Polymeric Gene Carriers Bearing Pendant ß-Cyclodextrin: The Relevance of Glycoside Permethylation on the "In Vitro" Cell Response.

The incorporation of cyclodextrins (CDs) to nonviral cationic polymer vectors is very attractive due to recent studies that report a clear improvement of their cytocompatibility and transfection efficiency. However, a systematic study on the influence of the CD derivatization is still lacking. In this work, the relevance of ß-CD permethylation has been addressed by preparing and evaluating two series of copolymers of the cationic N-ethyl pyrrolidine methacrylamide (EPA) and styrenic units bearing pendant hydroxylated and permethylated ß-CDs (HCDSt and MeCDSt, respectively). For both cell lines, CDs permethylation shows a strong influence on plasmid DNA complexation, "in vitro" cytocompatibility and transfection efficiency of the resulting copolymers over two murine cell lines. While the incorporation of the hydroxylated CD moiety increased the cytotoxicity of the copolymers in comparison with their homopolycationic counterpart, the permethylated copolymers have shown full cytocompatibility as well as superior transfection efficiency than the controls. This behavior has been related to the different chemical nature of both units and tentatively to a different distribution of units along the polymeric chains. Cellular internalization analysis with fluorescent copo-lymers supports this behavior.

Osseointegration improvement by plasma electrolytic oxidation of modified titanium alloys surfaces.

Titanium (Ti) is a material frequently used in orthopedic applications, due to its good mechanical properties and high corrosion resistance. However, formation of a non-adherent fibrous tissue between material and bone drastically could affect the osseointegration process and, therefore, the mechanical stability of the implant. Modifications of topography and configuration of the tissue/material interface is one of the mechanisms to improve that process by manipulating parameters such as morphology and roughness. There are different techniques that can be used to modify the titanium surface; plasma electrolytic oxidation (PEO) is one of those alternatives, which consists of obtaining porous anodic coatings by controlling parameters such as voltage, current, anodizing solution and time of the reaction. From all of the above factors, and based on previous studies that demonstrated that bone cells sense substrates features to grow new tissue, in this work commercially pure Ti (c.p Ti) and Ti6Al4V alloy samples were modified at their surface by PEO in different anodizing solutions composed of H2SO4 and H3PO4 mixtures. Treated surfaces were characterized and used as platforms to grow osteoblasts; subsequently, cell behavior parameters like adhesion, proliferation and differentiation were also studied. Although the results showed no significant differences in proliferation, differentiation and cell biological activity, overall results showed an important influence of topography of the modified surfaces compared with polished untreated surfaces. Finally, this study offers an alternative protocol to modify surfaces of Ti and their alloys in a controlled and reproducible way in which biocompatibility of the material is not compromised and osseointegration would be improved.

In vivo comparison of the effects of rhBMP-2 and rhBMP-4 in osteochondral tissue regeneration.

The aim of this work is to investigate the use of bone morphogenetic proteins (rhBMP-2, rhBMP-4) alone or in combination with cells delivered in a calcium alginate gel for the treatment of osteochondral defects. For this purpose, alginate gels were prepared mixing a 2% sodium alginate solution and a 200 mM calcium chloride solution (1:1). Osteochondral defects were created (4 mm wide, 5 mm deep) in the internal femoral condyle of rabbit knee and gels were directly formed into the defects. 3 months after surgery samples were harvested, gross morphology was documented and histological appearance was evaluated. The performed histological observations revealed subchondral bone regeneration in rhBMP-2 samples and moderate hyaline cartilage regeneration in rhBMP-4 samples. Thus, results indicate that alginate gel may serve as an appropriate delivery vehicle for rhBMP-2, rhBMP-4 and stromal cells. With this carrier material, differential behaviour between the evaluated proteins was observed. rhBMP-2 shows better restoration of subchondral bone in contrast to the superior efficiency of rhBMP-4 for hyaline cartilage repair.

Chitosan film as rhBMP2 carrier: delivery properties for bone tissue application.

