Bioprinting: From Technique to Application in Tissue Engineering and Regenerative Medicine.

Among the different approaches present in regenerative medicine and tissue engineering, the one that has attracted the most interest in recent years is the possibility of printing functional biological tissues. Bioprinting is a technique that has been applied to create cellularized three-dimensional structures that mimic biological tissues and thus allow their replacement. Hydrogels are interesting materials for this type of technique. Hydrogels based on natural polymers are known due to their biocompatible properties, in addition to being attractive biomaterials for cell encapsulation. They provide a threedimensional aqueous environment with biologically relevant chemical and physical signals, mimicking the natural environment of the extracellular matrix (ECM). Bioinks are ink formulations that allow the printing of living cells. The controlled deposition of biomaterials by bioinks needs to maintain cell viability and offer specific biochemical and physical stimuli capable of guiding cell migration, proliferation, and differentiation. In this work, we analyze the theoretical and practical issues of bioprinting, citing currently used methods, their advantages, and limitations. We present some important molecules that have been used to compose bioinks, as well as the cellular responses that have been observed in different tissues. Finally, we indicate future perspectives of the method.

Morphological and biochemical analysis of cells cultured on fibrous poly(ε-caprolactone) scaffolds with different degrees of fiber alignment produced by solution blow spinning.

BACKGROUND: Bioresorbable materials are compounds that decompose in physiological mediums both in vitro and in vivo and are used as an alternative to temporary implants in injured tissues. The aim of this study was to analyze the morphology and cytochemistry of cells grown on fibrous poly(ε-caprolactone) (PCL) scaffolds and to measure cell metabolism parameters by biochemical analysis of the conditioned culture medium from cells grown on the scaffolds. METHODS: Fibrous PCL scaffolds were used under the following conditions: unaligned fibers (NA), fibers aligned at 150 rpm (A150), and fibers aligned at 300 rpm (A300). Vero cells were cultured on these scaffolds for 24 h, 48 h, and 72 h. Samples were analyzed by SEM, MicroCT, cytochemistry, and culture medium biochemistry. RESULTS: The results of the cytochemical analysis showed cells were confluent and well spread on the culture plate, while cells grown on the polymeric scaffold, exhibited an elongated morphology. In the biochemical analyses, no significant differences were observed in the expression of alkaline phosphatase or in the levels of cholesterol or total protein in the culture medium. The different materials do not seem to promote changes in the expression or metabolism of these molecules. Only glucose was markedly reduced in the culture medium of cells grown on either aligned or unaligned scaffolds for 48 h or 72 h. This finding indicates the intense energy requirements of cells grown on these scaffolds. CONCLUSION: PCL fibers showed a great capacity to support cell growth. These data reinforce the interpretation that cells grow satisfactorily on PCL scaffolds.

Tissue Engineering Applied to Skeletal Muscle: Strategies and Perspectives.

Muscle tissue is formed by elongated and contractile cells with specific morphofunctional characteristics. Thus, it is divided into three basic types: smooth muscle tissue, cardiac striated muscle tissue and skeletal striated muscle tissue. The striated skeletal muscle tissue presents high plasticity, regeneration and growth capacity due to the presence of satellite cells, quiescent myoblasts that are activated in case of injury to the tissue and originate new muscle fibers when they differentiate. In more severe deficiencies or injuries there is a loss of their regenerative capacity, thus compromising the body's functionality at different levels. Tissue engineering studies the development of biomaterials capable of stimulating the recovery of cellular activity in injured body tissues, as well as the activity of cells with muscle differentiation potential in injury repair. However, the need for three-dimensional re-assembly in a complex organization makes it difficult to mimic this tissue and fully regenerate it for the sake of precise and effective movements. Thus, this article aims to provide a narrative review of tissue engineering strategies applied to the regeneration of skeletal muscle, in a critical evaluation of research, whether aimed at injury or atrophies such as spinal muscular atrophy.

Poly (ε-caprolactone)/Poly (lactic acid) fibers produced by rotary jet spinning for skin dressing with antimicrobial activity.

