Cryogelation for preparation of novel biodegradable tissue-engineering scaffolds

2-Hydroxyethyl methacrylate-L-lactate (HEMA-LLA) and HEMA-L-lactate-dextran (HEMA-LLA-D) were synthesized. ¹H-NMR confirmed the formation of these oligomers and macromers. Cryogels with different pore structures were prepared using different amounts of HEMA, HEMA-LLA and HEMA-LLA-D by a cryogelation technique. SEM micrographs exhibited pore morphologies. Cryogels were highly porous with interconnected pore structures, opaque, spongy and highly elastic. It was possible to compress them to remove the water in the pores and to return to their original form just by immersing them in water in few minutes, which was quite reproducible. Their swelling abilities, compressive strengths and degradation in buffer solutions were found to be related with their structural properties which was controlled by changing the cryogelation recipe.     

Novel Melt-Processable Chitosan–Polybutylene Succinate Fibre Scaffolds for Cartilage Tissue Engineering

Novel chitosan/polybutylene succinate fibre-based scaffolds (C-PBS) were seeded with bovine articular chondrocytes in order to assess their suitability for cartilage tissue engineering. Chondrocytes were seeded onto C-PBS scaffolds using spinner flasks under dynamic conditions, and cultured under orbital rotation for a total of 6 weeks. Non-woven polyglycolic acid (PGA) felts were used as reference materials. Tissue-engineered constructs were characterized by scanning electron microscopy (SEM), hematoxylin–eosin (H&E), toluidine blue and alcian blue staining, immunolocalization of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification at different time points. SEM showed the chondrocytes' typical morphology, with colonization at the surface and within the pores of the C-PBS scaffolds. These observations were supported by routine histology. Toluidine blue and alcian blue stains, as well as immunohistochemistry for collagen types I and II, provided qualitative information on the composition of the engineered extracellular matrix. More pronounced staining was observed for collagen type II than collagen type I. Similar results were observed with constructs engineered on PGA scaffolds. These also exhibited higher amounts of matrix glycosaminoglycans and presented a central region which contained fewer cells and little matrix, a feature that was not detected with C-PBS constructs.     

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.     

In Vitro Osteogenesis of Synovium Mesenchymal Cells Induced by Controlled Release of Alendronate and Dexamethasone from a Sintered Microspherical Scaffold

In vitro osteogenesis was successfully achieved with synovium-derived mesenchymal stem cells (SMSCs), which intrinsically have a strong chondrogenic tendency, by in situ release of alendronate (AL) and dexamethasone (Dex) from poly(lactic-co-glycolic acid) (PLGA)/hydroxyapatite (HA) sintered microspherical scaffold (PLGA/HA-SMS). Cumulative release profiles of AL and Dex from PLGA/HA-SMS and the influence on SMSCs osteogenic commitment were investigated. SMSCs seeded in Al-/Dex-loaded PLGA/HA-SMS (PLGA/HA-Com-SMS) exhibited significant osteogenic differentiation, as indicated by high yields of alkaline phosphatase (ALP) and bone calcification. In addition, mechanical properties (compressional) of PLGA/HA-Com-SMSs were also evaluated and approved. In conclusion, by promoting osteogenic commitment of SMSCs in vitro, this newly designed controlled-release system opens a new door to bone reparation and regeneration.     

A Calcium-Cross-Linked Hydrogel Based on Alginate-Modified Atelocollagen Functions as a Scaffold Material

We have developed a scaffold material consisting of a covalently-bonded structure of alginate and atelocollagen (AtCol). Addition of calcium ions caused the material to form a hydrogel (alginate-modified AtCol gel). The condition of the alginate-modified AtCol gel could be controlled by the feed ratio of alginate and the activating reagent. Measurement of temporal stability in culture medium suggested that covalent bonding between alginate and AtCol might contribute to the structural stability of the alginate-modified AtCol gel. Culture with endothelial cells indicated that cell adhesiveness on the alginate-modified AtCol gel was similar to that on native collagen. Collagenase digestion revealed that the alginate-modified AtCol gel had considerable ability to retain basic fibroblast growth factor. Additionally, active cell migration into alginate-modified AtCol was detected by in vitro assay using endothelial cells. These findings indicate that this gel material can be expected to function as a scaffold for inducing vascular in-growth.     

