THERMAL BEHAVIOR OF IN VITRO MINERALIZED ANIONIC COLLAGEN MATRICES

In the present study porcine skin and bovine pericardium were used as a source of type I collagen. Both were submitted to an alkaline treatment and mineralized by the alternate soaking method. Thermal stability and extent of mineralization have been investigated using DSC and TG. After alkaline hydrolysis there is a decrease in thermal stability but mineralization stabilizes collagen structure. Thermogravimetric data have shown that the amount of hydroxyapatite present in bovine pericardium matrix (45%) was greater than on porcine skin matrix (20%). Presence of hydroxyapatite was confirmed by EDX.

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

Estudo da descelularização tecidual na produção de arcabouços biológicos para enxertos

The regeneration of bone defects with loss of substance remains as a therapeutic challenge in the medical field. There are basically four types of grafts: autologous, allogenic, xenogenic and isogenic. It is a consensus that autologous bone is the most suitable material for this purpose, but there are limitations to its use, especially the insufficient amount in the donor. Surveys show that the components of the extracellular matrix (ECM) are generally conserved between different species and are well tolerated even in xenogenic recipient. Thus, several studies have been conducted in the search for a replacement for autogenous bone scaffold using the technique of decellularization. To obtain these scaffolds, tissue must undergo a process of cell removal that causes minimal adverse effects on the composition, biological activity and mechanical integrity of the remaining extracellular matrix. There is not, however, a conformity among researchers about the best protocol for decellularization, since each of these treatments interfere differently in biochemical composition, ultrastructure and mechanical properties of the extracellular matrix, affecting the type of immune response to the material. Further down the arsenal of research involving decellularization bone tissue represents another obstacle to the arrival of a consensus protocol. The present study aimed to evaluate the influence of decellularization methods in the production of biological scaffolds from skeletal organs of mice, for their use for grafting. This was a laboratory study, sequenced in two distinct stages. In the first phase 12 mice hemi-calvariae were evaluated, divided into three groups (n = 4) and submitted to three different decellularization protocols (SDS [group I], trypsin [Group II], Triton X-100 [Group III]). We tried to identify the one that promotes most efficient cell removal, simultaneously to the best structural preservation of the bone extracellular matrix. Therefore, we performed quantitative analysis of the number of remaining cells and descriptive analysis of the scaffolds, made possible by microscopy. In the second stage, a study was conducted to evaluate the in vitro adhesion of mice bone marrow mesenchymal cells, cultured on these scaffolds, previously decellularized. Through manual counting of cells on scaffolds there was a complete cell removal in Group II, Group I showed a practically complete cell removal, and Group III displayed cell remains. The findings allowed us to observe a significant difference only between Groups II and III (p = 0.042). Better maintenance of the collagen structure was obtained with Triton X-100, whereas the decellularization with Trypsin was responsible for the major structural changes in the scaffolds. After culture, the adhesion of mesenchymal cells was only observed in specimens deccelularized with Trypsin. Due to the potential for total removal of cells and the ability to allow adherence of these, the protocol based on the use of Trypsin (Group II) was considered the most suitable for use in future experiments involving bone grafting decellularized scaffolds

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prototyping for Surgical and Prosthetic Treatment

Techniques of rapid prototyping were introduced in the 1980s in the field of engineering for the fabrication of a solid model based on a computed file. After its introduction in the biomedical field, several applications were raised for the fabrication of models to ease surgical planning and simulation in implantology, neurosurgery, and orthopedics, as well as for the fabrication of maxillofacial prostheses. Hence, the literature has described the evolution of rapid prototyping technique in health care, which allowed easier technique, improved surgical results, and fabrication of maxillofacial prostheses. Accordingly, a literature review on MEDLINE ( PubMed) database was conducted using the keywords rapid prototyping, surgical planning, and maxillofacial prostheses and based on articles published from 1981 to 2010. After reading the titles and abstracts of the articles, 50 studies were selected owing to their correlations with the aim of the current study. Several studies show that the prototypes have been used in different dental-medical areas such as maxillofacial and craniofacial surgery; implantology; neurosurgery; orthopedics; scaffolds of ceramic, polymeric, and metallic materials; and fabrication of personalized maxillofacial prostheses. Therefore, prototyping has been an indispensable tool in several studies and helpful for surgical planning and fabrication of prostheses and implants.