The rotary jet spinning technique permits the production of biomaterials that can be used as devices that come into contact with biological systems (including biological fluids) for diagnostic or surgical applications. These materials are composed of synthetic or natural compounds and allow the incorporation of drugs for therapeutic purposes. Two solutions containing 50% poly(lactic acid) (PLA) and 50% poly(ε-caprolactone) (PCL) diluted in three different solvents were prepared for rotary jet spinning (RJS) process. Vancomycin, an antibiotic indicated for the treatment of severe staphylococcal infections in patients with penicillin allergy, was added in the polymer solutions, to obtain drug-loaded fibrous mats. Morphological surface characterization by scanning electron microscopy revealed heterogeneous pores in the microfibers. Vancomycin loading interfered with the morphology of all samples in terms of fiber size, leading to smaller diameter fibers. Attenuated total reflectance/Fourier transform infrared spectroscopy was used for identification of the samples. The vibrational characteristics of PCL/PLA and vancomycin were consistent with expectations. Vero cell culture assays by the extract dilution and direct contact methods revealed the absence of cytotoxicity, except for the sample prepared with 50% of PCL and of a 9/2 (V/V) vancomycin content, with the growth of confluent and evenly spread cells on the fibrous mats surface. Microbiological analysis, performed on Staphylococcus aureus by the halo inhibition test and by the broth dilution method, showed that the antibacterial activity of vancomycin was maintained by the loading process in the polymer fibers. The results showed that rotary jet spinning produces satisfactory amounts of Vancomycin-loaded fibers, as potential web dressing for wound repair.

Osteogenic differentiation of rat bone mesenchymal stem cells cultured on poly (hydroxybutyrate-co-hydroxyvalerate), poly (ε-caprolactone) scaffolds.

Bioresorbable biomaterials can fill bone defects and act as temporary scaffold to recruit MSCs to stimulate their differentiation. Among the different bioresorbable polymers studied, this work focuses on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL). Were prepared blends of PHBV and PCL to obtain PHBV based biomaterials with good tenacity, important for bone tissue repair, associated with biocompatible properties of PCL. This study assesses the viability of Vero cells on scaffolds of PHBV, PCL, and their blends and the osteogenic differentiation of mesenchymal stem cells (MSCs). Materials were characterized in surface morphology, DSC and Impact Strength (IS). Vero cells and MSCs were assessed by MTT assay, cytochemical and SEM analysis. MSC osteogenic differentiation was evaluated through alizarin red staining and ALP activity. We found some roughness onto surface materials. DSC showed that the blends presented two distinct melting peaks, characteristic of immiscible blends. IS test confirmed that PHBV-PCL blends is an alternative for increase the tenacity of PHBV. MTT assay showed cells with high metabolic activities on extract toxicity test, but with low activity in the direct contact test. SEM analysis showed spreading cells with irregular and flattened morphology on different substrates. Cytochemical study revealed that MSCs maintained their morphology, although in smaller number for MSCs. The development of nodules of mineralized organic matrix in MSC cultures was identified by alizarin red staining and osteogenic differentiation was confirmed by the quantification of ALP activity. Thus, our scaffolds did not interfere on viability of Vero cells or the osteogenic differentiation of MSCs.

Development of a chitosan and hyaluronic acid hydrogel with potential for bioprinting utilization: A preliminary study.

Bioprinting is a technique that has been applied in the areas of tissue engineering and regenerative medicine (TERM). Natural polymer-based hydrogels are known for their favorable biocompatible properties, as well as attractive biomaterials for cell encapsulation. These hydrogels provide an aqueous three-dimensional environment with biologically relevant chemical and physical signals, mimicking the natural environment of the extracellular matrix (ECM). Chitosan (CHI) and hyaluronic acid (HA) have been widely researched for biomedical applications. Bioinks are "ink" formulations, usually hydrogels, that allow the printing of living cells. This work proposes the development of a low cost and simple chitosan CHI-AH hydrogel with potential to become a bioink. At physiological temperature, the biomaterials form a hydrogel. The material developed was characterized by the analysis of morphology, cytotoxicity, and cell viability. FTIR showed the characteristic vibrational bands of chitosan and HA. No difference in swelling was observed between the different formulations studied, although SEM showed architectural differences between the hydrogels obtained. Extract cytotoxicity testing showed that the hydrogel is not cytotoxic. The direct toxicity test also revealed the absence of toxicity, but the cells had difficulty migrating into the gel, probably because of its density. These data were confirmed by SEM. Further testing are ongoing to better understand the gel's characteristics to improve the limitations found so far.

Effects of the combination of low-level laser therapy and anionic polymer membranes on bone repair.

