Muhammad Suhaeri

Fibronectin-tethered graphene oxide as an artificial matrix for osteogenesis

An artificial matrix (Fn-Tigra), consisting of graphene oxide (GO) and fibronectin (Fn), is developed on pure titanium (Ti) substrates via an electrodropping technique assisted with a custom-made coaxial needle. The morphology and topography of the resulting artificial matrix is orderly aligned and composed of porous microcavities. In addition, Fn is homogenously distributed and firmly bound onto GO as determined via immunofluorescence and elemental mapping, respectively. The artificial matrix is moderately hydrophobic (63.7°), and exhibits an average roughness of 546 nm and a Youngs modulus (E) of approximately 4.8 GPa. The biocompatibility, cellular behavior, and osteogenic potential of preosteoblasts on Fn-Tigra are compared to those of cells cultured on Ti and Ti-GO (Tigra). Cell proliferation and viability are significantly higher on Fn-Tigra and Tigra than that of cells grown on Ti. Focal adhesion molecule (vinculin) expression is highly activated at the central and peripheral area of preosteoblasts when cultured on Fn-Tigra. Furthermore, we demonstrate enhanced in vitro osteogenic differentiation of preosteoblasts cultured on Fn-Tigra over those cultured on bare Ti, as determined via Alizarin red and von Kossa staining, and the analysis of osteocalcin, type I collagen, alkaline phosphatase activity, and calcium contents. Finally, we investigate the biophysical and biomechanical properties of the cells using AFM. While the height and roughness of preosteoblasts increased with time, cell surface area decreased during in vitro osteogenesis over 2 weeks. In addition, the E of cells cultured on Tigra and Fn-Tigra increase in a statistically significant and time-dependent manner by 30%, while those cultured on bare Ti retain a relatively consistent E. In summary, we engineer a biocompatible artificial matrix (Fn-Tigra) capable of osteogenic induction and consequently demonstrate its potential in bone tissue engineering applications.

Tunable Crosslinked Cell-Derived Extracellular Matrix Guides Cell Fate.

Extracellular matrix (ECM), comprised of multiple cues (chemical, physiomechanical), provides a niche for cell attachment, migration, and differentiation. Given that different cells give rise to distinct physiological milieus, the role of such microenvironmental cues on various cells has been well-studied. Particularly, the effect of various physiomechanical factors on stem cell lineage has been resolved into individual variables via ECM protein-coated polymeric systems. Such platforms, while providing a reductionist approach as a means to remove any confounding factors, unfortunately fall short of capturing the full biophysical scope of the natural microenvironment. Herein, the use of a cell-derived ECM platform is reported in which its crosslinking density is tunable; varying concentrations (0, 0.5, 1, 2% w/v) of genipin (GN), a naturally derived crosslinker with low toxicity, are used to form inter- and intrafibril crosslinks. ECM crosslinking produces GN concentration-dependent changes in ECM stiffness (<0.1-9.4 kPa), roughness (96-280 nm), and chemical composition (100-60% amine content). The effect of the various crosslinked ECM profiles on human mesenchymal stem cell differentiation, vascular morphogenesis, and cardiomyogenesis are then evaluated. Taken together, this study demonstrates that tunable crosslinked cell-derived ECM platform is capable of providing a comprehensive physiological platform, and envisions its use in future tissue engineering applications.

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

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which 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.

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

BackgroundTopography, 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.ResultsOur 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.ConclusionsThe 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.

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

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°C, maintained its original properties, was considered natural FDM, whereas the ones stored at -20°C or -80°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.

