A cell-free approach with a supporting biomaterial in the form of dispersed microspheres induces hyaline cartilage formation in a rabbit knee model.

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage.

Biostable scaffolds of polyacrylate polymers implanted in the articular cartilage induce hyaline-like cartilage regeneration in rabbits.

PURPOSE: To study the influence of scaffold properties on the organization of in vivo cartilage regeneration. Our hypothesis was that stress transmission to the cells seeded inside the pores of the scaffold or surrounding it, which is highly dependent on the scaffold properties, determines the differentiation of both mesenchymal cells and dedifferentiated autologous chondrocytes. METHODS: 4 series of porous scaffolds made of different polyacrylate polymers, previously seeded with cultured rabbit chondrocytes or without cells, were implanted in cartilage defects in rabbits. Subchondral bone was injured during the surgery to allow blood to reach the implantation site and fill the scaffold pores. RESULTS: At 3 months after implantation, excellent tissue regeneration was obtained, with a well-organized layer of hyaline-like cartilage at the condylar surface in most cases of the hydrophobic or slightly hydrophilic series. The most hydrophilic material induced the poorest regeneration. However, no statistically significant difference was observed between preseeded and non-preseeded scaffolds. All of the materials used were biocompatible, biostable polymers, so, in contrast to some other studies, our results were not perturbed by possible effects attributable to material degradation products or to the loss of scaffold mechanical properties over time due to degradation. CONCLUSIONS: Cartilage regeneration depends mainly on the properties of the scaffold, such as stiffness and hydrophilicity, whereas little difference was observed between preseeded and non-preseeded scaffolds.

Time evolution of in vivo articular cartilage repair induced by bone marrow stimulation and scaffold implantation in rabbits.

PURPOSE: Tissue engineering techniques were used to study cartilage repair over a 12-month period in a rabbit model. METHODS: A full-depth chondral defect along with subchondral bone injury were originated in the knee joint, where a biostable porous scaffold was implanted, synthesized of poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymer. Morphological evolution of cartilage repair was studied 1 and 2 weeks, and 1, 3, and 12 months after implantation by histological techniques. The 3-month group was chosen to compare cartilage repair to an additional group where scaffolds were preseeded with allogeneic chondrocytes before implantation, and also to controls, who underwent the same surgery procedure, with no scaffold implantation. RESULTS: Neotissue growth was first observed in the deepest scaffold pores 1 week after implantation, which spread thereafter; 3 months later scaffold pores were filled mostly with cartilaginous tissue in superficial and middle zones, and with bone tissue adjacent to subchondral bone. Simultaneously, native chondrocytes at the edges of the defect started to proliferate 1 week after implantation; within a month those edges had grown centripetally and seemed to embed the scaffold, and after 3 months, hyaline-like cartilage was observed on the condylar surface. Preseeded scaffolds slightly improved tissue growth, although the quality of repair tissue was similar to non-preseeded scaffolds. Controls showed that fibrous cartilage was mainly filling the repair area 3 months after surgery. In the 12-month group, articular cartilage resembled the untreated surface. CONCLUSIONS: Scaffolds guided cartilaginous tissue growth in vivo, suggesting their importance in stress transmission to the cells for cartilage repair.

Implantation of a polycaprolactone scaffold with subchondral bone anchoring ameliorates nodules formation and other tissue alterations.