In view of the limitations of bone reconstruction surgeries using autologous grafts as a gold standard, tissue engineering is emerging as an alternative, which permits the fabrication and improvement of scaffolds to stimulate osteogenesis and angiogenesis, processes that are essential for bone repair. Polymers are used to mimic the extracellular bone matrix and support cell growth. In addition, bone neoformation can be induced by external factors such as laser irradiation, which stimulates bone metabolism. The objective of this study was to evaluate the regeneration of bone defects using collagen and elastin membranes derived from intestinal serosa and bovine auricular cartilage combined with low-level laser application. Thirty-six Wistar rats were operated to create a 3-mm defect in the distal metaphysis of the left femur and divided into six groups: G1 (control, no treatment); G2 (laser); G3 (elastin graft), G4 (elastin+laser); G5 (collagen graft); G6 (collagen+laser). The animals were sacrificed 6 weeks after surgery and the femurs were removed for analysis of bone repair. Macroscopic and radiological results showed the absence of an infectious process in the surgical area. This was confirmed by histological analysis, which revealed no inflammatory infiltrate. Histomorphometry showed that the formation of new bone started from the margins of the bone defect and its volume was greater in elastin+laser and collagen+laser. We conclude that newly formed bone in the graft area was higher in the groups that received the biomaterials and laser. The collagen and elastin matrices showed biocompatibility.

Comparative study of aligned and nonaligned poly(ε-caprolactone) fibrous scaffolds prepared by solution blow spinning

Fibrous scaffolds have become popular in tissue engineering (TE) due to their morphological resemblance to extracellular matrix components. While electrospinning is the most common technique in the field, solution blow spinning is an emerging technique with great potential. One of its many advantages is that it can produce aligned fibers with a very simple experimental setup. This work aimed to fabricate poly (ε-caprolactone) mats with aligned fibers and compare them to nonaligned ones. For that, samples were produced using three rotational speeds of a cylindrical collector and characterized in terms of fiber alignment and diameter, mechanical properties, wettability, and biological response. Results showed that with a static collector, fibers were randomly deposited and nonaligned. As the speed was increased, the fibers began to align (as proven by image analysis), resulting in a change in mechanical behavior, but no differences in fiber diameter. Cells cultured on aligned samples were more elongated, and a higher alignment degree seemed to favor cellular growth. The results confirmed the potential of this up-and-coming technique to produce aligned fibers for TE. (AU)

Comparative study of aligned and nonaligned poly(ε-caprolactone) fibrous scaffolds prepared by solution blow spinning.

Fibrous scaffolds have become popular in tissue engineering (TE) due to their morphological resemblance to extracellular matrix components. While electrospinning is the most common technique in the field, solution blow spinning is an emerging technique with great potential. One of its many advantages is that it can produce aligned fibers with a very simple experimental setup. This work aimed to fabricate poly(ε-caprolactone) mats with aligned fibers and compare them to nonaligned ones. For that, samples were produced using three rotational speeds of a cylindrical collector and characterized in terms of fiber alignment and diameter, mechanical properties, wettability, and biological response. Results showed that with a static collector, fibers were randomly deposited and nonaligned. As the speed was increased, the fibers began to align (as proven by image analysis), resulting in a change in mechanical behavior, but no differences in fiber diameter. Cells cultured on aligned samples were more elongated, and a higher alignment degree seemed to favor cellular growth. The results confirmed the potential of this up-and-coming technique to produce aligned fibers for TE. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1462-1470, 2019.

Factors and molecules that could impact cell differentiation in the embryo generated by nuclear transfer.

Somatic cell nuclear transfer is a technique to create an embryo using an enucleated oocyte and a donor nucleus. Nucleus of somatic cells must be reprogrammed in order to participate in normal development within an enucleated egg. Reprogramming refers to the erasing and remodeling of cellular epigenetic marks to a lower differentiation state. Somatic nuclei must be reprogrammed by factors in the oocyte cytoplasm to a rather totipotent state since the reconstructed embryo must initiate embryo development from the one cell stage to term. In embryos reconstructed by nuclear transfer, the donor genetic material must respond to the cytoplasmic environment of the cytoplast and recapitulate this normal developmental process. Enucleation is critically important for cloning efficiency because may affect the ultrastructure of the remaining cytoplast, thus resulting in a decline or destruction of its cellular compartments. Nonetheless, the effects of in vitro culturing are yet to be fully understood. In vitro oocyte maturation can affect the abundance of specific transcripts and are likely to deplete the developmental competence. The epigenetic modifications established during cellular differentiation are a major factor determining this low efficiency as they act as epigenetic barriers restricting reprogramming of somatic nuclei. In this review we discuss some factors that could impact cell differentiation in embryo generated by nuclear transfer.

