Halime Kenar

Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for bone tissue engineering

Macroporous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) matrices were prepared after solvent evaporation and solute leaching. PHBV solutions with different concentrations were prepared in chloroform: dichloromethane (1:2, v/v). In order to create a matrix with high porosity and uniform pore sizes, sieved sucrose crystals (75-300 or 300-500 microm) were used. PHBV foams were treated with rf-oxygen plasma to modify their surface chemistry and hydrophilicity with the aim of increasing the reattachment of osteoblasts. Surface characteristics, pore sizes and their distribution on PHBV surface were studied by scanning electron microscopy (SEM) and Scion Image Analysis Program. Void volume, pore sizes and density of foams were found to be significantly affected by foam preparation conditions. Stability of PHBV foams in aqueous media was studied. Their weight and density were unchanged for a period of 120 days and then a significant decrease was observed for the rest of the study (60 days). Osteoblasts were seeded onto the foams and their proliferation inside the matrices was also determined by SEM. After 29 and 60 days of incubation, growth of osteoblasts on matrices was observed.

A 3D aligned microfibrous myocardial tissue construct cultured under transient perfusion

The goal of this study was to design and develop a myocardial patch to use in the repair of myocardial infarctions or to slow down tissue damage and improve long-term heart function. The basic 3D construct design involved two biodegradable macroporous tubes, to allow transport of growth media to the cells within the construct, and cell seeded, aligned fiber mats wrapped around them. The microfibrous mat housed mesenchymal stem cells (MSCs) from human umbilical cord matrix (Whartons Jelly) aligned in parallel to each other in a similar way to cell organization in native myocardium. Aligned micron-sized fiber mats were obtained by electrospinning a polyester blend (PHBV (5% HV), P(L-D,L)LA (70:30) and poly(glycerol sebacate) (PGS)). The micron-sized electrospun parallel fibers were effective in Whartons Jelly (WJ) MSCs alignment and the cells were able to retract the mat. The 3D construct was cultured in a microbioreactor by perfusing the growth media transiently through the macroporous tubing for two weeks and examined by fluorescence microscopy for cell distribution and preservation of alignment. The fluorescence images of thin sections of 3D constructs from static and perfused cultures confirmed enhanced cell viability, uniform cell distribution and alignment due to nutrient provision from inside the 3D structure.

Novel surface patterning approaches for tissue engineering and their effect on cell behavior

Methods for the creation of specially designed surfaces for use in the preparation of tailor-made tissue constructs with the ultimate aim of tissue engineering are reviewed here. Fundamental aspects of cell adhesion, proliferation and differentiation and the parameters involved in these processes are discussed. A survey of recent micro- and nano-technological methods for creating physical and chemical cues on tissue engineering carriers is presented. This overview is supported with data from the literature on various applications of different cells on materials with widely differing chemistries and physical properties. Interactions between different cell types and micro- and nano-fabricated substrates are summarized.

Effect of low level laser therapy and zoledronate on the viability and ALP activity of Saos-2 cells

A limited number of clinical studies indicate the supportive role of low level laser therapy (LLLT) on medical and/or surgical approaches carried out in treatment modalities for bisphosphonate related necrosis of jaws (BRONJ), the most common side effect of bisphosphonates used to inhibit bone resorption. The purpose of this study was to investigate the effects of LLLT on cell proliferation and alkaline phosphatase (ALP) activity of human osteoblast-like cells (Saos-2) treated with different doses of zoledronate, the most potent bisphosphonate. Saos-2 cells were treated with different concentrations of zoledronate and were irradiated with diode laser (wavelength 808 nm, 10 s, 0.25 or 0.50 W). Cell numbers and ALP activity of the cells were determined. LLLT mildly increased the proliferation rate or ALP activity, while zoledronate reduced both. When applied together, LLLT lessened the detrimental effects of zoledronate and improved cell function and/or proliferation. Based on the results of this study, it was concluded that LLLT has biostimulative effects on Saos-2 cells, even after treatment with zoledronate. LLLT may serve as a useful supportive method for BRONJ treatment through enhancement of healing by osteoblasts.