Gel de plaquetas: arcabouço 3D para cultura celular

INTRODUÇÃO: O reparo tissular é o objetivo final da cirurgia. A cultura celular requer arcabouço mecânico que dê suporte ao crescimento celular e difusão dos nutrientes. O uso do plasma rico em plaquetas (PRP) como um arcabouço 3D possui diversas vantagens: é material biológico, de fácil absorção pós-transplante, rico em fatores de crescimento, em especial PDGF- ββ e TGF-β que estimula síntese de matriz extracelular na cartilagem. OBJETIVO: Desenvolver arcabouço 3D à base de PRP. MATERIAIS E MÉTODOS: Duas formas foram idealizadas: Sphere e Carpet. Condições estéreis foram utilizadas. O gel de plaquetas permaneceu em cultura celular, observado diariamente em microscópio invertido. RESULTADOS: Ambos arcabouços obtiveram sucesso, com aspectos positivos e negativos. DISCUSSÃO: A forma Sphere não aderiu ao plástico. Observou-se retração do gel e investigação ao microscópio dificultada devido às áreas opacas no campo visual. A forma Carpet não aderiu ao plástico e apresentou-se translúcida. O tempo de estudo foi de 20 dias. CONCLUSÕES: A produção de um arcabouço 3D PRP foi um sucesso, e trata-se de uma alternativa que necessita ser mais utilizado e investigado para que se consolide em uma rota eficiente e confiável na tecnologia de engenharia tissular, particularmente em cultura de tecido cartilaginoso.

Evolutionary dynamics of rRNA gene clusters in cichlid fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Porous titanium for biomedical applications: An experimental study on rabbits

Objective: The aim of this study was to carry out an in vivo assessment of bone ingrowth in two different types of porous titanium -the first being completely porous, and the second with a porous surface and dense nucleus, manufactured by powder metallurgy- and to evaluate their mechanical properties. Study design: Ten scaffolds from each group were submitted to metallographic analysis and compression tests. Next, two scaffolds of each type were inserted into 14 rabbits, which were sacrificed 8 weeks after surgery. The samples were submitted for histological examination. Results: Metallographic analysis revealed interconnected pores, and the average interconnected pore diameter was about 360 mm, with 36% total porosity. The totally porous titanium samples and the titanium samples with porous surface and dense nucleus showed an average compressive strength of 16.19 MPa and 69.27 MPa, respectively. After 8 weeks, the animals showed bone ingrowth, even into the most internal pores. Conclusions: The pore morphology was effective in permitting bone ingrowth in both groups. Titanium scaffolds with a porous surface and dense nucleus showed the best mechanical properties and most adequate interface.

Polímeros biorreabsorvíveis como substrato para cultura de células e engenharia tecidual

Biomateriais poliméricos são desenvolvidos para uso como substitutos de tecidos danificados e/ou estimular sua regeneração. Uma classe de biomateriais poliméricos são os biorreabsorvíveis, compostos que se decompõem tanto in vitro quanto in vivo. São empregados em tecidos que necessitam de um suporte temporário para sua recomposição tecidual. Dentre os vários polímeros biorreabsorvíveis, destacam-se os alfa-hidróxi ácidos, entre eles, diferentes composições do poli(ácido lático) (PLA), como o poli(L-ácido lático) (PLLA), poli(D-ácido lático) (PDLA), poli(DL-ácido lático) (PDLLA), além do poli(ácido glicólico) (PGA) e da policaprolactona (PCL). Estes polímeros são considerados biorreabsorvíveis por apresentarem boa biocompatibilidade e os produtos de sua decomposição serem eliminados do corpo por vias metabólicas. Diversas linhas de pesquisa mostram que os diferentes substratos à base de PLA estudados não apresentam toxicidade, uma vez que as células são capazes de crescer e proliferar sobre eles. Além disso, diversos tipos de células cultivadas sobre diferentes formas de PLA são capazes de se diferenciarem sobre os diferentes polímeros e passar a produzir componentes de matriz extracelular. Neste trabalho, é revisada a utilização de substratos à base de alfa-hidróxi ácidos, com destaque para diferentes formas de PLA, utilizados como substratos para cultura de células, bem como suas aplicações.

Conditional reasoning: A key to assessing computer-based knowledge-building communication processes

This article describes a methodological approach to conditional reasoning in online asynchronous learning environments such as Virtual-U VGroups, developed by SFU, BC, Canada, consistent with the notion of meaning implication: If part of a meaning C is embedded in B and a part of a meaning B is embedded in A, then A implies C in terms of meaning [Piaget 91]. A new transcript analysis technique was developed to assess the flows of conditional meaning implications and to identify the occurrence of hypotheses and connections among them in two human science graduate mixed-mode online courses offered in the summer/spring session of 1997 by SFU. Flows of conditional meaning implications were confronted with Virtual-U VGroups threads and results of the two courses were compared. Findings suggest that Virtual-U VGroups is a knowledge-building environment although the tree-like Virtual-U VGroups threads should be transformed into neuronal-like threads. Findings also suggest that formulating hypotheses together triggers a collaboratively problem-solving process that scaffolds knowledge-building in asynchronous learning environments: A pedagogical technique and an built-in tool for formulating hypotheses together are proposed. © Springer Pub. Co.

