Soner Çakmak

A novel dermal substitute based on biofunctionalized electrospun PCL nanofibrous matrix

In this study, nanofibrous matrices of polycaprolactone (PCL) and PCL/collagen with immobilized epidermal growth factor (EGF) were successfully fabricated by electrospinning for the purpose of damaged skin regeneration. Nanofiber diameters were found to be 284 ± 48 nm for PCL and 330 ± 104 nm for PCL/collagen matrices. The porosities were calculated as 85% for PCL and 90% for PCL/collagen matrices. The covalent immobilization of EGF onto the nanofibrous matrices was verified by the increase of surface atomic nitrogen ratio from 1.0 to 2.4% for PCL and from 3.7 to 4.7% for PCL/collagen. Moreover, EGF immobilization efficiencies of PCL and PCL/collagen matrices were determined as 98.5 and 99.2%, respectively. Human dermal keratinocytes (HS2) were cultivated on both neat and EGF immobilized PCL and PCL/collagen matrices to investigate the effects of matrix chemical composition and presence of EGF on cell proliferation and differentiation. EGF immobilized PCL/collagen matrices exerted early cell spreading and rapid proliferation. Statistically high expression levels of loricrin in HS2 cells cultivated on EGF immobilized PCL/collagen matrices were (p < 0.001) regarding superior differentiation ability of these cells compared to HS2 cells cultured on neat PCL and PCL/collagen matrices. In conclusion, this novel EGF immobilized PCL/collagen nanofibrous matrix could potentially be considered as an alternative dermal substitutes and wound healing material for skin tissue engineering applications.

Random/aligned electrospun PCL/PCL-collagen nanofibrous membranes: comparison of neural differentiation of rat AdMSCs and BMSCs

In this study, the aligned (A) and randomly oriented (R) polycaprolactone (PCL-A and PCL-R) and PCL/collagen (PCL/Col-A and PCL/Col-R) nanofibers were electrospun onto smooth PCL membranes (PCLMs) prepared by solvent casting. In order to investigate the effects of chemical composition and nanotopography of fibrous surfaces on proliferation and on neural differentiation of mesenchymal stem cells (MSCs), adipose and bone marrow-derived rat MSCs (AdMSCs and BMSCs) were cultivated in suitable media i.e. inducing medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), and cell maintenance medium (CMM). BMSCs adhered and proliferated on all nanofibrous membranes more efficiently than AdMSCs. PCL/Col-A was found as the most convenient surface supporting proliferation in both cell types. Immunofluorescence staining indicated that BMSCs and AdMSCs are prone for differentiation to oligodendrocytes more than they differentiate to other neuronal cell types. PCL-A nanofibrous membranes supported differentiation of MSCs to O4(+) (an oligodendrocytes surface antigen) cells in both culture media. The intensity of immunoreactivity of O4(+) cells differentiated from BMSCs on PCL-A was highest when compared with the other groups (p < 0.001). Some BIII-T signed neural cells were investigated on PCL-A nanofibrous membranes, but the intensity of immunoreactivity was lower than that of O4(+) cells. In conclusion, this study can be evaluated to establish the cell therapy strategies in neurodegenerative disorders, which are relevant to oligodendrocyte abstinence using BMSCs or AdMSCs on aligned nanofibrous membranes.

Thermosensitive PHEMA microcarriers: ATRP synthesis, characterization, and usabilities in cell cultures

In this study, we developed a novel microcarrier to enhance the production of anchorage-dependent mammalian cells in large scale by preserving them from the effects of shear forces and to enhance their removal from the surface without using proteolytic enzymes and chelating agents. This ‘thermosensitive microcarrier’ was synthesized by the grafting thermoresponsive molecule, N-isopropylacrylamide (NIPAAm), to the crosslinked poly(2-hydroxyethyl methacrylate) (PHEMA) beads by surface-initiated atom transfer radical polymerization. NIPAAm was polymerized on bromine-activated beads’ surfaces to prepare PHEMA-g-PNIPAAm microcarriers. Then, they were chemically characterized by attenuated total reflectance Fourier transform infrared and electron spectroscopy for chemical analysis. Surface morphologies were further investigated by scanning electron microscope and atomic force microscopy techniques. The results of characterization studies confirmed that PNIPAAm was successfully grafted onto PHEMA beads by the means of atom transfer radical polymerization reaction. The cellular activities of PHEMA-g-PNIPAAm microcarriers were evaluated at static and dynamic culture conditions by using two types of cell lines with different morphology, i.e. L929 mouse fibroblasts and HS2 epithelial human keratinocytes. The microcarriers exhibited better cell adhesion and proliferation characteristics for both cell lines. Although their thermally induced cell detachment efficiencies are lower than that of trypsinization, thermally harvested cells preserved their surface morphologies and proliferation characteristics.

