Effect of electrical stimulation on chondrogenic differentiation of mesenchymal stem cells cultured in hyaluronic acid - Gelatin injectable hydrogels.

Electrical stimulation (ES) has provided enhanced chondrogenesis of mesenchymal stem cells (MSCs) cultured in micro-mass without the addition of exogenous growth factors. In this study, we demonstrate for the first time that ES of MSCs encapsulated in an injectable hyaluronic acid (HA) - gelatin (GEL) mixture enhances the chondrogenic potential of the hydrogel. Samples were stimulated for 21 days with 10 mV/cm at 60 kHz, applied for 30 min every 6 h a day. Mechanical properties of hydrogels were higher if the precursors were dissolved in Calcium-Free Krebs Ringer Buffer (G' = 1141 ± 23 Pa) compared to those diluted in culture media (G' = 213 ± 19 Pa). Cells within stimulated hydrogels were rounder (55%) than non-stimulated cultures (32%) (p = 0.005). Chondrogenic markers such as SOX-9 and aggrecan were higher in stimulated hydrogels compared to controls. The ES demonstrated that normalized content of glycosaminoglycans and collagen to DNA was slightly higher in stimulated samples. Additionally, collagen type II normalized to total collagen was 2.43 times higher in stimulated hydrogels. These findings make ES a promising tool for enhancing articular cartilage tissue engineering outcomes by combining hydrogels and MSCs.

Chitosan-silica hybrid porous membranes.

Chitosan-silica porous hybrids were prepared by a novel strategy in order to improve the mechanical properties of chitosan (CHT) in the hydrogel state. The inorganic silica phase was introduced by sol-gel reactions in acidic medium inside the pores of already prepared porous scaffolds. In order to make the scaffolds insoluble in acidic media chitosan was cross-linked by genipin (GEN) with an optimum GEN concentration of 3.2 wt.%. Sol-gel reactions took place with Tetraethylorthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane (GPTMS) acting as silica precursors. GPTMS served also as a coupling agent between the free amino groups of chitosan and the silica network. The morphology study of the composite revealed that the silica phase appears as a layer covering the chitosan membrane pore walls. The mechanical properties of the hybrids were characterized by means of compressive stress-strain measurements. By immersion in water the hybrids exhibit an increase in elastic modulus up to two orders of magnitude.

Comparative study of PCL-HAp and PCL-bioglass composite scaffolds for bone tissue engineering.

The aim of this work is to compare the effect of hydroxyapatite (HAp) or bioglass (BG) nanoparticles in a polycaprolactone composite scaffold aimed to bone regeneration. To allow a comparison of the influence of both types of fillers, scaffolds made of PCL or composites containing up to 20 % by weight HAp or BG were obtained. Scaffolds showed acceptable mechanical properties for its use and high interconnected porosity apt for cellular colonization. To study the effect of the different materials on pre-osteoblast cells differentiation, samples with 5 % mineral reinforcement, were cultured for up to 28 days in osteogenic medium. Cells proliferated in all scaffolds. Nevertheless, differentiation levels for the selected markers were higher in pure PCL scaffolds than in the composites; inclusion of bioactive particles showed no positive effects on cell differentiation. In osteogenic culture conditions, the presence of bioactive particles is thus not necessary in order to observe good differentiation.

Different hyaluronic acid morphology modulates primary articular chondrocyte behavior in hyaluronic acid-coated polycaprolactone scaffolds.

Scaffolds for cartilage tissue engineering should promote both adequate biomechanical environment and chondrogenic stimulation. Hyaluronic acid (HA) has been used in cartilage engineering for its chondrogenic and chondroprotective properties, nevertheless its mechanical properties are limited. Influence of HA microstructure in chondrocyte response has not been addressed yet. In this work, polycaprolactone (PCL) scaffolds were modified using HA following two coating strategies: coating in one step (PCL-HA1s) yields a gel-like phase within the scaffold, whereas a two-step reaction (PCL-HA2s) yields a thin HA layer coating internal surfaces of PCL structure. Chondrocytes were seeded in the scaffolds and cultured in dedifferentiating conditions up to 3 weeks and analyzed using a total DNA assay and sulfated glycosaminoglycan (sGAG) determination assay; cell morphology and extracellular matrix secretion were assessed by electron microscopy as well as immunofluorescent imaging (collagen I, collagen II, aggrecan, CD44). Cells proliferate in all samples and no cytotoxicity is observed. PCL-HA1s shows higher sGAG production per cell than PCL and PCL-HA2s at all times. Presence of hyaluronic acid promotes qualitative expression of CD44 surface markers and aggrecan (more visible in PCL-HA1s than PCL-HA2s), whereas in dedifferentiating conditions, expression of CD44 and aggrecan can hardly be detected in pure PCL scaffolds. Collagen type II seems more prominent in PCL-HA2s; although PCL-HA2s shows markers for COL II, aggrecan and CD44, quantitative ECM production is not improved with respect to PCL. It is thus likely that CD44 activation is not sufficient for explaining the better response in PCL-HA1s.