A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells

Objectives: The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation. Methods: DPSC cells were grown in 0.05-0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1. Results: DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation. Significance: Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics. © 2012 Academy of Dental Materials.

Bionanocomposites from electrospun PVA/pineapple nanofibers/Stryphnodendron adstringens bark extract for medical applications

Tissue engineering has been defined as an interdisciplinary field that applies the principles of engineering and life sciences for the development of biological substitutes to restore, maintain or improve tissue function. This area is always looking for new classes of degradable biopolymers that are biocompatible and whose activities are controllable and specific, more likely to be used as cell scaffolds, or in vitro tissue reconstruction. In this paper, we developed a novel bionanocomposite with homogeneous porous distribution and prospective natural antimicrobial properties by electrospinning technique using Stryphodedron barbatimao extract (Barbatimão). SEM images showed equally distribution of nanofibres. DSC and TGA showed higher thermal properties and change crystallinity of the developed bionanocomposite mainly because these structural modification. © 2012 Elsevier B.V.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: An improved approach in cartilage tissue engineering

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n=5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1×105) were than encapsulated inside 60μl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI. © 2013 Informa UK Ltd.

Tissue-engineered blood vessel substitute by reconstruction of endothelium using mesenchymal stem cells induced by platelet growth factors

Background: Cardiovascular diseases remain leaders as the major causes of mortality in Western society. Restoration of the circulation through construction of bypass surgical treatment is regarded as the gold standard treatment of peripheral vascular diseases, and grafts are necessary for this purpose. The great saphenous vein is often not available and synthetic grafts have their limitations. Therefore, new techniques to produce alternative grafts have been developed and, in this sense, tissue engineering is a promising alternative to provide biocompatible grafts. This study objective was to reconstruct the endothelium layer of decellularized vein scaffolds, using mesenchymal stem cells (MSCs) and growth factors obtained from platelets. Methods: Fifteen nonpregnant female adult rabbits were used for all experiments. Adipose tissue and vena cava were obtained and subjected to MSCs isolation and tissue decellularization, respectively. MSCs were subjected to differentiation using endothelial inductor growth factor (EIGF) obtained from human platelet lysates. Immunofluorescence, histological and immunohistochemical analyses were employed for the final characterization of the obtained blood vessel substitute. Results: The scaffolds were successfully decellularized with sodium dodecyl sulfate. MSCs actively adhered at the scaffolds, and through stimulation with EIGF were differentiated into functional endothelial cells, secreting significantly higher quantities of von Willebrand factor (0.85 μg/mL; P < .05) than cells cultivated under the same conditions, without EIGF (0.085 μg/mL). Cells with evident morphologic characteristics of endothelium were seen at the lumen of the scaffolds. These cells also stained positive for fascin protein, which is highly expressed by differentiated endothelial cells. Conclusions: Taken together, the use of decellularized bioscaffold and subcutaneous MSCs seems to be a potential approach to obtain bioengineered blood vessels, in the presence of EIGF supplementation. © 2013 Society for Vascular Surgery.

Biocompatibility of a porous alumina ceramic scaffold coated with hydroxyapatite and bioglass

This study aimed to evaluate the osteointegration and genotoxic potential of a bioactive scaffold, composed of alumina and coated with hydroxyapatite and bioglass, after their implantation in tibias of rats. For this purpose, Wistar rats underwent surgery to induce a tibial bone defect, which was filled with the bioactive scaffolds. Histology analysis (descriptive and morphometry) of the bone tissue and the single-cell gel assay (comet) in multiple organs (blood, liver, and kidney) were used to reach this aim after a period of 30, 60, 90, and 180 days of material implantation. The main findings showed that the incorporation of hydroxyapatite and bioglass in the alumina scaffolds produced a suitable environment for bone ingrowth in the tibial defects and did not demonstrate any genotoxicity in the organs evaluated in all experimental periods. These results clearly indicate that the bioactive scaffolds used in this study present osteogenic potential and still exhibit local and systemic biocompatibility. These findings are promising once they convey important information about the behavior of this novel biomaterial in biological system and highlight its possible clinical application. © 2013 Wiley Periodicals, Inc.