Cardiomyoblast (H9c2) Differentiation on Tunable Extracellular Matrix Microenvironment

Suhaeri, Muhammad,Subbiah, Ramesh,Van, Se Young,Du, Ping,Kim, In Gul,Lee, Kangwon,Park, Kwideok Mary Ann Liebert 2015 Tissue engineering. Part A Vol.21 No.11

<P>Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as α-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5?kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for β1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis.</P>

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber

Suhaeri, Muhammad,Subbiah, Ramesh,Kim, Su-Hyun,Kim, Chong-Hyun,Oh, Seung Ja,Kim, Sang-Heon,Park, Kwideok American Chemical Society 2017 ACS APPLIED MATERIALS & INTERFACES Vol.9 No.1

<P>For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in winch anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly(L-lactide-co-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5 7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types.</P>

Elasticity Modulation of Fibroblast-Derived Matrix for Endothelial Cell Vascular Morphogenesis and Mesenchymal Stem Cell Differentiation

Du, Ping,Suhaeri, Muhammad,Subbiah, Ramesh,Van, Se Young,Park, Jimin,Kim, Sang Heon,Park, Kwideok,Lee, Kangwon Mary Ann Liebert 2016 Tissue engineering. Part A Vol.22 No.5

<P>Biophysical properties of the microenvironment, including matrix elasticity and topography, are known to affect various cell behaviors; however, the specific role of each factor is unclear. In this study, fibroblast-derived matrix (FDM) was used as cell culture substrate and physically modified to investigate the influence of its biophysical property changes on human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) behavior in vitro. These FDMs were physically modified by simply storing them at different temperatures: the one stored at 4 degrees C, maintained its original properties, was considered natural FDM, whereas the ones stored at -20 degrees C or -80 degrees C, exhibited a distinct surface morphology, were considered physically modified FDM. Physical modification induced matrix fiber rearrangement in FDM, forming different microstructures on the surface as characterized by focused ion beam (FIB)-cryoSEM. A significant increase of matrix elasticity was found with physically modified FDMs as determined by atomic force microscopy. HUVEC and hMSC behaviors on these natural and physically modified FDMs were observed and compared with each other and with gelatin-coated coverslips. HUVECs showed a similar adhesion level on these substrates at 3 h, but exhibited different proliferation rates and morphologies at 24 h; HUVECs on natural FDM proliferated relatively slower and assembled to capillary-like structures (CLSs). It is observed that HUVECs assembled to CLSs on natural FDMs are independent on the exogenous growth factors and yet dependent on nonmuscle myosin II activity. This result indicates the important role of matrix mechanical properties in regulating HUVECs vascular morphogenesis. As for hMSCs multilineage differentiation, adipogenesis is improved on natural FDM that with lower matrix elasticity, while osteogenesis is accelerated on physically modified FDMs that with higher matrix elasticity, these results further confirm the crucial role of matrix elasticity on cell fate determination.</P>

Investigation of the changes of biophysical/mechanical characteristics of differentiating preosteoblasts in vitro

Ramesh Subbiah,Muhammad Suhaeri,황민태,Woojun Kim,박귀덕 한국생체재료학회 2015 생체재료학회지 Vol.19 No.4

Background: Topography, stiffness, and composition of biomaterials play a crucial role in cell behaviors. In this study, we have investigated biochemical (gene markers), biophysical (roughness), and biomechanical (stiffness) changes during the osteogenic differentiation of preosteoblasts on gelatin matrices. Results: Our results demonstrate that gelatin matrices offer a favorable microenvironment for preosteoblasts as determined by focal adhesion and filopodia formation. The osteogenic differentiation potential of preosteoblasts on gelatin matrices is confirmed by qualitative (Alizarin red, von kossa staining, immunofluorescence, and gene expression) and quantitative analyses (alkaline phosphatase activity and calcium content). The biomechanical and biophysical properties of differentiating preosteoblasts are analyzed using atomic force microscopy (AFM) and micro indentation. The results show sequential and significant increases in preosteoblasts roughness and stiffness during osteogenic differentiation, both of which are directly proportional to the progress of osteogenesis. Cell proliferation, height, and spreading area seem to have no direct correlation with differentiation; however, they may be indirectly related to osteogenesis. Conclusions: The increased stiffness and roughness is attributed to the mineralized bone matrix and enhanced osteogenic extracellular matrix protein. This report indicates that biophysical and biomechanical aspects during in vitro cellular/extracellular changes can be used as biomarkers for the analysis of cell differentiation.

Temperature-mediated elasticity modulation of fibroblast-derived matrix induces control of stem cell fate

Ping Du,Muhammad Suhaeri,Ramesh Subbiah,반세영,박지민,김상헌,박귀덕,이강원 한국고분자학회 2016 한국고분자학회 학술대회 연구논문 초록집 Vol.41 No.1

Integration of cell-derived ECM and polymer toward therapeutic applications

박귀덕,김인걸,Muhammad Suhaeri,Ramesh Subbiah,김종현,최동훈 한국고분자학회 2017 한국고분자학회 학술대회 연구논문 초록집 Vol.42 No.1