RESUMEN
Here, we investigated the synergistic effect of electrospun nanofibrous scaffolds made of gelatin /sulfated hyaluronan (sHA) or native hyaluronan (HA)/chondroitin sulfate (CS) and, keratinocytes (HaCaT)-human mesenchymal stem cells (hMSCs) contact co-culture on epithelial differentiation of hMSCs. The hMSCs were co-cultured in contact with HaCaT cells for 5 days on electrospun scaffold. Results show that electrospun scaffolds containing sulfated glycosaminoglycans (GAGs) stimulate epithelial differentiation in terms of various protein expression markers (keratin 14, ΔNp63α and Pan-cytokeratin) and gene expression of several dermal proteins (keratin 14, ΔNp63α). Electrospun scaffold independent of GAGs alone did not affect the epithelial differentiation of hMSCs but combination of keratinocyte-hMSC contact co-culture and electrospun scaffold promotes the epithelial differentiation of hMSCs.
Asunto(s)
Comunicación Celular/fisiología , Queratinocitos/citología , Queratinocitos/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Andamios del Tejido , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo/instrumentación , Técnicas de Cocultivo/métodos , Galvanoplastia/métodos , Regulación de la Expresión Génica , Humanos , Ensayo de Materiales , Nanofibras/química , Andamios del Tejido/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Incorporation of bioactive components like glycosaminoglycans (GAGs) into tissue engineering scaffolds, is a promising approach towards developing new generation functional biomaterial. Here, we have designed electrospun nanofibrous scaffolds made of gelatin and different concentrations of chemically sulfated or non-sulfated hyaluronan (sHA or HA) and chondroitin sulfate (CS). Evenly distributed fiber morphology was observed with no differences between varying concentrations and types of GAGs. In vitro release kinetics revealed that GAGs release is driven by diffusion. The effects of these scaffolds were analyzed on human keratinocyte (HaCaT), fibroblast (Hs27) and mesenchymal stem cells (hMSCs) adhesion and proliferation. A significant increase in cell number (~5 fold) was observed when cultivating all three cell types alone on scaffolds containing sHA and CS. These findings suggest that sulfated GAG-containing electrospun nanofibrous scaffolds might be beneficial for the development of effective skin tissue engineered constructs by stimulating cellular performance and therefore accelerate epidermal-dermal regeneration processes.
Asunto(s)
Ingeniería de Tejidos , Biomimética , Proliferación Celular , Células Cultivadas , Sulfatos de Condroitina , Matriz Extracelular , Humanos , Ácido Hialurónico , Piel , Andamios del TejidoRESUMEN
The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)-10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.
Asunto(s)
Compuestos de Anilina/farmacología , Microambiente Celular/efectos de los fármacos , Conductividad Eléctrica , Células Madre Mesenquimatosas/citología , Osteogénesis/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Animales , Biomarcadores/metabolismo , Calcio/farmacología , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Colágeno/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Estimulación Eléctrica , Elementos Químicos , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Perfilación de la Expresión Génica , Humanos , Ácido Hialurónico/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Osteogénesis/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría por Rayos XRESUMEN
Osteoarthritis (OA) is a multifactorial, often progressive, painful disease. OA often progresses with an apparent irreversible loss of articular cartilage, exposing underlying bone, resulting in pain and loss of mobility. This cartilage loss is thought to be permanent due to ineffective repair and apparent lack of stem/progenitor cells in that tissue. However, the adjacent synovial lining and synovial fluid are abundant with mesenchymal progenitor/stem cells (synovial mesenchymal progenitor cells [sMPCs]) capable of differentiating into cartilage both in vitro and in vivo. Previous studies have demonstrated that MPCs can home to factors such as monocyte chemotactic protein 1 (MCP-1/CCL2) expressed after injury. While MCP-1 (and its corresponding receptors) appears to play a role in recruiting stem cells to the site of injury, in this study, we have demonstrated that MCP-1 is upregulated in OA synovial fluid and that exposure to MCP-1 activates sMPCs, while concurrently inhibiting these cells from undergoing chondrogenesis in vitro. Furthermore, exposure to physiological (OA knee joint synovial fluid) levels of MCP-1 triggers changes in the transcriptome of sMPCs and prolonged exposure to the chemokine induces the expression of MCP-1 in sMPCs, resulting in a positive feedback loop from which sMPCs cannot apparently escape. Therefore, we propose a model where MCP-1 (normally expressed after joint injury) recruits sMPCs to the area of injury, but concurrently triggers changes in sMPC transcriptional regulation, leading to a blockage in the chondrogenic program. These results may open up new avenues of research into the lack of endogenous repair observed after articular cartilage injury and/or arthritis.