Collagen scaffolds with in situ-grown calcium phosphate for osteogenic differentiation of Wharton's jelly and menstrual blood stem cells.

The aim of this research was to investigate the osteogenic differentiation potential of non‐invasively obtained human stem cells on collagen nanocomposite scaffolds with in situ‐grown calcium phosphate crystals. The foams had 70% porosity and pore sizes varying in the range 50–200 µm. The elastic modulus and compressive strength of the calcium phosphate containing collagen scaffolds were determined to be 234.5 kPa and 127.1 kPa, respectively, prior to in vitro studies. Mesenchymal stem cells (MSCs) obtained from Whartons jelly and menstrual blood were seeded on the collagen scaffolds and proliferation and osteogenic differentiation capacities of these cells from two different sources were compared. The cells on the composite scaffold showed the highest alkaline phosphatase activity compared to the controls, cells on tissue culture polystyrene and cells on collagen scaffolds without in situ‐formed calcium phosphate. MSCs isolated from both Whartons jelly and menstrual blood showed a significant level of osteogenic activity, but those from Whartons jelly performed better. In this study it was shown that collagen nanocomposite scaffolds seeded with cells obtained non‐invasively from human tissues could represent a potential construct to be used in bone tissue engineering. Copyright

Skeletal Muscle Patch Engineering on Synthetic and Acellular Human Skeletal Muscle Originated Scaffolds

The reconstruction of skeletal muscle tissue is currently performed by transplanting a muscle tissue graft from local or distant sites of the patients body, but this practice leads to donor site morbidity in case of large defects. With the aim of providing an alternative treatment approach, skeletal muscle tissue formation potential of human myoblasts and human menstrual blood derived mesenchymal stem cells (hMB-MSCs) on synthetic [poly(l-lactide-co-caprolactone), 70:30] scaffolds with oriented microfibers, human muscle extracellular matrix (ECM), and their hybrids was investigated in this study. The reactive muscle ECM pieces were chemically crosslinked to the synthetic scaffolds to produce the hybrids. Cell proliferation assay WST-1, scanning electron microscopy (SEM), and immunostaining were carried out after culturing the cells on the scaffolds. The ECM and the synthetic scaffolds were effective in promoting spontaneous myotube formation from human myoblasts. Anisotropic muscle patch formation was more successful when human myoblasts were grown on the synthetic scaffolds. Nonetheless, spontaneous differentiation could not be induced in hMB-MSCs on any type of the scaffolds. Human myoblast-synthetic scaffold combination is promising as a skeletal muscle patch, and can be improved further to serve as a fast integrating functional patch by introducing vascular and neuronal networks to the structure.

An in vitro human skeletal muscle model: coculture of myotubes,neuron-like cells, and the capillary network

This study reports the generation of a new human muscle tissue equivalent from skeletal muscle-derived stem cells and human umbilical vein endothelial cells (HUVECs). Skeletal muscle stem cells were isolated by the preplate technique and differentiated into neuron-like cells that were positive for neuronal beta-tubulin3 and nestin and negative for the astrocyte marker glial fibrillary acidic protein (GFAP). Coculture of skeletal muscle stem cells with the HUVECs under optimized fetal bovine serum and media conditions resulted in formation of a capillary network among the multinucleated myotubes. The neuron-like cells derived from the human skeletal muscle stem cells were seeded onto vascularized myotubes to obtain the neuromuscular junctions in the coculture. At the end of 24 h of coculture, the neuron-like cells were found to be in association with the myotubes. This model represents a novel complex in vitro human skeletal muscle model containing advanced capillary networks and interacting myotubes and neurons, and it can be used for in vitro drug testing or for skeletal muscle regeneration either through application of cellular therapy or cell-laden tissue-engineered muscle constructs.