RGD-bearing peptide-amphiphile-hydroxyapatite nanocomposite bone scaffold: an in vitro study

In this study, a fibrous nanocomposite scaffold was developed by combining hydroxyapatite (HA) fibers produced by electrospinning method and arginine-glycine-aspartic acid (RGD)-bearing peptide-amphiphile (PA) gel (PA-RGD) produced by self-assembly and gelation induced by calcium ions. Scanning electron microscope, transmission electron microscope and atomic force microscopy imaging confirmed the successful production of inorganic and organic components of this nanocomposite material. Within the HA, the presence of a CaCO3 phase, improving biodegradation, was shown by x-ray diffraction analysis. The in vitro effectiveness of the PA-RGD/HA scaffold was determined on MC3T3-E1 preosteoblast cultures in comparison with HA matrix and PA-RGD gel. The highest cellular proliferation was obtained on PA-RGD gel, however, alkaline phosphatase activity results denoted that osteogenic differentiation of the cells is more favorable on HA containing matrices with respect to PA-RGD itself. Microscopic observations revealed that all three matrices support cell attachment and proliferation. Moreover, cells form bridges between the HA and PA-RGD components of the nanocomposite scaffold, indicating the integrity of the biphasic components. According to the real time-polymerase chain reaction (RT-PCR) analyses, MC3T3-E1 cells expressed significantly higher osteocalcin on all matrices. Bone sialoprotein (BSP) expression level is ten-fold higher on PA-RGD/HA nanocomposite scaffolds than that of HA and PA-RGD scaffolds and the elevated expression of BSP on PA-RGD/HA nanocomposite scaffolds suggested higher mineralized matrix on this novel scaffold. Based on the results obtained in this study, the combination of HA nanofibers and PA-RGD gel takes advantage of good structural integrity during the cell culture, besides the osteoinductive and osteoconductive properties of the nanofibrous scaffold.

A Silk Fibroin and Peptide Amphiphile-Based Co-Culture Model for Osteochondral Tissue Engineering

New biomaterials with the properties of both bone and cartilage extracellular matrices (ECM) should be designed and used with co-culture systems to address clinically applicable osteochondral constructs. Herein, a co-culture model is described based on a trilayered silk fibroin-peptide amphiphile (PA) scaffold cultured with human articular chondrocytes (hACs) and human bone marrow mesenchymal stem cells (hBMSCs) in an osteochondral cocktail medium for the cartilage and bone sides, respectively. The presence of hACs in the co-cultures significantly increases the osteogenic differentiation potential of hBMSCs based on ALP activity, RT-PCR for osteogenic markers, calcium analyses, and histological stainings, whereas hACs produces a significant amount of glycosaminoglycans (GAGs) for the cartilage region, even in the absence of growth factor TGF-β family in the co-culture medium. This trilayered scaffold with trophic effects offers a promising strategy for the study of osteochondral defects.

Synergistic effect of exogeneous and endogeneous electrostimulation on osteogenic differentiation of human mesenchymal stem cells seeded on silk scaffolds

Bioelectrical regulation of bone fracture healing is important for many cellular events such as proliferation, migration, and differentiation. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk scaffolds in response to different modes of electrostimulation (e.g., exogeneous and/or endogeneous). Endogeneous electrophysiology was altered through the use of monensin (10 nM) and glibenclamide (10 μM), along with external electrostimulation (60 kHz; 100–500 mV). Monensin enhanced the expression of early osteogenic markers such as alkaline phosphatase (ALP) and runt‐related transcription factor 2 (RUNX‐2). When exogeneous electrostimulation was combined with glibenclamide, more mature osteogenic marker upregulation based on bone sialoprotein expression (BSP) and mineralization was found. These results suggest the potential to exploit both exogeneous and endogeneous biophysical control of cell functions towards tissue‐specific goals.

Poly(butylene adipate-co-terephthalate) scaffolds: processing, structural characteristics and cellular responses

ABSTRACT The aliphatic-aromatic copolyester, poly(butylene adipate-co-terephthalate) (PBAT) combines good mechanical and thermal properties with biodegradation ability. However, until now, researches in its potential medical use remain limited. Only in a few studies blends of PBAT with routinely used biocompatible polymers had been prepared and investigated regarding tissue engineering applications. Therefore, in this study, we decided to determine processability of neat PBAT as a scaffold material for bone tissue by using different fabrication methods i.e. solvent evaporation, electrospinning, solvent casting-particulate leaching (SCPL) and melt molding-particulate leaching. The results of physicochemical characterizations and cell culture studies with MC3T3-E1 preosteoblasts confirmed that neat PBAT has favorable characteristics for bone tissue engineering, however, fabrication method strongly affects the cellular responses. Regarding to the characterizations and cell cultures, PBAT scaffolds produced by SCPL and electrospinning are proposed to be used for bone tissue engineering.