Layer-by-layer paper-stacking nanofibrous membranes to deliver adipose-derived stem cells for bone regeneration

Bone tissue engineering through seeding of stem cells in three-dimensional scaffolds has greatly improved bone regeneration technology, which historically has been a constant challenge. In this study, we researched the use of adipose-derived stem cell (ADSC)-laden layer-by-layer paper-stacking polycaprolactone/gelatin electrospinning nanofibrous membranes for bone regeneration. Using this novel paper-stacking method makes oxygen distribution, nutrition, and waste transportation work more efficiently. ADSCs can also secrete multiple growth factors required for osteogenesis. After the characterization of ADSC surface markers CD29, CD90, and CD49d using flow cytometry, we seeded ADSCs on the membranes and found cells differentiated, with significant expression of the osteogenic-related proteins osteopontin, osteocalcin, and osteoprotegerin. During 4 weeks in vitro, the ADSCs cultured on the paper-stacking membranes in the osteogenic medium exhibited the highest osteogenic-related gene expressions. In vivo, the paper-stacking scaffolds were implanted into the rat calvarial defects (5 mm diameter, one defect per parietal bone) for 12 weeks. Investigating with microcomputer tomography, the ADSC-laden paper-stacking membranes showed the most significant bone reconstruction, and from a morphological perspective, this group occupied 90% of the surface area of the defect, produced the highest bone regeneration volume, and showed the highest bone mineral density of 823.06 mg/cm(3). From hematoxylin and eosin and Masson staining, the new bone tissue was most evident in the ADSC-laden scaffold group. Using quantitative polymerase chain reaction analysis from collected tissues, we found that the ADSC-laden paper-stacking membrane group presented the highest osteogenic-related gene expressions of osteocalcin, osteopontin, osteoprotegerin, bone sialoprotein, runt-related transcription factor 2, and osterix (two to three times higher than the control group, and 1.5 times higher than the paper-stacking membrane group in all the genes). It is proposed that ADSC-laden layer-by-layer paper-stacking scaffolds could be used as a way of promoting bone defect treatment.