Effect of 3D bioprinting on osteogenic differentiation of gelatin/sodium alginate/laponite composite cellular hydrogel scaffolds / 中华创伤杂志

ABSTRACT

Objective:To prepare biomimetic tissue engineering scaffolds of gelatin/sodium alginate/laponite composite hydrogel loaded with BMSCs by 3D biological printing technique，and explore the osteogenic effect of 3D printing on hydrogel scaffolds containing bone marrow mesenchymal stem cells（BMSCs）.Methods:BMSCs were routinely extracted and identified by flow cytometry. Gelatin，sodium alginate and laponite were mixed and then BMSCs were added to prepare cell-containing composite hydrogel scaffolds using 3D bioprinting. Non-printed scaffolds containing cells were prepared by injection molding method. In vitro，the prepared scaffolds were divided into the printing group with cells and non-printing group with cells according to whether they were printed，with 12 samples per group. Another simple cell culture group was set as control. Then，the internal structure of the composite hydrogel was observed by scanning electron microscope，and the expansion rate and water content of the scaffolds were measured by freeze-drying method. At day 3 after culture，the growth status of BMSCs was observed by phalloidine staining. cell counting kit（CCK）-8 assay was used to detect cell activity in scaffolds at days 1，3，and 7 after culture and RT-PCR to detect the expression of osteogenesis related genes Osterix，osteocalcin（OCN）and collagen I at days 7 and 14 ofter culture. In vivo，four groups were set according to printing or not and whether containing cells or not：printing implant group with cells，non-printing implant group with cells，printing implant group without cells and non-printing implant group without cells，with 9 samples per group. Scaffolds in four groups were implanted to the posterior gluteal muscle pouches（random on left or right）of 36 8-week-old SD rats，respectively. The samples were taken X-ray images at 2，4 and 8 weeks after operation，respectively. The osteogenic differentiation of tissues at 8 weeks was observed by HE and Masson staining. Results:The flow cytometry showed that the cells were BMSCs. Internal pores of hydrogels were obvious，and cells stretched freely in the pores. Differences of the swelling rate and water content were not statistically significant between printing group with cells［（1，039.37±30.66）%，（91.21±0.26）%］and non-printing group with cells［（1，032.38±35.05）%，（91.16±0.28）%］（ P>0.05）. At day 3 after culture in vitro，the cells grew well in the hydrogel. After culturing for 1 day in vitro，there was no significant difference in absorbance between printing group with cells and non-printing group with cells（ P>0.05）. At day 3 after culture，there was no significant difference in absorbance between printing group with cells and non-printing group with cells，but both groups showed a higher level than simple cell culture group（ P<0.05）. At day 7 after culture，the absorbance in printing group with cells（2.72±0.17）was higher than that in non-printing group with cells（2.35±0.11），and both of which were higher than that in simple cell culture group（1.95±0.12）（ P<0.05）. At day 7 after culture in vitro，there was no statistically significant difference in the expression of osteogenic differentiation-related genes between printing group with cells and the non-printing group with cells（ P>0.05），but they were all higher than those in simple cell culture group（ P<0.05）. At day 14 after culture in vitro，the expression of osteogenesis-related genes Osterix（1.650±0.095），OCN（2.725±0.091），collagen I（2.024±0.091）in printing group with cells were higher than those in non-printing group with cells（1.369±0.114，2.174±0.198，1.617±0.082，respectively）and those in simple cell culture group（1.031±0.094，1.116±0.092，0.736±0.140，respectively）（ P<0.05）. After implantation for 2 weeks in vivo，with no statistically significant difference in the gray values of X-ray films in each group（ P>0.05）. At weeks 4 and 8 after implantation，the gray values of X-ray films in printing implant group with cells and non-printing implant group with cells were higher than those in printing implant group without cells and non-printing implant group without cells（ P<0.01）. At 8 weeks after implantation，HE staining showed that the scaffolds were degraded in different degrees and immersed with cells，with collagen production seen in Masson staining as well. Conclusions:Composite hydrogel scaffolds can provide a good three-dimensional environment for BMSCs growth. 3D bioprinting can promote the proliferation and osteogenic differentiation of BMSCs in hydrogel scaffolds. In addition，BMSCs-loaded scaffolds can be degraded slowly in vivo with good ectopic osteogenic ability.