3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

Abstract

Despite the promising neuro-regenerative capacities of stem cells, there is currently no licensed stem cell-based product in the repair and regeneration of peripheral nerve injuries. Here, we explored the potential use of human gingiva-derived mesenchymal stem cells (GMSCs) as the only cellular component in 3D bio-printed scaffold-free neural constructs that were transplantable to bridge facial nerve defects in rats. We showed that GMSCs have the propensity to aggregate into compact 3D-spheroids that could produce their own matrix. When cultured under either 2D- or 3D-collagen scaffolds, GMSC spheroids were found to be more capable of differentiating into both neuronal and Schwann-like cells than their adherent counterparts. Using a scaffold-free 3D bio-printer system, nerve constructs were printed from GMSC spheroids in the absence of exogenous scaffolds and allowed to mature in a bioreactor. In vivo transplantation of the GMSC-laden nerve constructs promoted regeneration and functional recovery when used to bridge segmental defects in rat facial nerves. Our findings suggest that GMSCs represent an easily accessible source of MSCs for 3D bio-printing of scaffold-free nervous tissue constructs with promising potential application for repair and regeneration of peripheral nerve defects.

A scaffold‐free Bio 3D nerve conduit for repair of a 10‐mm peripheral nerve defect in the rats

“…After a specific period of time, adjacent spheroids spontaneously fuse with each other and their 3D structure and form is maintained even when the needle array is decannulated (Moldovan et al, ). Tubular materials such as vessels (Itoh et al, ), tracheae (Taniguchi et al, ), and nerve conduits (Yurie et al, ; Zhang et al, ) have been fabricated using this “Bio‐3D printer.” The cell types used to form these structures have included fibroblasts, endothelial cells or mesenchymal stromal cells of human origin, thereby confirming that these structures can be created using a patient's own cells.…”

“…Various tissues other than tubular structures, such as cartilage or myocardium, have also been fabricated using Bio 3D printing technology. The unique aspect of our method of 3D printing is the use of spheroids as the “building blocks” (Moldovan et al, ) or “bio ink” (Zhang et al, ) and the placement of them into a pre‐determined needle array. Inserted spheroids spontaneously fuse with neighboring spheroids, and their structure is maintained after removal from the needle array.…”

“…The Bio 3D conduits were fabricated by using a “Bio‐3D printer (Regenova®, Cyfuse, Tokyo, Japan)” to assemble the spheroids for constructing a tubular structure, as previously described (Itoh et al, ; Moldovan, Hibino, & Nakayama, ; Ong, Zhou, Han, et al, ; Takeoka, Matsumoto, Taniguchi, et al, ; Taniguchi et al, ; Yurie et al, ; Zhang et al, ). Briefly, spheroids were robotically picked out from the 96‐well plate using a 25‐gauge suction nozzle and inserted into a needle array unit according to a 3‐dimensional computer‐aided design that specified the shape of the tubular construct (Figure b).…”

“…A “Bio‐3D printer (Regenova®, Cyfuse, Tokyo, Japan)” can fabricate tubular materials such as vessels (Itoh, Nakayama, Noguchi, et al, ), tracheae (Taniguchi, Matsumoto, Tsuchiya, et al, ), and nerve conduits (Yurie, Ikeguchi, Aoyama, et al, ; Zhang et al, ) exclusively from cells without added foreign materials. The Bio 3D nerve conduit was fabricated by placing spheroids, which were generated from normal human dermal fibroblasts, into a needle array to form a tubular structure using a “Bio‐3D printer.” Biomaterial was not used during its fabrication.…”

“…We previously reported that nerve regeneration was promoted by a Bio 3D printed nerve conduit without cytotoxic inflammation; the substantial cellular composition and presence of ECM components aided the regenerative process through its biocompatibility. However, only a 5 mm nerve gap length was reported to be regenerated with a Bio 3D printed nerve conduit (Yurie et al, ; Zhang et al, ). The purpose of the present study was to fabricate Bio 3D conduits for a 10 mm nerve gap and to evaluate their capacity for nerve regeneration in a rat sciatic nerve defect model.…”