Chondrogenesis in perfusion bioreactors using porous silk scaffolds and hESC-derived MSCs.

Tissue engineered cartilage can be grown in vitro with the use of cell-scaffold constructs and bioreactors. The present study was designed to investigate the effects of perfusion bioreactors on the chondrogenic potential of engineered constructs prepared from porous silk fibroin scaffolds seeded with human embryonic stem cell (hESC)-derived mesencyhmal stem cells (MSCs). After four weeks of incubation, constructs cultured in perfusion bioreactors showed significantly higher amounts of glycosaminoglycans (GAGs) (p < 0.001), DNA (p < 0.001), total collagen (p < 0.01), and collagen II (p < 0.01) in comparison to static culture. Mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions and RT-PCR results concluded that cells cultured in perfusion bioreactors highly expressed (p < 0.001) cartilage-related genes when compared with static culture. Distinct differences were noted in tissue morphology, including polygonal extracellular matrix structure of engineered constructs in thin superficial zones and an inner zone under static and dynamic conditions, respectively. The results suggest that the utility of perfusion bioreactors to modulate the growth of tissue-engineered cartilage and enhance tissue growth in vitro.

Cellular Behavior on Epidermal Growth Factor (EGF)-Immobilized PCL/Gelatin Nanofibrous Scaffolds.

Nano-scaled poly(ε-caprolactone) (PCL) and PCL/gelatin fibrous scaffolds with immobilized epidermal growth factor (EGF) were prepared for the purpose of wound-healing treatments. The tissue scaffolds were fabricated by electrospinning and the parameters that affect the electrospinning process were optimized. While the fiber diameters were 488 ± 114 nm and 663 ± 107 nm for PCL and PCL/gelatin scaffolds, respectively, the porosities were calculated as 79% for PCL and 68% for PCL/gelatin scaffolds. Electrospun PCL and PCL/gelatin scaffolds were first modified with 1,6-diaminohexane to introduce amino groups on their surfaces, then EGF was chemically conjugated to the surface of nanofibers. The results obtained from Attenuated Total Reflectance Fourier Transform Infrared (ATR-FT-IR) spectroscopy and quantitative measurements showed that EGF was successfully immobilized on nanofibrous scaffolds. L929 mouse fibroblastic cells were cultivated on both neat and EGF-immobilized PCL and PCL/gelatin scaffolds in order to investigate the effect of EGF on cell spreading and proliferation. According to the results, especially EGF-immobilized PCL/gelatin scaffolds exerted early cell spreading and superior and rapid proliferation compared to EGF-immobilized PCL scaffolds and neat PCL, PCL/gelatin scaffolds. Consequently, EGF-immobilized PCL/gelatin scaffolds could potentially be employed as novel scaffolds for skin tissueengineering applications.

Computational fluid dynamics modeling of momentum transport in rotating wall perfused bioreactor for cartilage tissue engineering.

In this study, computational fluid dynamics (CFD) analysis of a rotating-wall perfused-vessel (RWPV) bioreactor is performed to characterize the complex hydrodynamic environment for the simulation of cartilage development in RWPV bioreactor in the presence of tissue-engineered cartilage constructs, i.e., cell-chitosan scaffolds. Shear stress exerted on chitosan scaffolds in bioreactor was calculated for different rotational velocities in the range of 33-38 rpm. According to the calculations, the lateral and lower surfaces were exposed to 0.07926-0.11069 dyne/cm(2) and 0.05974-0.08345 dyne/cm(2), respectively, while upper surfaces of constructs were exposed to 0.09196-0.12847 dyne/cm(2). Results validate adequate hydrodynamic environment for scaffolds in RWPV bioreactor for cartilage tissue development which concludes the suitability of operational conditions of RWPV bioreactor.

bFGF-loaded HA-chitosan: a promising scaffold for periodontal tissue engineering.

A scaffold containing growth factors promoting regeneration may be a useful device to maintain periodontal regeneration when applied with appropriate cells. The aim of this study is to evaluate the convenience of chitosan and hydroxyapatite (HA)-chitosan scaffolds loaded with basic fibroblast growth factor (bFGF) for periodontal tissue engineering applications. Scaffolds were fabricated by freeze-drying technique using 2 and 3% chitosan gel in the absence or presence of HA particles. Addition of HA beads to chitosan gels produced a novel scaffold in which the pore sizes and interconnectivity were preserved. The scaffolds were loaded with 100 ng bFGF by embedding technique. HA-chitosan scaffolds provide better controlled release kinetics for bFGF compared with chitosan scaffolds and total release continued up to 168 h. Cell culture studies were carried out with periodontal ligament (PDL) cells and cementoblasts. Both 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) assay and confocal laser scanning microscope analysis revealed cells proliferating inside the scaffolds. The results demonstrated that bFGF-loaded HA-chitosan scaffolds provide a suitable three-dimensional environment supporting the cellular structure, proliferation, and mineralization.

In vitro release of dexamethasone or bFGF from chitosan/hydroxyapatite scaffolds.

