Ana Civantos

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.

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 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.

Urea assisted hydroxyapatite mineralization on MWCNT/CHI scaffolds

Urea assisted hydroxyapatite (HAp) mineralization was performed on scaffolds composed of a major fraction of multiwall carbon nanotubes (MWCNT, 85 wt.%) and a minor one of chitosan (CHI, 15 wt.%). The MWCNT/CHI scaffolds were synthesized through a cryogenic process (so called ISISA, ice segregation induced self-assembly) that allowed the achievement of macroporous monoliths whose structure resembled a chamber-like architecture in the form of interconnected MWCNT/CHI sheets arranged in parallel layers crossed by pillars. The mineralized architectures were composed of flower like hydroxyapatite (HAp) crystalline clusters of ca. 1 µm, homogeneously distributed throughout the internal surface of the scaffold macrostructure. HAp mineralized MWCNT/CHI scaffolds were characterized by X-ray diffraction (XRD), infrared spectroscopy (FTIR) and scanning and transmission electron microscopy (SEM and TEM, respectively). Calibrated energy dispersion X-ray spectroscopy (EDS) and selected-area electron diffraction (SAED) were also performed in the transmission electron microscope to further HAp characterization. Preliminary in vitro experiments demonstrated the suitability of HAp mineralized MWCNT/CHI scaffolds for bone tissue growth.

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.

Preparation, characterization and in vitro osteoblast growth of waste-derived biomaterials

Renewable raw biocompatible materials can be prepared from beer production waste, that due to their nature contain the main chemical components present in bone (phosphorous, silicon, magnesium and calcium). Their characteristics can be tailored for use as replacement candidates in osteoporotic treatments, coatings for prostheses, bone grafts and odontoestomatologi implants, for example, with greater cost effectiveness than conventional scaffolds and eliminating the use of non-renewable raw materials or toxic substances in their preparation.

Sustainable Materials and Biorefinery Chemicals from Agriwastes

This is an open access chapter distributed under the terms of the Creative Commons Attribution License.-- et al.

Macroporous Calcium Phosphate/Chitosan Composites Prepared via Unidirectional Ice Segregation and Subsequent Freeze-Drying

Calcium phosphate chitosan-based composites have gained much interest in recent years for biomedical purposes. In this paper, three-dimensional calcium phosphate chitosan-based composites with different mineral contents were produced using a green method called ice segregation induced self-assembly (ISISA). In this methodology, ice crystals were used as a template to produce porous structures from an aqueous solution of chitosan (CS) and hydroxyapatite (Hap) also containing acetic acid (pH = 4.5). For better characterization of the nature of the inorganic matter entrapped within the resulting composite, we performed either oxygen plasma or calcination processes to remove the organic matter. The nature of the phosphate salts was studied by XRD and NMR studies. Amorphous calcium phosphate (ACP) was identified as the mineral phase in the composites submitted to oxygen plasma, whereas crystalline Hap was obtained after calcination. SEM microscopy revealed the formation of porous structures (porosity around 80–85%) in the original composites, as well as in the inorganic matrices obtained after calcination, with porous channels of up to 50 µm in diameter in the former case and of up to 20 µm in the latter. The biocompatibility of the composites was assessed using two different cell lines: C2C12GFP premyoblastic cells and MC3T3 preosteoblastic cells.

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.

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films.

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.