PURPOSE: Articular cartilage has limited repair capacity. Two different implant devices for articular cartilage regeneration were tested in vivo in a sheep model to evaluate the effect of subchondral bone anchoring for tissue repair. METHODS: The implants were placed with press-fit technique in a cartilage defect after microfracture surgery in the femoral condyle of the knee joint of the sheep and histologic and mechanical evaluation was done 4.5 months later. The first group consisted of a biodegradable polycaprolactone (PCL) scaffold with double porosity. The second test group consisted of a PCL scaffold attached to a poly(L-lactic acid) (PLLA) pin anchored to the subchondral bone. RESULTS: For both groups most of the defects (75%) showed an articular surface that was completely or almost completely repaired with a neotissue. Nevertheless, the surface had a rougher appearance than controls and the repair tissue was immature. In the trials with solely scaffold implantation, severe subchondral bone alterations were seen with many large nodular formations. These alterations were ameliorated when implanting the scaffold with a subchondral bone anchoring pin. DISCUSSIONS: The results show that tissue repair is improved by implanting a PCL scaffold compared to solely microfracture surgery, and most importantly, that subchondral bone alterations, normally seen after microfracture surgery, were partially prevented when implanting the PCL scaffold with a fixation system to the subchondral bone.

Biointegration of corneal macroporous membranes based on poly(ethyl acrylate) copolymers in an experimental animal model.

Currently available keratoprosthesis models (nonbiological corneal substitutes) have a less than 75% graft survival rate at 2 years. We aimed at developing a model for keratoprosthesis based on the use of poly(ethyl acrylate) (PEA)-based copolymers, extracellular matrix-protein coating and colonization with adipose-derived mesenchymal stem cells. Human adipose tissue derived mesenchymal stem cells (h-ADASC) colonization efficiency of seven PEA-based copolymers in combination with four extracellular matrix coatings were evaluated in vitro. Then, macroporous membranes composed of the optimal PEA subtypes and coating proteins were implanted inside rabbit cornea. After a 3-month follow-up, the animals were euthanized, and the clinical and histological biointegration of the implanted material were assessed. h-ADASC adhered and survived when cultured in all PEA-based macroporous membranes. The addition of high hydrophilicity to PEA membranes decreased h-ADASC colonization in vitro. PEA-based copolymer containing 10% hydroxyethyl acrylate (PEA-HEA10) or 10% acrylic acid (PEA-AAc10) monomeric units showed the best cellular colonization rates. Collagen plus keratan sulfate-coated polymers demonstrated enhanced cellular colonization respect to fibronectin, collagen, or uncoated PEAs. In vivo implantation of membranes resulted in an extrusion rate of 72% for PEA, 50% for PEA-AAc10, but remarkably of 0% for PEA-HEA10. h-ADASC survival was demonstrated in all the membranes after 3 months follow-up. A slight reduction in the extrusion rate of h-ADASC colonized materials was observed. No significant differences between the groups with and without h-ADASC were detected respect to transparency or neovascularization. We propose PEA with low hydroxylation as a scaffold for the anchoring ring of future keratoprosthesis.

New porous polycaprolactone-silica composites for bone regeneration.

Polycaprolactone porous membranes were obtained by freeze extraction of dioxane from polycaprolactone-dioxane solid solutions. Porosities as high as 90% with interconnected structures were obtained by this technique. A silica phase was synthesized inside the pores of the polymer membrane by sol-gel reaction using tetraethylorthosilicate (TEOS) as a silica precursor and catalyzed in acidic and basic conditions. Two different morphologies of the inorganic phase were obtained depending on the type of catalyst. In acid catalyzed sol-gel reaction, a homogeneous layer of silica was deposited on the pores, and discrete microspheres were synthesized on the pore walls when a basic catalyst was used. The morphology of the inorganic phase influenced the mechanical and thermal behavior, as well as the hydrophilic character of the composites. Bioactivity of the porous materials was tested in vitro by measuring the deposition of hydroxyapatite on the surfaces of the porous composite membranes. Polycaprolactone/silica composites revealed a superior bioactivity performance compared with that of the pure polymer; evidenced by the characteristic cauliflower structures on the material surface, increase in weight and Ca/P ratio of the hydroxyapatite layer. Also, the acid catalyzed composites presented better bioactivity than the base catalyzed composites, evidencing the importance in the morphology of the silica phase.

Molecular dynamics of ethylene glycol dimethacrylate glass former: influence of different crystallization pathways.

