Composite Films of Gelatin and Hydroxyapatite/Bioactive Glass for Tissue-Engineering Applications

Cross-linked gelatin/hydroxyapatite/bioactive glass (G/HA/CEL2) films with different compositions (100:0:0 (G1); 30:70:0 (G2); 30:0:70 (G3); 30:35:35 (G4) (%, w/w/w)) were prepared as scaffold materials for tissue-engineering applications, particularly in the field of bone repair. A bioactive glass with 45% SiO₂, 3% P₂O₅, 26% CaO, 7% MgO, 15% Na₂O and 4% K₂O molar composition was selected (CEL2). Genipin was used as a cross-linker for the gelatin component. Samples were characterized in terms of their bioactivity, thermal properties, mechanical behaviour and cell compatibility. After only 3 days of incubation in simulated body fluid (SBF) at 37°C, calcium phosphate crystals precipitated on G3 and G4 surfaces, due to the high CEL2 bioactivity. Cross-linking increased the thermal stability of the gelatine component as indicated by thermal analysis (denaturation temperature was 92.3°C and 97.6°C for not cross-linked and cross-linked gelatin, respectively). Furthermore, tensile modulus of samples increased with increasing the inorganic phase amount (from 4.72 ± 0.23 MPa for G1 to 6.46 ± 0.05 MPa for G4). The adhesion and proliferation of human primary osteoblasts on composite films was evaluated. Cell viability was high with respect to the control for all samples and the presence of hydroxyapatite exerted an important role in the ability of mineralization.     

Electrospun PLGA nanofiber scaffolds for articular cartilage reconstruction: mechanical stability,degradation and cellular responses under mechanical stimulation in vitro

We investigated the potential of a nanofiber-based poly(DL-lactide-co-glycolide) (PLGA) scaffold to be used for cartilage reconstruction. The mechanical properties of the nanofiber scaffold, degradation of the scaffold and cellular responses to the scaffold under mechanical stimulation were studied. Three different types of scaffold (lactic acid/glycolic acid content ratio = 75 : 25, 50 : 50, or a blend of 75 : 25 and 50 : 50) were tested. The tensile modulus, ultimate tensile stress and corresponding strain of the scaffolds were similar to those of skin and were slightly lower than those of human cartilage. This suggested that the nanofiber scaffold was sufficiently mechanically stable to withstand implantation and to support regenerated cartilage. The 50 : 50 PLGA scaffold was degraded faster than 75 : 25 PLGA, probably due to the higher hydrophilic glycolic acid content in the former. The nanofiber scaffold was degraded faster than a block-type scaffold that had a similar molecular weight. Therefore, degradation of the scaffold depended on the lactic acid/glycolic acid content ratio and might be controlled by mixing ratio of blend PLGA. Cellular responses were evaluated by examining toxicity, cell proliferation and extracellular matrix (ECM) formation using freshly isolated chondrocytes from porcine articular cartilage. The scaffolds were non-toxic, and cell proliferation and ECM formation in nanofiber scaffolds were superior to those in membrane-type scaffolds. Intermittent hydrostatic pressure applied to cell-seeded nanofiber scaffolds increased chondrocyte proliferation and ECM formation. In conclusion, our nanofiber-based PLGA scaffold has the potential to be used for cartilage reconstruction.     

Improvement of Stem Cell Viability in Hyaluronic Acid Hydrogels Using Dextran Microspheres