Channeled scaffolds implanted in adult rat brain.

Scaffolds with aligned channels based on acrylate copolymers, which had previously demonstrated good compatibility with neural progenitor cells were studied as colonizable structures both in vitro with neural progenitor cells and in vivo, implanted without cells in two different locations, in the cortical plate of adult rat brains and close to the subventricular zone. In vitro, neuroprogenitors colonize the scaffold and differentiate into neurons and glia within its channels. When implanted in vivo immunohistochemical analysis by confocal microscopy for neural and endothelial cells markers demonstrated that the scaffolds maintained continuity with the surrounding neural tissue and were colonized by GFAP-positive cells and, in the case of scaffolds implanted in contact with the subventricular zone, by neurons. Local angiogenesis was evidenced in the interior of the scaffolds' pores. New axons and neural cells from the adult neural niche abundantly colonized the biomaterial's inner structure after 2 months, and minimal scar formation was manifest around the implant. These findings indicate the biocompatibility of the polymeric material with the brain tissue and open possibilities to further studies on the relevance of factors such as scaffold structure, scaffold seeding and scaffold placement for their possible use in regenerative strategies in the central nervous system. The development of neural interfaces with minimized glial scar and improved tissue compatibility of the implants may also benefit from these results. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:3276-3286, 2012.

Three-dimensional scaffolds as a model system for neural and endothelial 'in vitro' culture.

Biomaterials based on the hydrophobic homopolymer poly(ethyl acrylate), PEA, and its copolymers with hydroxyethyl acrylate, p(EA-co-HEA) and methacrylic acid, p(EA-co-MAAc) were prepared as polymeric scaffolds with interconnected pores of 90 microns and tested in vitro as culture substrates and compared for their impact on the differentiation of neural stem cells (NSC) obtained from the subventricular zone (SVZ) of postnatal rats and human endothelial cells (HUVEC). Immunocytochemical staining assay for specific markers show that p(EA-co-MAAc) scaffolds were suitable substrates to promote cell attachment and differentiation of adult NSC and HUVEC cells.

In vivo evaluation of 3-dimensional polycaprolactone scaffolds for cartilage repair in rabbits.

BACKGROUND: Cartilage tissue engineering using synthetic scaffolds allows maintaining mechanical integrity and withstanding stress loads in the body, as well as providing a temporary substrate to which transplanted cells can adhere. PURPOSE: This study evaluates the use of polycaprolactone (PCL) scaffolds for the regeneration of articular cartilage in a rabbit model. STUDY DESIGN: Controlled laboratory study. METHODS: Five conditions were tested to attempt cartilage repair. To compare spontaneous healing (from subchondral plate bleeding) and healing due to tissue engineering, the experiment considered the use of osteochondral defects (to allow blood flow into the defect site) alone or filled with bare PCL scaffold and the use of PCL-chondrocytes constructs in chondral defects. For the latter condition, 1 series of PCL scaffolds was seeded in vitro with rabbit chondrocytes for 7 days and the cell/scaffold constructs were transplanted into rabbits' articular defects, avoiding compromising the subchondral bone. Cell pellets and bare scaffolds were implanted as controls in a chondral defect. RESULTS: After 3 months with PCL scaffolds or cells/PCL constructs, defects were filled with white cartilaginous tissue; integration into the surrounding native cartilage was much better than control (cell pellet). The engineered constructs showed histologically good integration to the subchondral bone and surrounding cartilage with accumulation of extracellular matrix including type II collagen and glycosaminoglycan. The elastic modulus measured in the zone of the defect with the PCL/cells constructs was very similar to that of native cartilage, while that of the pellet-repaired cartilage was much smaller than native cartilage. CONCLUSION: The results are quite promising with respect to the use of PCL scaffolds as aids for the regeneration of articular cartilage using tissue engineering techniques.

Chitosan microparticles as injectable scaffolds for tissue engineering.

The use of chitosan microparticles as injectable carriers for cell transplantation represents a promising alternative to avoid the drawbacks of the implantation of other forms of three-dimensional (3D) scaffolds seeded with cells. In this study, a 3D construct is obtained in vitro by combining chitosan microparticles crosslinked with genipin and goat bone marrow stromal cells (GBMCs). Cell viability and the morphology of GBMCs were evaluated after culture for 7 and 14 days. Our results show the feasibility of chitosan microparticles as potential injectable scaffolds for tissue engineering and regenerative medicine.