Human lung fibroblast-derived matrix facilitates vascular morphogenesis in 3D environment and enhances skin wound healing

Extracellular matrix (ECM) is crucial to many aspects of vascular morphogenesis and maintenance of vasculature function. Currently the recapitulation of angiogenic ECM microenvironment is still challenging, due mainly to its diverse components and complex organization. Here we investigate the angiogenic potential of human lung fibroblast-derived matrix (hFDM) in creating a three-dimensional (3D) vascular construct. hFDM was obtained via decellularization of in vitro cultured human lung fibroblasts and analyzed via immunofluorescence staining and ELISA, which detect multiple ECM macromolecules and angiogenic growth factors (GFs). Human umbilical vein endothelial cells (HUVECs) morphology was more elongated and better proliferative on hFDM than on gelatin-coated substrate. To prepare 3D construct, hFDM is collected, quantitatively analyzed, and incorporated in collagen hydrogel (Col) with HUVECs. Capillary-like structure (CLS) formation at 7day was significantly better with the groups containing higher doses of hFDM compared to the Col group (control). Moreover, the group (Col/hFDM/GFs) with both hFDM and angiogenic GFs (VEGF, bFGF, SDF-1) showed the synergistic activity on CLS formation and found much larger capillary lumen diameters with time. Further analysis of hFDM via angiogenesis antibody array kit reveals abundant biochemical cues, such as angiogenesis-related cytokines, GFs, and proteolytic enzymes. Significantly up-regulated expression of VE-cadherin and ECM-specific integrin subunits was also noticed in Col/hFDM/GFs. In addition, transplantation of Col/hFMD/GFs with HUVECs in skin wound model presents more effective re-epithelialization, many regenerated hair follicles, better transplanted cells viability, and advanced neovascularization. We believe that current system is a very promising platform for 3D vasculature construction in vitro and for cell delivery toward therapeutic applications in vivo. STATEMENT OF SIGNIFICANCE Functional 3D vasculature construction in vitro is still challenging due to the difficulty of recapitulating the complex angiogenic extracellular matrix (ECM) environment. Herein, we present a simple and practical method to create an angiogenic 3D environment via incorporation of human lung fibroblast-derived matrix (hFDM) into collagen hydrogel. We found that hFDM offers a significantly improved angiogenic microenvironment for HUVECs on 2D substrates and in 3D construct. A synergistic effect of hFDM and angiogenic growth factors has been well confirmed in 3D condition. The prevascularized 3D collagen constructs also facilitate skin wound healing. We believe that current system should be a convenient and powerful platform in engineering 3D vasculature in vitro, and in delivering cells for therapeutic purposes in vivo.

Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing

Skin injuries are frequently encountered in daily life, but deep wounds often poorly self-heal and do not recover completely. In this study, we propose a novel skin patch that combines antibiotic, cell-derived extracellular matrix (ECM) and biocompatible polyvinyl alcohol (PVA) hydrogel. Methods: Decellularized human lung fibroblast-derived matrix (hFDM) was prepared on tissue culture plate (TCP) and PVA solution was then poured onto it. After a freeze-thaw process, PVA was peeled off from TCP along with hFDM tightly anchored to PVA. Subsequently, ciprofloxacin (Cipro)-incorporated PVA/hFDM (PVA/Cipro/hFDM) was fabricated via diffusion-based drug loading. Results: In vitro analyses of PVA/Cipro/hFDM show little cytotoxicity of ciprofloxacin, stability of hFDM, rich fibronectin in hFDM, and good cell attachment, respectively. In addition, hFDM proved to be beneficial in promoting cell migration of dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) using transwell inserts. The antibacterial drug Cipro was very effective in suppressing colony growth of gram-negative and -positive bacteria as identified via an inhibition zone assay. For animal study, infected wound models in BALB/c mice were prepared and four test groups (control, PVA, PVA/Cipro, PVA/Cipro/hFDM) were administered separately and their effect on wound healing was examined for up to 21 days. The results support that Cipro successfully reduced bacterial infection and thus encouraged faster wound closure. Further analysis using histology and immunofluorescence revealed that the most advanced skin regeneration was achieved with PVA/Cipro/hFDM, as assessed via re-epithelialization, collagen texture and distribution in the epidermis, and skin adnexa (i.e., glands and hair follicles) regeneration in the dermis. Conclusion: This work demonstrates that our skin patch successfully consolidates the regenerative potential of ECM and the antibacterial activity of Cipro for advanced wound healing.