Although hyaluronic acid (HA) has been widely used in clinics as an injectable biomaterial, it may not be appropriate as an injectable stem cell carrier because highly hydrophilic HA hydrogels provide an unfavorable environment in which the encapsulated stem cells are likely to be constrained to a round shape, thereby losing their native morphology. Herein, we hypothesized that dextran microspheres (DMs) can improve stem cell viability in HA hydrogels because they can act as substrates for stem cell adhesion, spreading and proliferation. DMs with a mean diameter of 80 μm were mixed with HA hydrogels. Human adipose-derived stem cells (hASCs) were isolated from human adipose tissue and seeded into the DM-incorporated HA hydrogels. When compared with the hydrogels alone, the number of viable cells was significantly increased in the presence of the DMs. Initially, hASCs appeared to be round in the HA hydrogels. At 12 h after seeding, the hASCs apparently attached onto the DMs and became slightly flattened. One day after seeding, the hASCs seemed to spread onto the surface of the DMs. Fluorescence micrography of live and dead cells confirmed that the cell viability was significantly improved by use of the DMs in HA hydrogels. Overall results demonstrated that the microsphere/hydrogel composite supported stem cell survival and spreading. These characteristics show the potential for use of the composite in cell-delivery and tissue-engineering applications.     

Physiological and cell biological aspects of perfusion culture technique employed to generate differentiated tissues for long term biomaterial testing and tissue engineering

Optimal results in biomaterial testing and tissue engineering under in vitro conditions can only be expected when the tissue generated resembles the original tissue as closely as possible. However, most of the presently used stagnant cell culture models do not produce the necessary degree of cellular differentiation, since important morphological, physiological, and biochemical characteristics disappear, while atypical features arise. To reach a high degree of cellular differentiation and to optimize the cellular environment, an advanced culture technology allowing the regulation of differentiation on different cellular levels was developed. By the use of tissue carriers, a variety of biomaterials or individually selected scaffolds could be tested for optimal tissue development. The tissue carriers are to be placed in perfusion culture containers, which are constantly supplied with fresh medium to avoid an accumulation of harmful metabolic products. The perfusion of medium creates a constant microenvironment with serum-containing or serum-free media. By this technique, tissues could be used for biomaterial or scaffold testing either in a proliferative or in a postmitotic phase, as is observed during natural development. The present paper summarizes technical developments, physiological parameters, cell biological reactions, and theoretical considerations for an optimal tissue development in the field of perfusion culture.     

Investigative Study of Myofibroblasts and Cytokines in Peri-Implant Tissue of Silicone Breast Expander by RT-PCR in a Rat Model

The excessive collagen deposition around silicone breast implants followed by contracture and development of severe pain is a major clinical problem. This study was conducted to investigate the profibrotic and antifibrotic cytokines secreted by inflammatory cells and development of myofibroblasts at the tissue material interface around silicone breast expander and ultra-high-molecular-weight polyethylene (UHMWPE). Both materials were implanted in rats for 30, 90 and 180 days. Inflammatory cells and collagen deposition at the material–tissue interface were assessed with Haematoxylin-Eosin and Masson's Trichrome stain. Gene expression of TGFβ, IL-1β, IFNγ, IL10 and α-SMA was quantitated by real-time (RT)-PCR in the peri-implant tissue. Results indicate a difference in collagen deposition and myofibroblast development around both materials with involvement of TGFβ, IFNγ and IL10. The results emphasise the need for further investigation into the molecular mechanisms of protomyofibroblast and myofibroblast formation around silicone implants, which would provide information on these target cells for inhibitory therapy in the clinical situation.     

In Situ Fabrication of Nano-hydroxyapatite in a Macroporous Chitosan Scaffold for Tissue Engineering

A chitosan (CS)/hydroxyapatite (HAP) nanohybrid scaffold with high porosity and homogeneous nanostructure was fabricated through a bionic treatment combined with thermally-induced phase separation. The nano-HAP particles were formed in situ in the scaffold at room temperature instead of mechanically mixing the powders with the polymer component. The scaffold was macroporous with a pore size of about 100–136 μm. The nano-sized HAP particles with diameters of 90–200 nm were scattered homogeneously in the interactively connective pores. Both the improvement of the compressive modulus and yield strength of the scaffolds showed that the in situ nano-HAP particles reinforced the microstructure of the scaffold. The in vitro bioactivity study carried out in simulated body fluid (SBF) indicated good mineralization activity. The crystallization phenomenon suggested that the nano-HAP particles have positive impacts on directing apatite crystallization in the scaffold and led to the good bioactivity of the nanohybrid scaffold.     