Tissue engineering approaches need biomaterials with suitable properties to provide an appropriate environment for cell attachment and growth. The performance of these biomaterials can be greatly enhanced through the incorporation of bioactive agents. For this reason, we developed chitosan films with cell-attachment ability, rhBMP-2 carrier capacity, and good in vivo performance, and we employ them as covering for implantable materials. In this work, we have tried to explain how the rh-BMP2 is delivered to the surroundings from the development chitosan films. Protein diffusion from film, film stability versus in vitro dissolution, and biodegradation were evaluated to study rhBMP-2 delivery. Our results show that chitosan film has sufficiently good features to be used as an rhBMP-2 carrier. A low diffusion rate was observed, which was sufficient to quickly induce an in vitro differentiation stimulus, although heavily activated films retain more than 80-85% of the protein on the film. On the other hand, we estimated that chitosan film dissolution due to initial acidification in the wound environment is no more than 15-20%. We also estimated chitosan film response to lysozyme and concluded that degradation via this process proceeded at a slow kinetic rate. In addition, rhBMP-2 in vitro activity after film processing, as well as in vivo film behavior, were studied. We confirm that rhBMP-2 remains active on the film and after release, both in vitro and in vivo. These results support the conclusion that the developed chitosan film allows sustained release of the rhBMP-2 osteoinductive protein and could be used as an activated coat for implant and surgical prosthesis.

Gellan gum based physical hydrogels incorporating highly valuable endogen molecules and associating BMP-2 as bone formation platforms.

Physical hydrogels have been designed for a double purpose: as growth factor delivery systems and as scaffolds to support cell colonization and formation of new bone. Specifically, the polysaccharide gellan gum and the ubiquitous endogenous molecules chondroitin, albumin and spermidine have been used as exclusive components of these hydrogels. The mild ionotropic gelation technique was used to preserve the bioactivity of the selected growth factor, rhBMP-2. In vitro tests demonstrated the effective delivery of rhBMP-2 in its bioactive form. In vivo experiments performed in the muscle tissue of Wistar rats provided a proof of concept of the ability of the developed platforms to elicit new bone formation. Furthermore, this biological effect was better than that of a commercial formulation currently used for regenerative purposes, confirming the potential of these hydrogels as new and innovative growth factor delivery platforms and scaffolds for regenerative medicine applications.

Biocompatibility assessment of spark plasma-sintered alumina-titanium cermets.

Alumina-titanium materials (cermets) of enhanced mechanical properties have been lately developed. In this work, physical properties such as electrical conductivity and the crystalline phases in the bulk material are evaluated. As these new cermets manufactured by spark plasma sintering may have potential application for hard tissue replacements, their biocompatibility needs to be evaluated. Thus, this research aims to study the cytocompatibility of a novel alumina-titanium (25 vol. % Ti) cermet compared to its pure counterpart, the spark plasma sintered alumina. The influence of the particular surface properties (chemical composition, roughness and wettability) on the pre-osteoblastic cell response is also analyzed. The material electrical resistance revealed that this cermet may be machined to any shape by electroerosion. The investigated specimens had a slightly undulated topography, with a roughness pattern that had similar morphology in all orientations (isotropic roughness) and a sub-micrometric average roughness. Differences in skewness that implied valley-like structures in the cermet and predominance of peaks in alumina were found. The cermet presented a higher surface hydrophilicity than alumina. Any cytotoxicity risk associated with the new materials or with the innovative manufacturing methodology was rejected. Proliferation and early-differentiation stages of osteoblasts were statistically improved on the composite. Thus, our results suggest that this new multifunctional cermet could improve current alumina-based biomedical devices for applications such as hip joint replacements.

Effect on in vitro cell response of the statistical insertion of N-(2-hydroxypropyl) methacrylamide on linear pro-dendronic polyamine's gene carriers.

