Evaluation of human dental stem cell growth characteristics and cellular morphological changes in response to extracellular matrix nanotopography

Objetivo: Nanotopografia e fatores extracelulares solúveis estão presentes nas célulastroncoda polpa dentária Seu efeito na sobrevivência e diferenciação de células-tronco dentárias ainda não foi estabelecido. Nosso objetivo foi analisar os papéis individuais e combinados da nanotopografia e do soro (fatores solúveis) da matriz extracelular (MEC) no crescimento, potencial de diferenciação e características morfológicas das células-tronco da polpa dentária humana. Este estudo avaliou e comparou a resposta detais células-tronco a diferentes estímulos ambientais – nanofibras, soro e meios condicionados. Materiais e métodos: Neste estudo, nanofibras de PLLA fabricadas foram usadas como meio biomimético estrutural in vitro da nanotopografia nativa encontrada na polpa de células-tronco/MEC in vivo. O soro e o meio condicionado foram utilizados como o imitador in vitro dos fatores solúveis aos quais as células-tronco são expostas in vivo. As células-tronco foram cultivadas na presença e ausência de nanofibras de ácido poli-L lático biodegradáveis e soro. As características de crescimento das células-tronco da polpa dentária humana foram avaliadas em termos de viabilidade celular e tempo de duplicação no intervalo de cada passagem. As alterações morfológicas celulares foram estudadas usando microscopia invertida e coloração H&E. Como segunda parte do estudo, as células em todas as condições de cultura foram expostas ao Meio Condicionado para Polpa Dentária (MCPD) por um curto período de 3 dias. As características de crescimento e as alterações morfológicas das células foram avaliadas após a exposição curta ao MCPD. Além disso, a microscopia eletrônica de varredura foi utilizada para o estudo morfológico das células-tronco em nanofibras, expostas aos meios condicionados. As células diferenciadas foram analisadas por TR-PCR quantitativa quanto à expressão neurogênica e odontogênica dos genes RUNX2, osteopontina e β-tubulina III. Resultados: As células-tronco dentárias apresentaram melhor sobrevida e proliferação na presença de nanofibras e soro. A ausência de nanofibras ou soro alterou a sobrevivência e proliferação de células-tronco de forma significativa e indicou diferenciação. Além disso, observou-se que, após a exposição curta ao MCPD, a presença de nanofibras e soro de PLLA favoreceu maior potencial de diferenciação odontogênica e neurogênica, sem alterações morfológicas características da diferenciação terminal. Conclusão: Células-tronco da polpa dentária humana são capazes de detectar sinais geométricos em nanoescala de seu microambiente. Nanotopografia e fatores solúveis da matriz extracelular afetam as células-tronco. Estudos adicionais são essenciais para identificar os principais caminhos que desempenham um papel vital nessas interações.Objective: Nanotopography and soluble extracellular factors are present in the dental stem cell niche in the pulp. Their effect on dental stem cell survival and differentiation is yet to be established. We aimed to analyze the individual and combined roles of extracellular matrix (ECM) nanotopography and serum (soluble factors) on the growth, differentiation potential, and morphological characteristics of the human dental pulp stem cells (hDPSC). This study aimed to evaluate and compare the hDPSC response to different environmental cues – nanofibers, serum, and conditioned media. Materials and methods: In this study, fabricated PLLA nanofibers were used as the in vitro structural biomimetic of the native nanotopography found in the in vivo ECM/stem cell niche. Serum and conditioned media were used as the in vitro mimic of the soluble factors to which stem cells get exposed in vivo. hDPSC were grown in the presence and absence of biodegradablepoly-L-lactic-acid nanofibers and serum. The growth characteristics of hDPSC were assessed in terms of cell viability and doubling time at the interval of every passage. Cellular morphological changes were studied using inverted microscopy and H&E. As the second part of the study, hDPSC in all culture conditions were exposed to Dental Pulp Conditioned Media (DPCM) for a short duration of 3 days. After transient exposure to DPCM, the growth characteristics and the morphological changes of hDPSC were assessed. In addition, scanning electron microscopy was used for the morphological study of hDPSC on nanofibers, exposed to conditioned media. The differentiated cells were analyzed by qRT-PCR for neurogenic and odontogenic expression of RUNX2, osteopontin, and β-tubulin III genes. Results: hDPSC showed better survival and proliferation in the presence of nanofibers and serum. Absence of nanofibers or serum greatly altered stem cell survival and proliferation and also indicated differentiation. In addition, it was observed that after transient exposure to DPCM, the presence of both PLLA nanofiber and serum favoured higher odontogenic and neurogenic differentiation potential, without characteristic morphological changes of terminal differentiation. Conclusion: hDPSC has the ability to sense nanoscale geometric cues from their microenvironment. Nanotopography and soluble factors of the extracellular matrix both affect hDPSC. Further studies are essential to identify the key pathways that play a vital role in such interactions. The hDPSC demonstrated better survival and proliferation in the presence of nanofibers and serum. Absence of nanofibers or serum greatly altered stem cell survival and proliferation and also showed changes indicative of differentiation. The results were compared and analyzed using GraphPad Prism 5 Software. hDPSC possess the ability to sense nanoscale geometric cues from their microenvironment. Nanotopography and soluble factors of the Extracellular matrix together influence the fate of hDPSC. Further studies are essential to identify the key pathways that play a vital role in such interactions

Biodegradable nanofiber coated human umbilical cord as nerve scaffold for sciatic nerve regeneration in albino Wistar rats

Background: Human umbilical cord (hUC) is encompassed by a mucoid connective tissue called Wharton’s jelly (WJ), made of hyaluronic acid, collagen, and stromal cells to support the blood vessels of hUC. This study was aimed to determine the in vitro neuronal differentiation of Wharton’s jelly-derived Mesenchymal stem cells (WJMSCs), and in vivo axonal regeneration potential of nanofiber coated human Wharton’s jelly as a neuronal graft after sciatic nerve injury in immunosuppressed Albino Wistar rats. Materials and methods: WJMSCs could be differentiated to neuron-like cells by inducing with neuronic supplementing media. The test animal’s axotomized nerves were implanted with trimmed human umbilical cord devoid of vascularity and nanocoated with electro-spun PLLA nanofibers. The control animals were bridged with native sciatic nerve reversed and sutured. Post-surgical functional recovery was studied by walking track, pinprick, muscle weight, and sweating quantification. At the end of the 4th week, the animals were euthanized, and Magneto Neuro graph (MNG) was performed. The explanted grafts were quantified by immunohistochemistry for immuno-rejection, neural scarring, neural adhesion axon regeneration, fiber diameter, myelin thickness, and g-ratio. The sciatic function index (SFI) values were similar by walking track analysis for both the test and control groups. Results:  The animals had functional and sensation recovery by the end of two weeks. No mortality, signs of inflammation, and acute immune rejection were observed post-surgery. Conclusions: The hUCWJ devoid of vascular elements can be a perfect peripheral nerve graft, and we hypothesis that the cryopreserved hUC could be an ideal resource for axonal regeneration in the future