Long term culture of epithelia in a continuous fluid gradient for biomaterial testing and tissue engineering

Epithelia perform barrier functions being exposed to different fluids on the luminal and basal side. For long-term testing of new biomaterials as artificial basement membrane substitutes, it is important to simulate this fluid gradient. Individually-selected biomaterials can be placed in tissue carriers and in gradient containers, where different media are superfused. Epithelia growing on the tissue carriers form a physiological barrier during the whole culture period. Frequently however, pressure differences between the luminal and basal compartments occur. This is caused by a unilateral accumulation of gas bubbles in the container compartments resulting in tissue damage. Consequently, the occurence of gas bubbles has to be minimized. Air bubbles in the perfusion culture medium preferentially acumulate at sites where different materials come into contact. The first development is new screw caps for media bottles, specifically designed to allow fluid contact with only the tube and not the cap material. The second development is the separation of remaining gas bubbles from the liquid phase in the medium using newly-developed gas expander modules. By the application of these new tools, the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material can be significantly increased compared to earlier experiments.     

Adhesion and Proliferation of Human Adipo-Stromal Cells for Two- or Three-Dimensional Poly(ethylene terephthalate) Substrates with or without RGD Immobilization

The adhesion and proliferation of human adipo-stromal cells was investigated for poly(ethylene terephthalete) (PET) films of two-dimensional (2D) substrate and non-woven fabrics of three-dimensional (3D) substrate after seeding at the same cell density and culturing at the same medium volume to surface area of the substrates ratio. When seeded by a static seeding method, more cells adhered on the film than on the non-woven fabric. However, the number ratio of cells proliferated to those initially adhered was similar. For the non-woven fabric, cell proliferation was enhanced by an agitated culture method. NaOH treatment introduced carboxyl groups into the surface of substrates, irrespective of the substrate type. Cell adhesion of a peptide (Arg-Gly-Asp, RGD) was chemically immobilized through the carboxyl groups on the non-woven fabric surface at a density of 10 pmol/cm². RGD immobilization significantly increased the number of cells adhered after the agitated seeding method, but did not affect the cell proliferation. Phosphorylation of focal adhesion kinase (FAK) was also enhanced by the RGD immobilization on the PET non-woven fabrics.     

Regeneration of Achilles' Tendon: The Role of Dynamic Stimulation for Enhanced Cell Proliferation and Mechanical Properties

The tissue engineering of tendon was studied using highly elastic poly(L-lactide-co-ε-caprolactone) (PLCL) scaffolds and focusing on the effect of dynamic tensile stimulation. Tenocytes from rabbit Achilles tendon were seeded (1.0 × 10⁶ cells/scaffold) onto porous PLCL scaffolds and cultured for periods of 2 weeks and 4 weeks. This was performed in a static system and also in a bioreactor equipped with tensile modulation which mimicked the environmental surroundings of tendons with respect to tensile extension. The degradation of the polymeric scaffolds during the culture was relatively slow. However, there was an indication that cells accelerated the degradation of PLCL scaffolds. The scaffold/cell adducts from the static culture exhibited inferior strength (at 2 weeks 350 kPa, 4 weeks 300 kPa) compared to the control without cells (at 2 weeks 460 kPa, 4 weeks 340 kPa), indicating that the cells contributed to the enhanced degradation. On the contrary, the corresponding values of the adducts from the dynamic culture (at 2 weeks 430 kPa, 4 weeks 370 kPa) were similar to, or higher than, those from the control. This could be explained by the increased quantity of cells and neo-tissues in the case of dynamic culture compensating for the loss in tensile strength. Compared with static and dynamic culture conditions, mechanical stimulation played a crucial role in the regeneration of tendon tissue. In the case of the dynamic culture system, cell proliferation was enhanced and secretion of collagen type I was increased, as evidenced by DNA assay and histological and immunofluorescence analysis. Thus, tendon regeneration, indicated by improved mechanical and biological properties, was demonstrated, confirming the effect of mechanical stimulation. It could be concluded that the dynamic tensile stimulation appeared to be an essential factor in tendon/ligament tissue engineering, and that elastic PLCL co-polymers could be very beneficial in this process.     