Statistical copolymers of N-(2-hydroxypropyl) methacrylamide (HPMA) and the dendronic methacrylic monomer 2-(3-(Bis(2-(diethylamino)ethyl)amino)propanamido)ethyl methacrylate (TEDETAMA, derived from N,N,N',N'-tetraethyldiethylenetriamine, TEDETA), were synthesized through radical copolymerization and evaluated in vitro as non-viral gene carriers. Three copolymers with nominal molar percentages of HPMA of 25%, 50% and 75% were prepared and studied comparatively to the positive controls poly-TEDETAMA and hyperbranched polyethyleneimine (PEI, 25kDa). Their ability to complex DNA at different N/P molar ratios, from 1/1 up to 8/1, was determined through agarose gel electrophoresis and Dynamic Light Scattering. The resulting complexes (polyplexes) were characterized and evaluated in vitro as possible non-viral gene carriers for Swiss-3T3 fibroblasts, using luciferase as reporter gene and a calcein cytocompatibility assay. All the copolymers, except the one with highest HPMA proportion (75 molar %) at the lowest N/P ratio, condensed DNA to a particle size between 100 and 300 nm. The copolymers with 25 and 50 molar % of HPMA displayed higher transfection efficiency and cytocompatibility than the positive controls poly-TEDETAMA and PEI. A higher proportion of HPMA (75 molar %) led to copolymers that displayed very low transfection efficiency, despite their full cytocompatibility even at the highest N/P ratio. These results indicate that the statistical combination of TEDETAMA and HPMA and its fine compositional tuning in the copolymers may fulfill the fine balance of transfection efficiency and cytocompatibility in a superior way to the control poly-TEDETAMA and PEI.

Pseudo-double network hydrogels with unique properties as supports for cell manipulation.

Pseudo-double network hydrogels based on vinylpyrrolidone and anionic methacrylic units were prepared, for the first-time, via a simple one step radical polymerization procedure using thermal or photoinitiation. These networks showed improved mechanical properties, in the hydrated state, compared with their single network cousins and were capable of hosting cells to confluence. Rapid cell detachment can be induced through simple mechanical agitation and the cell sheets can be transplanted easily without the need for a cell superstrate. The results reported in this work suggest that these hydrogels could be used as support systems for cell manipulation and are candidates to compete with the conventionally used thermoresponsive cell platforms based on poly-N-isopropylacrylamide (pNIPAm).

Chitosan scaffolds containing calcium phosphate salts and rhBMP-2: in vitro and in vivo testing for bone tissue regeneration.

Numerous strategies that are currently used to regenerate bone depend on employing biocompatible materials exhibiting a scaffold structure. These scaffolds can be manufactured containing particular active compounds, such as hydroxyapatite precursors and/or different growth factors to enhance bone regeneration process. Herein, we have immobilized calcium phosphate salts (CPS) and bone morphogenetic protein 2 (BMP-2)--combined or alone--into chitosan scaffolds using ISISA process. We have analyzed whether the immobilized bone morphogenetic protein preserved its osteoinductive capability after manufacturing process as well as BMP-2 in vitro release kinetic. We have also studied both the in vitro and in vivo biocompatibility of the resulting scaffolds using a rabbit model. Results indicated that rhBMP-2 remained active in the scaffolds after the manufacturing process and that its release kinetic was different depending on the presence of CPS. In vitro and in vivo findings showed that cells grew more in scaffolds with both CPS and rhBMP-2 and that these scaffolds induced more bone formation in rabbit tibia. Thus chitosan scaffolds containing both CPS and rhBMP-2 were more osteoinductive than their counterparts alone indicating that could be useful for bone regeneration purposes, such as some applications in dentistry.

In vivo ectopic implantation model to assess human mesenchymal progenitor cell potential.

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies.

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation.

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.

Chitosan scaffolds for osteochondral tissue regeneration.

A variety of biomaterials have been introduced as potential substrates for cartilage repair. One such candidate is chitosan, which shares some characteristics with glycosaminoglycan and hyaluronic acid present in articular cartilage. Depending on chitosan source and preparation procedure, variations into its properties can be attained. Thus, the aim of this article is to study and select the most adequate chitosan properties for in vivo osteochondral tissue regeneration. In this work, chitosan molecular weight, deacetylation degree, and calcium content are tested as material variable properties. According to these properties, porous scaffolds were prepared, implanted in rabbit knee osteochondral defects, and evaluated 3 months after surgery. Results show in vitro a considerable influence of the material molecular weight on the scaffold structure. In vivo, different tissue responses were observed depending on the implanted chitosan properties. Some samples showed no material degradation, multiple adverse tissue responses, and no bone/cartilage tissue formation. Other samples showed no adverse responses and bone and cartilage tissue regeneration. The chitosan with intact mineral content (17.9 wt %), lowest molecular weight (11.49 KDa), and lowest deacetylation degree (83%) shows a well structured subchondral bone and noticeable cartilaginous tissue regeneration, being it the best one of those tested for osteochondral defect regeneration.