Is the FVB/N mouse strain truly resistant to diet-induced obesity?

C57Bl/6J mice are the gold standard animal model of diet-induced obesity. These animals become obese with higher adiposity, blood fasting glucose, triglycerides, and total cholesterol when fed a high-fat diet (HFD). Conversely, the FVB/N mouse line is thought to be resistant to diet-induced obesity, with low or no weight gain and adiposity in response to a HFD In this study, we investigated whether FVB/N mice are resistant or susceptible to metabolic disorder that is promoted by a HFD Biometric parameters and blood chemistry were analyzed in C57Bl/6J and FVB/N mice that were fed a chow diet or HFD Glucose and insulin sensitivity were assessed by performing the glucose tolerance test and measuring serum insulin/glucose and homeostasis model assessment-insulin resistance. Metabolism-related gene expression was investigated by real-time reverse transcription polymerase chain reaction. Adipocyte morphology and liver steatosis were evaluated using standard histology. FVB/N mice had higher adiposity than C57Bl/6J mice that were fed a chow diet and were glucose intolerant. FVB/N mice that were fed a HFD presented higher insulin resistance and greater liver steatosis. Epididymal white adipose tissue exhibited severe inflammation in FVB/N mice that were fed a HFD The FVB/N mouse strain is suitable for studies of diet-induced obesity, and the apparent lack of a HFD-induced response may reveal several strain-specific events that are triggered by a HFD Further studies of the FVB/N background may shed light on the complex multifactorial symptoms of obesity and metabolic syndrome.

Cellular and morphological aspects of fibrodysplasia ossificans progressiva. Lessons of formation, repair, and bone bioengineering.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disease that causes bone formation within the muscles, tendons, ligaments and connective tissues. There is no cure for this disorder and only treatment of the symptoms is available. The purpose of this study was to review the literature and describe the clinical, cellular and molecular aspects of FOP. The material used for the study was obtained by reviewing scientific articles published in various literature-indexed databases. In view of its rarity and of the lack of insightful information and the unpredictability of its course, FOP is a challenging disorder for professionals who are confronted by it. However, this rare disease raises a great deal of interest because understanding the mechanism of mature bone formation can encourage research lines related to bone regeneration and the prevention of heterotopic ossification.

Higher order aberrations in amblyopic children and their role in refractory amblyopia / Aberrações de alta ordem em crianças com ambliopia e seu papel na ambliopia refratária

Objective: Some studies have hypothesized that an unfavourable higher order aberrometric profile could act as an amblyogenic mechanism and may be responsible for some amblyopic cases that are refractory to conventional treatment or cases of “idiopathic” amblyopia. This study compared the aberrometric profile in amblyopic children to that of children with normal visual development and compared the aberrometric profile in corrected amblyopic eyes and refractory amblyopic eyes with that of healthy eyes. Methods: Cross-sectional study with three groups of children – the CA group (22 eyes of 11 children with unilateral corrected amblyopia), the RA group (24 eyes of 13 children with unilateral refractory amblyopia) and the C group (28 eyes of 14 children with normal visual development). Higher order aberrations were evaluated using an OPD-Scan III (NIDEK). Comparisons of the aberrometric profile were made between these groups as well as between the amblyopic and healthy eyes within the CA and RA groups. Results: Higher order aberrations with greater impact in visual quality were not significantly higher in the CA and RA groups when compared with the C group. Moreover, there were no statistically significant differences in the higher order aberrometric profile between the amblyopic and healthy eyes within the CA and RA groups. Conclusions: Contrary to lower order aberrations (e.g., myopia, hyperopia, primary astigmatism), higher order aberrations do not seem to be involved in the etiopathogenesis of amblyopia. Therefore, these are likely not the cause of most cases of refractory amblyopia. .