Preparation and Characterization of Collagen from Soft-Shelled Turtle (Pelodiscus Sinensis) Skin for Biomaterial Applications

Collagen was isolated from the skin of soft-shelled turtle (Pelodiscus sinensis) by acid solubilization with pepsin. The yield of soft-shelled turtle collagen (STC) was 12.1% on a dry weight basis. The electrophoresis assay showed that STC consisted of a α ₁ α ₂ heterodimer similar to porcine collagen (PC). Amino-acid composition analysis showed that the hydroxyproline content of STC was 7.8%, which was lower than that of PC (9.5%). The denaturation temperature of STC was 36°C from optical rotation analysis. An accelerated fibrillogenesis of STC was observed in phosphate-buffered saline at 25°C. The resulting STC fibrillar gel had microfibrillar network with fibril diameter of ca. 124 nm, as revealed by observation with scanning electron microscopy. The compressive moduli of the STC gel and the PC gel were 3.2 ± 0.8 kPa and 3.6 ± 0.3 kPa, respectively. The potential of the STC gel for biomaterial applications was investigated by in vitro cell culture. Human dermal fibroblasts were three-dimensionally cultured in the STC gel and their growth was evaluated by DNA content measurement. Steady growth was observed in the STC gel for a 6-day culture period, although the growth rate was slower than in the PC gel. In conclusion, STC could be used as a novel collagen source for biomaterial applications.     

Use of Fluorescent-Antibody Staining of Cytocentrifuge-Prepared Smears in Combination with Cell Culture for Direct Detection of Respiratory Viruses

Over a 3-year period, 1,003 respiratory samples were collected and examined for selected respiratory viruses with cytocentrifuged prepared smears stained with fluorescently labeled antibodies (IFA) in conjunction with cell culture. IFA results were compared with results obtained by cell culture. Viruses were isolated or detected by direct means in 401 samples. Agreement between culture and IFA was 90%.     

Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies

Bulletin of Experimental Biology and Medicine - We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures...     

Substrates for Human Pluripotent Stem Cell Cultures in Conditioned Medium of Mesenchymal Stem Cells

We aimed to establish a culture system of human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), free from xenogeneic proteins, Matrigel^? and conditioned medium of mouse embryonic fibroblasts. The conditioned culture medium consisted of mesenchymal stem cells derived from human bone marrow. We examined surface properties suitable for hPSC culture by using self-assembled monolayers (SAMs) of alkanethiols with four different functional groups: CH₃, OH, COOH and NH₂. hPSCs neither adhered nor proliferated on surfaces with a water contact angle higher than 40°. Based on this finding, the contact angle of a polystyrene (PSt) culture dish was reduced to less than 40°, and COOH and OH groups were introduced to its surface by oxygen plasma treatment, making the PSt dish suitable for hPSC culture. This combination of a PSt dish treated with oxygen plasma treatment and conditioned medium of mesenchymal stem cells achieved a long-term maintenance of hPSCs without differentiation.     

Characterization of the Apatite Crystals of Bone and their Maturation in Osteoblast Cell Culture: Comparison with Native Bone Crystals

Calcium phosphate crystals deposited in the organic matrix synthesized by chick bone osteoblasts in culture were studied by x-ray and electron diffraction, Fourier transform infrared spectroscopy and chemical composition. The amounts of mineral phase deposited with time and the extent of calcification (% of mineral phase in the tissue) were also determined as a function of time, as were the nature of the changes in the short range order of the crystals. The amount of mineral deposited and the extent of calcification increased with time; the tissue not only contained more crystals of apatite, but the extent of calcification also increased with time as it does in vivo. After 30 days of culture the extent of calcification in the cell culture matrix was similar to that in late chick embryonic and early postnatal chick tibiae. The nature of the CO₃ and HPD₄ environments were similar to those found in vivo although the concentrations of these ions and the changes in their concentrations with time appeared to develop more slowly in cell culture than they do in vivo. However, the general overall pathway of maturation was similar in cell culture to that observed in vivo.     