Chitosan scaffolds containing dexamethasone (Dex) or basic fibroblast growth factor (bFGF) were developed to create alternative drug-delivery systems for possible tissue-engineering applications such as periodontal bone regeneration. Chitosan solutions (2% and 3% (w/v) in acetic acid) were prepared from chitosan flakes with high deacetylation degree (>85%), then these solutions were freeze-dried at -80 degrees C to obtain scaffolds with interconnected pore structures. Dex and bFGF were incorporated into scaffolds by embedding method (solvent sorption method). The initial loading amounts were varied as 300, 600 and 900 ng Dex per dry scaffold (average dry weight is 3 mg) and 50 or 100 ng bFGF per dry scaffold to a range of deliverable doses. Release studies which were conducted in Dulbecco's phosphate-buffered saline (DPBS) showed that 900 ng Dex loaded chitosan scaffolds in both compositions released total Dex during a 5-day period at a nearly constant rate after the initial burst. However, bFGF release from all scaffolds with both loading amounts (50 ng or 100 ng) was completed in 10 or 20 h. In order to prolong the release period of bFGF, composite scaffolds were fabricated in the presence of hydroxyapatite (HA) beads with average particle size of 40 mum. Sustained release of bFGF up to 7 days was achieved due to the electrostatic interactions between HA and bFGF molecules. These results suggested that chitosan scaffolds can be suitable for Dex release; however, the presence of HA in the chitosan scaffold is necessary to achieve the desired release period for bFGF.

Chondrogenesis on BMP-6 loaded chitosan scaffolds in stationary and dynamic cultures.

We originally investigated the suitability of chitosan scaffolds loaded with bone morphogenetic protein 6 (BMP-6) in both stationary and dynamic conditions for cartilage tissue engineering. In the first part of the present study, ATDC5 murine chondrogenic cells were seeded in chitosan and BMP-6 loaded chitosan scaffolds and cultured for 28 days under static conditions. In the following part, we examined the influence of dynamic cultivation conditions over BMP-6 loaded chitosan scaffolds by using rotating bioreactor with perfusion (RCMW). Tissue engineered constructs were characterized by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and biochemical assays for glycosaminoglycans (GAG) deoxyribonucleic acid (DNA) and collagen Type II quantification. At the end of 4 weeks static incubation period high levels of GAG (21.22 mg/g dry weight), DNA amounts (1.37 mg/g dry weight) and collagen Type II amounts (1.94 microg/g dry weight) were achieved for BMP-6 loaded chitosan scaffolds compared to chitosan scaffolds. However, the results obtained from morphological observations suggested hypertrophic differentiation of ATDC5 cells in the presence of BMP-6 under stationary conditions. The influence of mechanical stimulation appeared significantly with differentiated cells, cultured under dynamic conditions, showing the effect of retaining their phenotypes without hypertrophy.

Evaluation of alginate-chitosan semi IPNs as cartilage scaffolds.

In this study, alginate and alginate:chitosan semi interpenetrating polymer network (IPN) scaffolds were prepared by freeze-drying process. Alginate scaffolds were crosslinked with different concentrations of CaCl(2), i.e. 0.5, 1 or 3% (w/v), in 96% (v/v) ethanol solutions for two different periods, i.e. 4 and 24 h, after freeze-drying. Scanning electron microscope (SEM)/ Energy Dispersive Analysis by X-ray (EDAX) analysis and swelling studies indicated that crosslinking of scaffolds with 3% (w/v) CaCl(2) for 24 h was effectively created suitable alginate scaffolds in terms of optimum porosity and mechanical stability. This is why, alginate:chitosan semi IPN scaffolds were prepared at the crosslinking condition mentioned above in 70:30, 60:40 and 50:50% (v/v) alginate:chitosan ratios. Besides the attachment and proliferation abilities of ATDC5 murine chondrogenic cells on alginate, 70:30% (v/v) alginate:chitosan and 50:50% (v/v) alginate:chitosan scaffolds, their cellular responses were assessed for chondrogenic potential. These structural and cellular outcomes demonstrate potential utility of chitosan semi IPNs in alginate scaffolds. Comparative results found in relation to alginate scaffolds, support the necessity for alginate:chitosan scaffolds for improved cartilage tissue engineering.

Evaluation of RGD- or EGF-immobilized chitosan scaffolds for chondrogenic activity.

Chitosan scaffolds were prepared by freeze-drying method and modified with Arg-Gly-Asp (RGD) sequence of fibronectin or epidermal growth factor (EGF) by covalent immobilization. The results obtained from FTIR-ATR, fluorescence visualization and quantitative measurements showed that biosignal molecules, RGD and EGF, were successfully immobilized on chitosan scaffolds. ATDC5 murine chondrogenic cells were seeded on both type of scaffolds, chitosan-RGD and chitosan-EGF, and cultured for 28 days in stationary conditions. According to the results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) test, considerable increase in cell proliferation was only detected on chitosan-EGF scaffolds. Biochemical analysis of the chondrocyte seeded scaffolds showed that glycosaminoglycan (GAG) and deoxyribonucleic acid (DNA) content of the scaffolds increases with time. In conclusion, EGF-modified chitosan scaffolds (containing 1.83 microg EGF/3 mg dry scaffold) have been proposed to promote chondrogenesis and to have potential for reticular cartilage regeneration.