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.

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.

Influence of the substrate's hydrophilicity on the in vitro Schwann cells viability.

A series of polymeric biomaterials including poly (methyl acrylate) (PMA), chitosan (CHT), poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate and hydroxyethyl acrylate monomeric units were tested in vitro as culture substrates and compared for their impact on the proliferation and expansion of Schwann cells (SCs). Immunocytochemical staining assay and scanning electron microscopy techniques were applied to perform a quantitative analysis to determine the correct maintenance of the cultured glial cells on the different biomaterials. The results strongly suggest that cell attachment and proliferation is influenced by the substrate's surface chemistry, and that hydrophobic biomaterials based on PMA, PEA, and the copolymers PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and proliferation of SCs in vitro.

Fibrin-chitosan composite substrate for in vitro culture of chondrocytes.

The aim of this study was to develop a biocompatible monolayer substrate based on fibrin and chitosan for in vitro culture of chondrocytes. Fibrin-chitosan composite substrates combined the proved cell adhesion properties of fibrin with the hydrophilicity and poor adhesion capacity of chitosan. Chitosan microspheres were produced by coacervation method, agglomerated within a fibrin network and subsequently crosslinked with genipin. The composite substrate was stable for 28 days of culture due to the high crosslinking density. Human chondrocytes cultured on the composite substrate were viable during the culture period. At the end of culture time (28 days) the composite substrate showed low cellular proliferation, 41% more collagen type II and 13% more production of sulfated glycosaminoglycans with respect to the amounts found at 14 days. The study revealed that dedifferentiated chondrocytes cultured in monolayer on the composite substrate can re-acquire characteristics of differentiated cells without using three-dimensional substrates or chondrogenic media.