Objetivo: Alguns estudos levantaram a hipótese de que um perfil aberrométrico de alta ordem desfavorável poderia ser um fator ambliogênico, responsável por certos casos de ambliopia “idiopática” ou refratária ao tratamento convencional. Este trabalho tem como objetivos: 1) comparar o perfil aberrométrico de crianças amblíopes com o de crianças com desenvolvimento visual normal; 2) comparar a aberrometria de olhos amblíopes tratados com sucesso/curados e olhos amblíopes refratários ao tratamento convencional com a aberrometria de olhos saudáveis. Métodos: Estudo transversal com três grupos de crianças: grupo CA (22 olhos de 11 crianças com ambliopia unilateral curada), grupo RA (24 olhos de 13 crianças com ambliopia unilateral refratária) e grupo C (28 olhos de 14 crianças com desenvolvimento visual normal). Avaliou-se a aberrometria ocular total utilizando o OPD Scan-III (NIDEK). Comparou-se o perfil aberrométrico dos três grupos de estudo bem como dentro dos grupos CA e RA, o olho amblíope com o saudável. Resultados: As aberrações de alta ordem com maior impacto na qualidade visual não foram significativamente superiores nos grupos CA e RA, comparativamente ao grupo C. Por outro lado, não se encontraram diferenças estatisticamente significativas entre o perfil aberrométrico de alta ordem dos olhos amblíopes e dos olhos sãos dentro dos grupos CA e RA. Conclusão: Contrariamente às aberrações de baixa ordem (miopia, hipermetropia, astigmatismo primário), as de alta ordem não parecem relacionar-se com a etiopatogênese da ambliopia. É também pouco provável que estas sejam a causa da maioria dos casos de ambliopia refratária. .

Novel hybrid membrane of chitosan/poly (ε-caprolactone) for tissue engineering.

We investigated the potential use of 3D hybrid membrane: poly (ε-caprolactone) (PCL) mesh using rotary jet spinning with subsequent chitosan (CH) coating. The morphological examinations by scanning electron microscopy (SEM) were proved the efficiency of this technique on obtaining relative homogeneous PCL fiber mats (15,49±4,1µm), with high surface porosity (1,06±0,41µm) and effective CH coating. The feasibility of rotary jet spinning allowed the solvent evaporation during the process; this fact was verified by differential scanning calorimetry (DSC), indeed also had verified changes in thermal properties on the hybrid membrane, since the present of CH. It was investigated the mechanical properties of the hybrid membrane and CH film, the data were that the samples presents good tensile modulus but low strain at the break. In addition, it was verified the biocompatibility properties in vitro using Vero cells. PCL mesh demonstrated cells more spread vastly in the pore surface, with attachments in between fibers indicating the potential for cell adhesion. The films samples (CH and hybrid membrane) resulted in a cells layer on the surfaces with an intense staining (metachromasy), which is the result of cells more active. The cell counting -5 days of culture- and the MTT assay -21 days of culture- demonstrated that the materials tested proved to be different from the positive control and equal to each other and this fact, in our view, this indicates a satisfactory proliferation. Thus, based on the results here, this novel hybrid membrane provides an attractive material for tissue engineering applications.

Bovine osteoblasts cultured on polyanionic collagen scaffolds: an ultrastructural and immunocytochemical study.

Collagen is the most abundant protein in the body and is also the most important component of the extracellular matrix. Collagen has several advantages as a biomaterial such as lack of toxicity, biocompatibility, biodegradability, and easy reabsorption. In this study, we examined bovine osteoblasts cultured on native or anionic collagen scaffolds prepared from bovine pericardium after selective hydrolysis of glutamine and asparagine side chain amides for periods from 24 (BP24) and 48 h (BP48). The cells were cultured in control and mineralization medium at 37 °C in the presence of 5% CO(2). Transmission and scanning electron microscopy, energy dispersive spectroscopy, and an immunocytochemical marker were used for analysis. Cells with an irregular morphology forming a confluent multilayer were observed on matrices kept in control medium. Most of these cells presented a polygonal or elongated flattened morphology. Several spherical deposits of calcium crystal associated with phosphorus were observed on the native and BP48 matrices. Similar results were observed in samples kept in control medium except with lower calcium/phosphorus ratio. Vesicles actively expelled from the cell membrane were also seen (do this vesicles corresponds to calcium/phosphorus deposits). Osteocalcin was clearly visible on matrices kept in mineralization medium and was more expression on the surface of BP48 matrices. The results showed that anionic collagen is able to support osteoblastic differentiation, regardless of the medium used. Finally, the BP48 matrix promoted better osteoblast differentiation than the native matrix.

