Gelatin microparticles aggregates as three-dimensional scaffolding system in cartilage engineering.

A three-dimensional (3D) scaffolding system for chondrocytes culture has been produced by agglomeration of cells and gelatin microparticles with a mild centrifuging process. The diameter of the microparticles, around 10 µ, was selected to be in the order of magnitude of the chondrocytes. No gel was used to stabilize the construct that maintained consistency just because of cell and extracellular matrix (ECM) adhesion to the substrate. In one series of samples the microparticles were charged with transforming growth factor, TGF-ß1. The kinetics of growth factor delivery was assessed. The initial delivery was approximately 48 % of the total amount delivered up to day 14. Chondrocytes that had been previously expanded in monolayer culture, and thus dedifferentiated, adopted in this 3D environment a round morphology, both with presence or absence of growth factor delivery, with production of ECM that intermingles with gelatin particles. The pellet was stable from the first day of culture. Cell viability was assessed by MTS assay, showing higher absorption values in the cell/unloaded gelatin microparticle pellets than in cell pellets up to day 7. Nevertheless the absorption drops in the following culture times. On the contrary the cell viability of cell/TGF-ß1 loaded gelatin microparticle pellets was constant during the 21 days of culture. The formation of actin stress fibres in the cytoskeleton and type I collagen expression was significantly reduced in both cell/gelatin microparticle pellets (with and without TGF-ß1) with respect to cell pellet controls. Total type II collagen and sulphated glycosaminoglycans quantification show an enhancement of the production of ECM when TGF-ß1 is delivered, as expected because this growth factor stimulate the chondrocyte proliferation and improve the functionality of the tissue.

Molecular mobility in biodegradable poly(ε-caprolactone)/poly(hydroxyethyl acrylate) networks.

Poly(ε-caprolactone)/poly(hydroxyethyl acrylate) networks have been investigated by thermally stimulated depolarization currents (TSDC) and differential scanning calorimetry (DSC). The introduction of hydrophilic units (HEA) in the system aiming at tailoring the hydrophilicity of the system results in a series of copolymer networks with microphase separation into hydrophobic/hydrophilic domains. Polycaprolactone (PCL) crystallization is prevented by the topological constraints HEA units imposed in such heterogeneous domains. Moreover, the mobility of the amorphous PCL chains is enhanced as revealed by the main relaxation process which becomes faster. The glass transition of PHEA-rich domains shifts to lower temperatures, as the total amount of PCL in the copolymer increases, due to the presence of PCL units within the same region. The behaviour of the copolymer networks swollen with different content of water has been investigated to analyze the interaction between water molecules and hydrophobic/hydrophilic domains and provide further insights into the molecular structure of the system.

Alkaline degradation study of linear and network poly(ε-caprolactone).

Alkaline hydrolysis of a polycaprolactone (PCL) network obtained by photopolymerization of a PCL macromer was investigated. The PCL macromer was obtained by the reaction of PCL diol with methacrylic anhydride. Degradation of PCL network is much faster than linear PCL; the weight loss rate is approximately constant until it reaches around 70%, which happens after approximately 60 h in PCL network and 600 h in linear PCL. Calorimetric results show no changes in crystallinity throughout degradation, suggesting that it takes place in the crystalline and amorphous phases simultaneously. Scanning electron microscopy microphotographs indicate that degradation is produced by a different erosion mechanism in both kinds of samples. The more hydrophilic network PCL would follow a bulk-erosion mechanism, whereas linear PCL would follow a surface-erosion mechanism. Mechanical testing of degraded samples shows a decline in mechanical properties due to changes in sample porosity as a consequence of the degradation process.

Regenerative and Resorbable PLA/HA Hybrid Construct for Tendon/Ligament Tissue Engineering.

Tendon and ligament shows extremely limited endogenous regenerative capacity. Current treatments are based on the replacement and or augmentation of the injured tissue but the repaired tissue rarely achieve functionality equal to that of the preinjured tissue. To address this challenge, tissue engineering has emerged as a promising strategy. This study develops a regenerative and resorbable hybrid construct for tendon and ligament engineering. The construct is made up by a hollow poly-lactic acid braid with embedded microspheres carrying cells and an anti-adherent coating, with all the parts being made of biodegradable materials. This assembly intends to regenerate the tissue starting from the interior of the construct towards outside while it degrades. Fibroblasts cultured on poly lactic acid and hyaluronic acid microspheres for 6 h were injected into the hollow braid and the construct was cultured for 14 days. The cells thus transported into the lumen of the construct were able to migrate and adhere to the braid fibers naturally, leading to a homogeneous proliferation inside the braid. Moreover, no cells were found on the outer surface of the coating. Altogether, this study demonstrated that PLA/HA hybrid construct could be a promising material for tendon and ligament repair.

Effect of poly(L-lactide) surface topography on the morphology of in vitro cultured human articular chondrocytes.

Human articular chondrocytes were cultured in vitro on poly(L-lactic) acid, PLLA, substrates. Influence of the surface topography on cell morphology was found. Different surface microtopographies were obtained on PLLA by crystallizing at 120 degrees C after nucleation treatments that include isothermal stages at temperatures just below (55 degrees C) and just above (75 degrees C) the glass transition temperature (T(g) = 65 degrees C). Isothermal crystallization from the melt gave rise to big spherulites (approx. 50 microm diameter) with approx. 1 microm depth. Crystallization after nucleation treatments results in smaller (approx. 5 microm)-difficult to distinguish-spherulites. Cell viability was excellent and not affected by the surface roughness. Cell population on the nucleated samples resembles the result of culture on the reference tissue culture polystyrene (TCPS). However, cells cultured on big spherulites (PLLA isothermally crystallized without nucleation treatment) show a peculiar morphology, with a more isolated disposition and growth oriented in a characteristic direction.

Dielectric relaxation spectrum of poly (epsilon-caprolactone) networks hydrophilized by copolymerization with 2-hydroxyethyl acrylate.

The dielectric relaxation spectrum of polycaprolactone (PCL) networks hydrophilized with different amounts of 2-hydroxyethyl acrylate (HEA) is investigated. PCL is a semicrystalline polyester with a complex relaxation spectrum that includes the main alpha relaxation and two secondary modes (beta, gamma) at lower temperatures. The overlapping of the different relaxational modes was split by using several Havriliak-Negami functions. Crosslinking the material modifies the dynamics of the main relaxation process as reflected by the parameters that characterize the Vogel behavior of the process and the dynamic fragility. The incorporation of HEA units in the network results in a material with microphase separation: two alpha processes are detected, the one corresponding to the PCL chains and the new one associated to nanometric regions that contain different amount of both comonomers. The incorporation of the HEA units in the system involves the presence of a new beta(sw) relaxation due to the link of two side chains by water molecules through hydrogen bonding.