Construction Of Cell Sheet Using Electrospun Fibrous Membrane For Bone Defect Regeneration

Periodontal and bone defects are remaining common clinical challenges, and guided tissue regeneration (GTR) is the most effective treatment to repair them. The key for GTR treatment is the use of GTR membrane, therefore, studies on improving the performance of GTR membrane is of great significance. Tissue engineering provides new approaches for periodontal and bone regeneration, but there are still many challenges to achieving satisfactory new bone formation. For instance, in the conventional process of cell harvest, the use of proteolytic enzymes would like to destroy the fibronectin on cell membrane and cell junction, which are vital for the following cell attachment, proliferation and differentiation. Besides, cell loss is frequently associated with the conventional procedures for cell transplantation. To ameliorate these issues, recently, cell sheet engineering has been highlighted for its advantages in preserving cell function and be used directly as implants.In this study, we provided a new method to construct cell sheet basing on electrospun fibrous membranes. Briefly, poly(L-lactide) (PLLA)/Gelatin fibrous meshes, with fibers arranged nest-like or randomly, were electrospun by using different collectors. Bone mesenchymal stem cells (BMSC) were then cultured on the two kinds of fibrous meshes, and the possibility of getting BMSC sheet/mesh complexes was investigated. The formation of cell sheet was confirmed by HE staining and immunofluorescence staining. The osteogenic differentiation of cell sheet was examined by ALP, Ca content and type I collagen assay, as well as expressions of osteogenic genes including ALP, BMP2, OPN and Runx2. Both the fibrous meshes demonstrated good cell affinity, enhanced both the proliferation and osteogenic differentiation of BMSC. Noticeably, the nest-like fibrous mesh displayed stronger ability in promoting the osteogenic differentiation of BMSC than the non-woven fibrous mesh.Multi-layered cell sheet constructs were obtained by stacking the aforementioned cell/mesh complexes layer-by-layer, and the structure was confirmed by HE staining and immunofluorescence staining. To evaluate the potential of using the multi-layered cell sheet construct in bone reparation, the construct was implanted into a rat critical cranial detect. At 4 and 12 weeks post-operation, micro-CT, histological staining and immumohistochemical staining were taken to evaluate the new formation. As the results shown, cell sheet technology could promote the calcification of bone matrix and the maturation of trabecular bone, and thus significantly enhanced the bone regeneraion. The in vivo results also demonstrated that cell constructs based on nest-like fibrous meshes were somewhat more efficient in enhancing bone regeneration than those based on non-woven fibrous meshes, however, no highly significant difference was identified between the group.In a whole, single cell sheet complex and multi-layered cell sheet construct can be easily prepared by using electrospun fibrous meshes. The fiber arrangement can have some effects on cell proliferation and differentiation. The obtained cell sheet complex and construct can be easily handled and implanted into bone defect area to induce bone regeneration. Promisingly, the novel cell sheet complex and construct demonstrated strong potentials as repairing substrates in periodontal and bone regeneration.