the tenogenic related genes ACTA2,THBS4,TNC,TGF β1,tendon related collagen genes COL1A1,COL6A1,transcription factor gene SCX and collagen cross-linking genes BGN,FN of the hTSPCs in anisotropy group expressed higher than the other groups(p<0.05)and no significate difference for the other differentiation related genes in all the three groups(p>0.05).Conclusion:Optimization of hBMSCs embedded Alg-Gel microspheres produced by electrospray has been performed.The Alg-Gel composition selected allows conservation of hBMSCs viability,support proliferation and matrix deposition.The possibility to seed and assemble microspheres in designed 3D printed PCL scaffolds for the fabrication of a mechanically stable and biological supportive tissue engineering cartilage construct was demonstrated.Induced anisotropic COL I-A/IOPs-hTSPCs hydrogel can significantly promote the hTSPCs of homogeneous linear array,make them more tendon cell-like with the contact to each other lengthways,and achieved higher cell viability and proliferation.Moreover,it also has higher in terms of tenogenic differentiation into tendon related gene expression,for combined with biological anisotropy and early repair of vital tissue and organ injuries,opening up new ideas,providing the theoretical support for clinical transformation.