Osteoconductive capacity of hydroxyapatite implanted into the skull of diabetics.

Diabetes mellitus can cause various diseases, including loss of bone mineral density as a characteristic manifestation of osteoporosis. In this condition, bone is more vulnerable to pathologic fractures that can be treated by implantation of biomaterial grafts. The aim of this study was to evaluate the osteogenic capacity of hydroxyapatite implanted into bone defects in the skull of nonobese diabetic mice. Fifteen nonobese diabetic mice were divided into 3 groups: control (nondiabetic), spontaneously diabetic, and spontaneously diabetic receiving insulin replacement applied subcutaneously into the dorsum. Defects were created experimentally in the skull with a surgical bur and filled with hydroxyapatite granules. The animals were killed 4 weeks after surgery, and samples were obtained for analysis. Quantitative methods were used for measurement of the new bone formation. Data were analyzed by analysis of variance followed by the Tukey test (P < 0.05). Radiographic results showed good radiopacity of the hydroxyapatite; however, radiolucent spots were seen between the hydroxyapatite granules in the diabetic groups, indicating infiltration of connective tissue. Microscopic results showed projections of newly formed bone from the margin of bone defect toward the implant. The quantity of newly formed bone was significantly higher (P < 0.05) than that observed in the diabetic groups. The recipient area of diabetic groups contained a larger amount of connective tissue as demonstrated by radiographic analyses. In conclusion, the osteogenesis guided by the properties of hydroxyapatite may even occur in bone suffering from the effects of diabetes, but the quantity of newly formed bone is lower, and the process is slower.

Poly(ε-caprolactone) and poly(D,L-lactic acid-co-glycolic acid) scaffolds used in bone tissue engineering prepared by melt compression-particulate leaching method.

Porous bioresorbable polymers have been widely used as scaffolds in tissue engineering. Most of the bioresorbable scaffolds are aliphatic polyesters and the methods employed to prepare the porous morphology may vary. This work describes and evaluates the in vitro degradation of porous and dense scaffolds of poly(ε-caprolactone) (PCL) and poly(D,L-lactic acid-co-glycolic acid) (50/50) (PLGA50) prepared by particulate leaching-melt compression process. Biological evaluation was carried out using osteoblast cell cultures. The results showed an autocatalytic effect on the dense samples. Osteoblasts presented intermediate adhesion and the cell morphology on the surface of these materials was dispersed, which indicated a good interaction of the cells with the surface and the material.

Odontologia estética: integração entre Prótese e Periodontia: relato de caso clínico / Esthetic Dentistry: integration between Prosthesis and Periodontics: a clinical case report

O tratamento restaurador estético deve restabelecer a função mastigatória, além de proporcionar um sorriso agradável. Um planejamento multidisciplinar favorece a obtenção de um resultado clínico mais satisfatório. Este trabalho tem por objetivo relatar um caso clínico em que se integraram as especialidades Prótese e Periodontia, com cirurgia de aumento de coroa clínica e coroas totais livres de metal.

Blends of poly(3-hydroxybutyrate) and poly(p-dioxanone): miscibility, thermal stability and biocompatibility.

The miscibility, the thermal degradation and biocompatibility of the blends of two biodegradable and bioreabsorbable polymers, brittle polyhydroxybutyric acid (PHB) and flexible poly (p-dioxanone) (PPD) are reported. The blends were prepared by casting from chloroform solutions and analyzed by differential scanning calorimetry, dynamic mechanical analysis, scanning electron microscopy and thermal gravimetric analysis. The blends are immiscible and present promising morphology of a dense phase and a microporous phase, one or the other being the matrix, depending on the composition. Despite the immiscibility, the thermal stability under an inert atmosphere is improved for both polymers. The results obtained from toxicity tests showed that PHB/PPD blends do not present indirect or direct cytotoxicity as a substrate for cellular growth.

Increased response of Vero cells to PHBV matrices treated by plasma.

The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is known that poly 3-hydroxybutyric acids (PHBs) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materials and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120 h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment decreased the contact angle and increased the surface roughness; it also changed surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. SEM analysis of Vero cells adhered to PHBV treated by plasma showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare an appropriate surface for tissue engineering application.