Degradation Behavior of 3D Porous Polydioxanone-b-Polycaprolactone Scaffolds Fabricated Using the Melt-Molding Particulate-Leaching Method

Recently, polydioxanone (PDO) and polycaprolactone (PCL) have been applied in applications for tissue engineering owing to their flexibility, as well as biocompatibility and biodegradability, even though their degradation rates are usually either too fast or too slow for many applications. In this study, we synthesized poly(dioxanone-b-caprolactone) co-polymers (PDOCLs) with different DO/CL ratio (0:10–10:0) by ring-opening polymerization. The synthesized co-polymers were characterized by ¹H-NMR, the measurement of inherent viscosity (IV), GPC and DSC. PDOCL scaffolds with different DO/CL ratio were fabricated by a melt-molding particulate-leaching method without using any organic solvents during the scaffold fabrication process. The degradation behavior (in vitro) of the PDOCL scaffolds was evaluated in PBS at 37°C for up to 56 days by the changes in molecular weight, mechanical strength, gross weight and pH. It was observed that the degradation rate of PDOCL scaffolds could be controlled by adjusting the DO/CL ratio of the co-polymers (increasing CL composition leads to slower degradation rate). The PDOCL scaffolds did not lead to a significant drop in pH during the degradation, not even for the PDO-dominant PDOCL scaffolds showing a fast degradation rate, indicating the formation of a small amount of acidic by-products compared to the PLGA scaffolds. From the results, it was expected that the PDOCLs can be a new flexible scaffolding material with different degradation rate for various tissue-engineering applications.     

Fabrication and Characterization of Porous Tubular Silk Fibroin Scaffolds

Silk fibroin (SF) has been one of promising resources of biotechnology and biomedical materials due to its unique properties. Here, different sizes of porous tubular scaffolds were fabricated from a SF aqueous solution with the addition of poly(ethylene glycol diglycidyl ether) (PGDE). The scaffolds were generally flexible and transparent at the wet state with a pore size of 81–128 μm and porosity of 90–96%, depending on the concentrations of SF and PGDE. The mechanical properties measurement showed that the tubular SF scaffolds had satisfying tensile and compression properties, especially the excellent deformation–recovery ability. FT-IR spectra indicated that the SF in the tubular scaffolds was in a β-sheet structure, and no PGDE characteristic band was observed, suggesting that the PGDE could be removed from the scaffolds by soaking in deionized water. The cell compatibility of scaffolds was evaluated, and no obvious cytotoxicity to mouse L-929 fibroblasts was detected.     

Cell growth on tissue-engineering scaffolds prepared by gamma irradiation grafting of N-vinyl-2-pyrrolidone onto polyvinyl alcohol

In the field of tissue engineering, promoting cell attachment and proliferation in polymer matrices is an attractive challenge for treating patients suffering from the loss or dysfunction of tissues or organs. In this study we have investigated the effect of grafting N-vinyl-2-pyrrolidone (NVP) by gamma irradiation onto polyvinyl alcohol (PVA), a highly hydrophilic and non-toxic material. PVA scaffolds were prepared by freeze–thaw and progen (glycerol) methods. In the first method, samples were freeze–thawed for three consecutive cycles at ?25°C (90 min) and room temperature (60 min); in the latter, 0–40% glycerol was used as progen. Gamma irradiation of the scaffolds in the presence of NVP was performed at different concentrations (2, 3, 4 and 6%) with 5, 10 and 15 kGy ⁶⁰Co. The highest percentage of grafting was obtained at 4% NVP solution and 15 kGy. Cell attachment was optimal for the scaffolds prepared using freeze–thaw and glycerol methods with 3.8% and 2.7% polyvinyl pyrrolidone (PVP), respectively.     

Chitosan tissue scaffolds by emulsion templating

A novel method for producing porous chitosan gels with controllable pore size and volume fraction has been developed. Complex-shaped 3D objects can be fabricated. Chitosan gels produced with the method are believed to be suitable candidates for tissue scaffolds. The method involves creating an oil-in-water emulsion in which the water phase contains chitosan and a temperature-activated cross-linking agent. The emulsion can then be poured or injected into a mould with the shape of the desired object. The chitosan is cross-linked by heating the emulsion to about 75°C for about 15 min. The gelled object is then washed to remove the oil phase and surfactant. The gels were then dried in air, and further washed in ethanol. Scanning electron microscopy was then used to observe the pore size and fraction. The amount of porosity is directly proportional to the amount of oil phase. The pore size is controlled by the size of the oil droplets which is controlled primarily by the amount of surfactant added.