Resazurin Dye as a Reliable Tool for Determination of Cell Number and Viability in Mesenchymal Stem Cell Culture

Human mesenchymal stem cells are a valuable cell source for tissue engineering. Determination of cell number and viability is crucial. However, this can be tested only at the end of cell culture. This study shows that Resazurin dye staining is a reliable tool for evaluation of cell number and viability in culture without cell perturbation.     

Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses

Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1- or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease.     

Stacks of Microfabricated Structures as Scaffolds for Cell Culture and Tissue Engineering

The fabrication of microscopic 3-dimensional structures for directing and inducing cell proliferation, migration and differentiation is a prevailing goal in tissue engineering. We have developed microfluidic molding methods to generate proof-of-principle polyurethane microstructures containing holes as models of synthetic tissue scaffolds. Molding is achieved by injecting sub-milliliter quantities of polyurethane precursor into a network of capped polydimethylsiloxane microchannels. After curing of the polyurethane, the microchannels can be opened to release a solid polyurethane structure which replicates the structure of the microchannel network. The microchannels can be reused to generate many exact replicas which can be stacked. We were able to design stacks that mimic various bone architectures. 3-dimensional microtomographic images were used for the first time for visualization and quality control of the stacked microfabricated structures. The polyurethane structures are amenable to cell culture. This technology could potentially be applied to microfabricate implanTable 3-dimensional scaffolds of specified architecture and composition for tissue growth and regeneration.     

Fabrication and Characterization of Waterborne Biodegradable Polyurethanes 3-Dimensional Porous Scaffolds for Vascular Tissue Engineering

In this study, a series of 3-D interconnected porous scaffolds with various pore diameters and porosities was fabricated by freeze-drying with non-toxic biodegradable waterborne polyurethane (WBPU) emulsions of different concentration. The structures of these porous scaffolds were characterized by scanning electron microscopy (SEM), and the pore diameters were calculated using CIAS 3.0 software. The pores obtained were 3-D interconnected in the scaffolds. The scaffolds obtained at different pre-freeze temperatures showed a pore diameter ranging from 2.8 to 99.9 μm with a pre-freezing temperature of ?60°C and from 13.1 to 229.1 μm with a pre-freezing temperature of ?25°C. The scaffolds fabricated with WBPU emulsions of different concentration at the same pre-freezing temperature (?25°C) had pores with mean pore diameter between 90.8 and 39.6 μm and porosity between 92.0 and 80.0%, depending on the emulsion concentration. The effect of porous structure of the scaffolds on adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) cultured in vitro was evaluated using the MTT assay and environmental scanning electron microscopy (ESEM). It was found that the better adhesion and proliferation of HUVECs on 3-D scaffolds of WBPU with relative smaller pore diameter and lower porosity than those on scaffolds with larger pore and higher porosity and film. Our work suggests that fabricating a scaffold with controllable pore diameter and porosity could be a good method to be used in tissue-engineering applications to obtain carriers for cell culture in vitro.     

测定细胞存活和增殖的MTT方法的建立

MTT方法具有简便,灵敏,稳定可靠,不需要同位素等优点。为了摸索测定细胞存活和增殖的MTT方法的各种最适条件,我们以Raji细胞为对象,比较了十二种不同的MTT甲(月替)溶解缓冲液。实验结果表明,20％SDS-50％DMF效果最好,溶解力强,溶解甲(月替)所需时间短,背景低,OD值高,不会引起培养基中小牛血清的絮状沉淀。其溶解的甲(月替)产物吸收峰在550—600nm之间。测定细胞范围为每孔500—400 000,这一范围能满足生物学和医学研究中多种测定的需要。