In vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells/osteoblasts combined with PLLA/HAp nanostructured aerogel scaffolds

Abstract

<h3>Objectives</h3> Osteoblast deficiency is a key problem in bone tissue engineering, and transplant of mesenchymal stem cells combined with osteoblasts can achieve ideal results. We sought to investigate the in vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells (ADSCs) and osteoblasts (OBs) combined with poly(l-lactic acid) (PLLA)/hydroxyapatite (HAp) nanostructured aerogel scaffolds. <h3>Methods</h3> We tested a new supercritical fluid-assisted technique for the formation of nanostructured aerogel scaffolds. We obtained PLLA aerogel scaffolds and PLLA/HAp aerogel scaffolds with high level of porosity (>90%), interconnectivity, and mechanical properties (compressive modulus up to 2 kPa). The fibrous nanostructure of these scaffolds was joined to micronic cells of controllable size. ADSCs and OBs were obtained by using an adherent method and an enzymatic digestion method. ADSCs, OBs, and the mixture of ADSCs and OBs (at a ratio of 1:1) were cultured with PLLA/HAp nanostructured aerogel scaffolds, respectively. After 48 hours of in vitro culture, cell-scaffold complexes were subcutaneously implanted into the back of Sprague-Dawley rats in corresponding groups, and PLLA/HAp nanostructured aerogel scaffolds without cells were implanted in a control group. The rats in each group were killed at 8 weeks postoperatively. The macroscopic and histopathological observations were performed to assess the ectopic osteogenesis potential. <h3>Results</h3> After adipogenic, chondrogenic, and osteogenic induction, ADSCs were positive for Oil Red O, toluidine blue, and alizarin red staining. Results of flow cytometry showed that ADSCs were positive for CD147, CD90, CD105, and CD44, with the rate of positivity being >80%, but negative for CD117, CD34, CD131, and CD45, with the rate of positivity being <5%. Passage 3 OBs were positive for both alizarin red staining and alkaline phosphatase staining. At 8 weeks after implantation, soft tissues grew into the complexes under gross observation. At 8 weeks after implantation, ectopic bone formation was visible in each group. The bone formation was more visible in the ADSC-PLLA/HAp nanostructured aerogel scaffold group than in the other groups with a significant difference (<i>P</i> < .05). <h3>Conclusions</h3> To conclude, ADSCs can promote the ectopic bone formation of OBs in vivo in combination with PLLA/HAp nanostructured aerogel scaffolds.