Cell-free cartilage engineering approach using hyaluronic acid-polycaprolactone scaffolds: a study in vivo.

Polycaprolactone scaffolds modified with cross-linked hyaluronic acid were prepared in order to establish whether a more hydrophilic and biomimetic microenvironment benefits the progenitor cells arriving from bone marrow in a cell-free tissue-engineering approach. The polycaprolactone and polycaprolactone/hyaluronic acid scaffolds were characterized in terms of morphology and water absorption capacity. The polycaprolactone and polycaprolactone/hyaluronic acid samples were implanted in a chondral defect in rabbits; bleeding of the subchondral bone was provoked to generate a spontaneous healing response. Repair at 1, 4, 12, and 24 weeks was assessed macroscopically using the International Cartilage Repair Society score and the Oswestry Arthroscopy Score and microscopically using immunohistological staining for collagen type I and type II, and for Ki-67. The presence of hyaluronic acid improves scaffold performance, which supports a good repair response without biomaterial pre-seeding.

Fibronectin-matrix sandwich-like microenvironments to manipulate cell fate.

Conventional 2D substrates fail to represent the natural environment of cells surrounded by the 3D extracellular matrix (ECM). We have proposed sandwich-like microenvironments as a versatile tool to study cell behaviour under quasi-3D conditions. This is a system that provides a broad range of dorsal and ventral independent spatio-temporal stimuli. Here, we use this sandwich technology to address the role of dorsal stimuli in cell adhesion, cell proliferation and ECM reorganisation. Under certain conditions, dorsal stimuli within sandwich microenvironments prevent the formation of focal plaques as well as the development of the actin cytoskeleton, whereas α5versusαv integrin expression is increased compared to the corresponding 2D controls. Cell signaling is similarly enhanced after dorsal stimuli (measured by the pFAK/FAK level) for cells sandwiched after 3 h of 2D ventral adhesion, but not when sandwiched immediately after cell seeding (similar levels to the 2D control). Cell proliferation, studied by the 5-bromo-2-deoxyuridine (BrdU) incorporation assay, was significantly reduced within sandwich conditions as compared to 2D substrates. In addition, these results were found to depend on the ability of cells to reorganise the dorsal layer of proteins at the material interface, which could be tuned by adsorbing FN on material surfaces that results in a qualitatively different conformation and distribution of FN. Overall, sandwich-like microenvironments switch cell behaviour (cell adhesion, morphology and proliferation) towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo.

Biomimetic hydroxyapatite coating on pore walls improves osteointegration of poly(L-lactic acid) scaffolds.

Polymer-ceramic composites obtained as the result of a mineralization process hold great promise for the future of tissue engineering. Simulated body fluids (SBFs) are widely used for the mineralization of polymer scaffolds. In this work an exhaustive study with the aim of optimizing the mineralization process on a poly(L-lactic acid) (PLLA) macroporous scaffold has been performed. We observed that when an air plasma treatment is applied to the PLLA scaffold its hydroxyapatite nucleation ability is considerably improved. However, plasma treatment only allows apatite deposition on the surface of the scaffold but not in its interior. When a 5 wt % of synthetic hydroxyapatite (HAp) nanoparticles is mixed with PLLA a more abundant biomimetic hydroxyapatite layer grows inside the scaffold in SBF. The morphology, amount, and composition of the generated biomimetic hydroxyapatite layer on the pores' surface have been analyzed. Large mineralization times are harmful to pure PLLA as it rapidly degrades and its elastic compression modulus significantly decreases. Degradation is retarded in the composite scaffolds because of the faster and extensive biomimetic apatite deposition and the role of HAp to control the pH. Mineralized scaffolds, covered by an apatite layer in SBF, were implanted in osteochondral lesions performed in the medial femoral condyle of healthy sheep. We observed that the presence of biomimetic hydroxyapatite on the pore's surface of the composite scaffold produces a better integration in the subchondral bone, in comparison to bare PLLA scaffolds.

In vitro 3D culture of human chondrocytes using modified ε-caprolactone scaffolds with varying hydrophilicity and porosity.