Gene expression profile on chitosan/rhBMP-2 films: A novel osteoinductive coating for implantable materials.

This study focusses on the gene expression profile related to a new rhBMP-2 carrier material, chitosan film. This film could be suitable for use as an osteoinductive coating of commercially available titanium implants. The developed material was characterized, biocompatibility was tested and the cellular response was extensively characterized by transcriptional expression studies. Finally, in vivo studies were carried out to confirm the osteoinductivity of the developed coating. Results show good material properties for cell adhesion and proliferation. Presented data show cellular differentiation to the osteoblastic phenotype due to rhBMP-2, with a 90% common transcriptional response between the control rhBMP-2 treatment and the developed chitosan/rhBMP-2 film. The growing surface also had an influence on the observed cellular response and was quantified as 7% of the total. These results indicate that both the growth factor and the material induce a cell response, but this is mainly driven by the osteoinductor factor. In vivo, new bone formation and early vascularization was observed around chitosan/rhBMP-2 coated titanium pieces implanted in mouse muscle. In contrast, control implants did not induce this reaction. This work, therefore, shows both in vitro and in vivo that chitosan/rhBMP-2 film is a promising osteoinductive coating for titanium implantable materials.

Improvement of porous beta-TCP scaffolds with rhBMP-2 chitosan carrier film for bone tissue application.

Ceramic materials are osteoconductive matrices extensively used in bone tissue engineering approaches. The performance of these types of biomaterials can be greatly enhanced by the incorporation of bioactive agents and materials. It is previously reported that chitosan is a biocompatible, biodegradable material that enhances bone formation. In the other hand, bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive factor. In this work we coated porous beta-tricalcium phosphate (beta-TCP) scaffolds with recombinant human BMP-2 (rhBMP-2) carrier chitosan films and studied how they could modify the ceramic physicochemical properties, cellular response, and in vivo bone generation. Initial beta-TCP disks with an average diameter of 5.78 mm, 2.9 mm thickness, and 53% porosity were coated with a chitosan film. These coating properties were studied by X-ray diffraction, Fourier transform-infrared analysis, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray analysis (EDX). Treatment modified the scaffold porous distribution and increased the average hardness. The biocompatibility did not seem to be altered. In addition, adhered C2C12 cells expressed alkaline phosphatase activity, related to cell differentiation toward osteogenic lineage, due to the incorporation of rhBMP-2. On the other hand, in vivo observations showed new bone formation 3 weeks after surgery, a much shorter time than control beta-TCP ceramics. These results suggest that developed coating improved porous beta-TCP scaffold for bone tissue applications and added osteoinductive properties.

Cryoprotectant permeation through human articular cartilage.

OBJECTIVE: The cryopreservation of intact articular cartilage is constrained by minimal chondrocyte survival. It was the aim of the present study to gain an insight into the permeation kinetics of cryoprotectants through cartilage. This knowledge is essential for achieving adequate tissue permeation prior to cooling. DESIGN: The diffusion coefficients and penetration rates through human articular cartilage of dimethyl sulfoxide (Me(2)SO) and glycerol at different temperatures (4 degrees C, 17 degrees C, 27 degrees C and 37 degrees C) and at two concentrations [10% (v/v) and absolute state] were measured using diffusion nuclear magnetic imaging. Deuterated water (D(2)O) was used as a control substance. RESULTS: Glycerol penetrated faster than Me(2)SO at all temperatures and at rates that were comparable to those for D(2)O. The penetration rate of each agent increased with increasing temperature. The diffusion coefficients for glycerol and Me(2)SO increased with increasing temperature and decreased at the higher concentration, but the differences between each agent were not significant. CONCLUSIONS: The classical cryopreservation protocols expose cartilage samples to Me(2)SO at a too low temperature and/or for an insufficient time period for optimal cell survival. When considering the penetration rate, glycerol appears to be a more efficient cryoprotective agent than Me(2)SO. The present study demonstrates the power of nuclear magnetic resonance technology to elucidate key physiological factors in cryobiology.