Zhang, QunzhouNguyen, PhuongBurrell, Justin CZeng, JinchengShi, ShihongShanti, Rabie M.Kulischak, GraceLe, Anh DCullen, D Kacy2023-05-222023-05-222021-12-012022-06-08https://repository.upenn.edu/handle/20.500.14332/9071Achieving a satisfactory functional recovery after severe peripheral nerve injuries (PNI) remains one of the major clinical challenges despite advances in microsurgical techniques. Nerve autografting is currently the gold standard for the treatment of PNI, but there exist several major limitations. Accumulating evidence has shown that various types of nerve guidance conduits (NGCs) combined with post-natal stem cells as the supportive cells may represent a promising alternative to nerve autografts. In this study, gingiva-derived mesenchymal stem cells (GMSCs) under 3D-culture in soft collagen hydrogel showed significantly increased expression of a panel of genes related to development/differentiation of neural crest stem-like cells (NCSC) and/or Schwann cell precursor-like (SCP) cells and associated with NOTCH3 signaling pathway activation as compared to their 2D-cultured counterparts. The upregulation of NCSC-related genes induced by 3D-collagen hydrogel was abrogated by the presence of a specific NOTCH inhibitor. Further study showed that GMSCs encapsulated in 3D-collagen hydrogel were capable of transmigrating into multilayered extracellular matrix (ECM) wall of natural NGCs and integrating well with the aligned matrix structure, thus leading to biofabrication of functionalized NGCs. In vivo, implantation of functionalized NGCs laden with GMSC-derived NCSC/SCP-like cells (designated as GiSCs), significantly improved the functional recovery and axonal regeneration in the segmental facial nerve defect model in rats. Together, our study has identified an approach for rapid biofabrication of functionalized NGCs through harnessing 3D collagen hydrogel-directed conversion of GMSCs into GiSCs. © 2021, The Author(s).Cell cultureCell signalingCollagenGenesMolecular biologyNeuronsStem cells5' nucleotidasebrain derived neurotrophic factorcell surface markercollagencollagen gelcomplementary DNAcytoskeleton proteindll1 proteindll4 proteinepidermal growth factor receptorerbb receptor feedback inhibitor 1F actinglial cell derived neurotrophic factorglial cell line derived neurotrophic factorglial fibrillary acidic proteinHermes antigenhuman insulinhydrogelinhibitor of differentiation 1neurotrophic factorNotch3 receptorprotein Jagged 2protein p75protein S100BThy 1 membrane glycoproteintranscription factor Ets 1transcription factor Ets 2transcription factor HES 1transcription factor HES 4transcription factor hes 7transcription factor hey 1transcription factor juntranscription factor Slugtranscription factor snail1transcription factor Sox10transcription factor Sox9transcription factor tbx3transcription factor Twistunclassified drugvinculinBiofabricationFunctional recoveryFunctionalizedGold standardsMesenchymal stem cellNerve conduitsNerve injuriesNeural crestsPeripheral nervesSchwann cellsanimal cellanimal experimentanimal modelArticlecell differentiationcell encapsulationcell migrationcomparative studycontrolled studyextracellular matrixfacial nervefacial nerve injuryfemalegene expressiongingivagingiva derived mesenchymal stem cellgingival tissuehumanhuman cellhuman tissuein vivo studymesenchymal stem cellmuscle action potentialnerve conduction velocitynerve fiber regenerationneural crest cellnonhumanprotein expressionratreal time polymerase chain reactionSchwann cellsignal transductionthree dimensional cell culturetwo dimensional cell cultureupregulationHydrogelsDentistryOral and Maxillofacial SurgeryOther DentistryArticle