Two series of 3D scaffolds based on ε-caprolactone were synthesized. The pore size and architecture (spherical interconnected pores) was the same in all the scaffolds. In one of the series of scaffolds, made of pure ε-polycaprolactone, the volume fraction of pores varied between 60% and 85% with the main consequence of varying the interconnectivity between pores since the pore size was kept constant. The other scaffolds were prepared with copolymers made of a ε-caprolactone-based hydrophobous monomer and hydroxyethyl acrylate, as the hydrophilic component. Thus, the hydrophilicity and, presumably, the adhesion properties varied monotonously in the copolymer series while porosity was kept constant. A suspension of human chondrocytes in culture medium was injected in the 3D scaffolds and cultured in static conditions up to 28 days. SEM and immunofluorescence assays allowed characterizing cells and extracellular matrix inside the scaffolds after different culture times. To do that, cross sections of the scaffolds were observed by SEM and confocal microscopy. The quantity of cells inside the scaffolds decreases with a decrease of the volume fraction of pores, due to the lack of interconnectivity between the cavities. The scaffolds up to a 30% of hydrophilicity behave in a similar way than the hydrophobous; a further increase of the hydrophilicity rapidly decreases cell viability. In all the experiments production of collagen type I, type II, and aggrecan was found, and some cells were Ki-67 positive, showing that some cells are adhered to the pore walls and maintain their dedifferentiated phenotype even when cultured in three-dimensional conditions.

Hybrid structure in PCL-HAp scaffold resulting from biomimetic apatite growth.

Polymer-ceramic composites are favourite candidates when aiming to replace bone tissue. We present here scaffolds made of polycaprolactone-hydroxyapatite (PCL-HAp) composites, and investigate in vitro mineralisation of the scaffolds in SBF after or without a nucleation treatment. In vitro bioactivity is enhanced by HAp incorporation as well as by nucleation treatment, as demonstrated by simulated body fluid (SBF) mineralization. Surprisingly, we obtained a hybrid interconnected organic-inorganic structure, as a result of micropore invasion by biomimetic apatite, which results in a mechanical strengthening of the material after two weeks of immersion in SBF92. The presented scaffolds, due to their multiple qualities, are expected to be valuable supports for bone tissue engineering.

Biodegradable polycaprolactone scaffold with controlled porosity obtained by modified particle-leaching technique.

Scaffold with controlled porosity constitute a cornerstone in tissue engineering, as a physical support for cell adhesion and growth. In this work, scaffolds of polycaprolactone were synthesized by a modified particle leaching method in order to control porosity and pore interconnectivity; the aim is to observe their influence on the mechanical properties and, in the future, on cell adhesion and proliferation rates. Low molecular weight PEMA beads with an average size of 200 microm were sintered with various compression rates in order to obtain the templates (negatives of the scaffolds). Then the melt polycaprolactone was injected into the porous template under nitrogen pressure in a custom made device. After cooling and solidifying of the melt polymer, the porogen was removed by selective dissolution in ethanol. The porosity and morphology of the scaffold were studied as well as the mechanical properties. Porosities from 60% to 85% were reached; it was found that pore interconnectivity logically increases with increasing porosity, and that mechanical strength decreases with increasing porosity. Because of their interesting properties and interconnected structure, these scaffolds are expected to find useful applications as a cartilage or bone repair material.

Analysis of twenty-eight recurrent pseudarthroses of the carpal navicular after the Matti-Russe operation.

The authors present a review of 28 cases of failure of consolidation of the carpal navicular after the Matti-Russe operation (multicentric study of 106 cases). The causes of the failures are analysed. Among the non significant factors, we find the delay, the radiographic character of the pseudarthrosis, the donor site of the graft, the time of immobilization and the site of the fracture. The superior polar fractures do not have a bad prognosis in this series. Five causes seem to be in direct relation with the failure of consolidation: the age, the failure of an initial surgery procedure, the presence of radiologic "necrosis", the quality of the technical performance of the Matti-Russe operation, and finally dorsal carpal instability that seems to be a fundamental cause in the failure of bony union. The future of these pseudarthroses is analysed concerning: the functional results (64% of poor results); the evolution towards the pseudarthrosis (30%); the surgical reoperations (12 cases out of 28) with eight repeat Matti-Russe operations that finished in five consolidations.