The crystallization induced by different thermal treatments of a low molecular weight glass former, ethylene glycol dimethacrylate (EGDMA), was investigated by dielectric relaxation spectroscopy (DRS) and differential scanning calorimetry (DSC). The fully amorphous material, dielectrically characterized for the first time, exhibits three relaxation processes: the alpha-relaxation related to dynamic glass transition whose relaxation rate obeys a Vogel-Fulcher-Tamman-Hesse (VFTH) law and two secondary processes (beta and gamma) with Arrhenius temperature dependence. Therefore, the evaluation of distinct crystallization pathways driven by different thermal histories was accomplished by monitoring the mobility changes in the multiple dielectric relaxation processes. Besides isothermal cold-crystallization, nonisothermal crystallizations coming from both the melt and the glassy states were induced. While an amorphous fraction, characterized by a glass transition, remains subsequent to crystallization from the melt, no alpha-relaxation is detected after the material undergoes nonisothermal cold-crystallization. In the latter, the secondary relaxations persist with a new process that evolves at low frequencies, designated as alpha' that was also detected at advanced crystallization states under isothermal cold-crystallization. Under the depletion of the alpha-relaxation, the beta-process when detected becomes better resolved keeping the same location prior to crystallization leading to a decoupled temperature dependence relative to the alpha-process.

Blending polysaccharides with biodegradable polymers. I. Properties of chitosan/polycaprolactone blends.

Blends of polycaprolactone (PCL) and chitosan (CHT) were prepared by casting from a solution. CHT and PCL were dissolved by using acetic acid/water mixtures. Both solutions were slowly mixed to cast blend films containing 10%, 20%, 30%, and 40% by weight of CHT. PCL and CHT form phase separated blends. The phase morphology is in large extent controlled by the casting procedure. Even if casting of the film starts from a clear solution, the solvent composition determines the form in which phase separation takes place and consequently the morphology of the resulting blend after solvent evaporation. The blend containing 20% CHT presents cocontinuous phases. The sample presents a high elastic modulus even at temperatures above melting of PCL. Blends with higher CHT contents consist of disperse PCL domains in a CHT matrix and the contrary occurs in the blend containing 10% CHT in which disperse CHT domains with a network morphology appear inside the spherulites of PCL. In all the blends, the nucleation effect of CHT accelerates the crystallization of PCL from the melt, although in the blends with high CHT contents a part of the PCL mass included in large domains might not be affected by the presence of CHT. The sample containing 20% CHT has a peculiar behavior with respect to the crystallization of PCL, only a small part of PCL crystallizes in isothermal treatments although this fraction crystallizes faster than in the rest of the blends.

Future design of a new keratoprosthesis. Physical and biological analysis of polymeric substrates for epithelial cell growth.

One of the main issues in the development of new biocolonizable materials is to understand the influence of the synthetic material on the biological response in terms of cellular adhesion, proliferation, and differentiation. In this study, we characterized different polymeric materials (with different hydrophobicity/hydrophilicity ratios and electrical charges) using dynamic-mechanical analysis, equilibrium water content, and surface energy. Cell adhesion, viability, morphology, and proliferation studies were conducted with these materials using a conjunctival epithelial cell line (IOBA-NHC). The biological data regarding physicochemical parameters of the materials were also correlated. When conjunctival epithelial cells were grown on poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymers, P(EA-co-HEA), samples with up to 20% hydrophilic groups on their polymeric chain showed adhesion, viability, and proliferation, although these three factors decreased as the hydrophilic group content increased. The poly(ethyl acrylate-co-methacrylic acid) 90/10 copolymer, P(EA-co-MAAc) 90/10, showed better results than poly(ethyl acrylate-co-hydroxyethyl acrylate) copolymers and were even better than tissue control polystyrene (TCPS). This feature is explained by the presence of electrical charges on the surface of the poly(ethyl acrylate-co-methacrylic acid) 90/10 copolymer. The fact that the ionic groups are configured in domains structured in nanophases as happens in this copolymer improves cell adhesion even further.