We end the year 2009, our fourth year of existence, with three state-of-the-art papers and two technical papers. In the first formal paper, Liebschner describes current efforts in identifying mechano-biological principles that are believed to guide tissue formation based on biomechanical loading. The goal of the research is to discuss a reverse engineering solution in a strategy that starts with the final product, in this case, a human trabecular bone tissue, and determines the required scaffold micro-architecture and features in order to achieve that goal. Computer models of dissected human trabecular bone samples are generated, their micro-mechanical environment analysed and results used as design goals. In the second paper, Pouzada proposes hybrid moulds as a case of integration of alternative materials and rapid prototyping for tooling. This work reviews some of the outcomes of research activity on specific issues of manufacturing and utilisation of these tools. Most of the results refer to moulding blocks produced in epoxy composites that were manufactured using the vacuum casting technique. Some references are also made to rapid tooling techniques based on laser sintering. Biomanufacturing in tissue engineering is the topic of review by Bartolo et al. In recent years, the use of rapid prototyping techniques in the fabrication of tissue engineering scaffolds has generated a lot of interest. This paper presents a state-of-the-art review of all such research activities and attempts to give a crystal ball forecast of the future trends in this growing field.

The purpose of this study was to evaluate hybrid poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/chitosan nanofibrous mats as scaffolds for skin engineering. In vitro studies were carried out to test the potential of the scaffolds for fibroblasts adhesion, viability, and proliferation (L929 cell line). The in vivo performance was also studied in a full-thickness wound healing model. PHBV/chitosan 4:1 w/w exhibited a higher in vitro biocompatibility and a better ability for cell adhesion and growth, compared to PHBV/chitosan 2:3 w/w. The in vivo assay also revealed the better performance of this scaffold, improving the wound healing process in rats.

Highly porous three-dimensional biodegradable scaffolds was obtained from beta-tricalcium phosphate-hydroxyapatite bioceramic (BCP), PCL, and Angiotensin-(1-7). We used the solvent casting and particulate leaching methods (SC/PL). The processed scaffolds were characterized by X-ray microtomography (µ-CT). Biocompatibility tests in vitro were performed during three and seven days using MTT and Alkaline Phosphatase Activity (APA) assays. Both the MTT activity and APA were evaluated using a one-way ANOVA test. The µ-CT results showed that the increase of the PCL:BCP weight ratio leads to structures with lower pore sizes. The pore interconnectivity of the processed scaffolds was evaluated in terms of the fragmentation index (FI). We observed that the obtained composites present poorly connected structures, with close values of FI. However, as the polymer phase is almost transparent to the X-rays, it was not taken into consideration in the µ-CT tests. The MTT activity assay revealed that scaffolds obtained with and without Angiotensin-(1-7) present mild and moderate cytotoxic effects, respectively. The APA assay showed that the rat osteoblasts, when in contact for three days with the PCL composites, presented an APA similar to that observed for the control cells. Nevertheless, for an incubation time of seven days we observed a remarkable decrease in the alkaline phosphatase activity. In conclusion, using the solvent casting and salt leaching method we obtained 3D porous that are composites of PCL, BC and Ang-(1-7), which have suitable shapes for the bone defects, a high porosity and interconnect pores. Furthermore, the viability in vitro showed that the scaffolds have potential for drug delivery system and could be used in future in vivo tests.

Abstract Autologous fibrin matrices derived from the Leukocyte and Platelet Rich Plasma (L-PRP) and Leukocyte and Platelet Rich Fibrin (L-PRF) techniques present great potential to act as a bioactive scaffold in regenerative medicine, contributing to the maintenance of cell viability, proliferation stimulus and differentiation. In contrast, there are few studies that characterize the bioactive potential of these fibrin scaffolds by considering the process of production. The objective of this work was to characterize the intrinsic potential of maintaining cell viability of different fibrin scaffolds containing platelets and leukocytes. In order to achieve that, blood samples from a volunteer were collected and processed to obtain fibrin clots using the suggested techniques. To characterize the potential for in vitro viability, mesenchymal stem cells from human infrapatellar fat were used. The scaffolds were cellularized (1x105 cells/scaffolds) and maintained for 5 and 10 days under culture conditions with Dulbecco's Modified Eagle Medium, without addition of fetal bovine serum, and subsequently subjected to analyses by Fourrier transform infra-red spectroscopy, circular dichroism and fluorescence microscopy. The results demonstrated distinct intrinsic potential viability between the scaffolds, and L-PRP was responsible for promoting higher levels of viability in both periods of analysis. No viable cells were identified in the fibrin matrix used as controls. These results allow us to conclude that both fibrin substrates have presented intrinsic potential for maintaining cell viability, with superior potential exhibited by L-PRP scaffold, and represent promising alternatives for use as bioactive supports in musculoskeletal regenerative medicine.

AbstractScaffolds featuring spatiotemporal control of drug release is highly desirable for bone tissue regeneration. The objective of this study was to construct a scaffold with gradient porosity and drug distribution and evaluate the effect of scaffold structure on drug release kinetics and cell bioactivity. Nano-hydroxyapatite/collagen/phosphatidylserine scaffolds embedded with steroidal saponin loaded collagen microparticles were prepared using a porogen leaching protocol. Morphological characterization showed that the scaffolds consisted of dense layer and loose layer, and pores were interconnective. The microparticles were entrapped at the center of the scaffolds follow a gradient distribution. Release kinetics correlated with the structure. The loose layer showed greater drug release amount as compared to the dense layer. Such differences in release kinetics have distinct effects on cell bioactivity. Cell proliferated much more in loose layer than that in the dense layer. Such spatial and temporal control over drug deposition and delivery within the scaffolds could provide opportunities for tissue regeneration associated with optimum drug doses at wound site, and lessen undesirable drug release and side-effects at uninjured site.

Bone ingrowth requires materials with the existence of open and interconnected pores with diameters larger than 150 µm for proper circulation of nutrients. Such materials must possess enough mechanical strength to avoid failure whilst offering a bioactive surface for bone regeneration. We have developed porous ceramic alumina scaffold with compressive strength that achieves 3.3 MPa by replication method by using the network structure of cellular polymer foam. However, the biocompatibility of ceramics based on Al2O3 requires further improvement so that it could have strong bonding to natural bone tissue. To address this problem of the interface between alumina and bone, we have developed a novel calcium phosphate with Zn2+ (CaP-Zn) coating onto porous alumina ceramic scaffold by impregnating with calcium phosphate/poly(vinyl alcohol) slurry. The tri-dimensional alumina scaffold coated with CaP-Zn was extensively characterized by SEM, EDS and FTIR.

Scaffolds of PDLLA were produced to be implemented in maxilofacial surgeries inducing bone repair and regeneration. To prepare these scaffolds, bioglass (BG58S) was synthesized by sol-gel method, in order to be applied as osteoconductive dispersed particles in PDLLA matrix. Once presenting greater facility on parts fabrication, this polymeric matrix enables complex geometries production besides presenting compatible degradation rate for scaffold absorption and bone regeneration. Scaffolds production was performed by selective laser sintering in order to obtain tailored-made parts. FTIR and XRD analyses were carried out to observe the composition and evaluate the presence of crystalized phases in bioglass, obtaining Wollastonite. SEM was used to observe the BG particle distribution in PDLLA matrix and flexural test was performed to evaluate the composite mechanical properties. Results showed that was possible to obtain pieces using SLS method and with addition of 10%wt BG to polymeric matrix, flexural modulus and strength increased regarding to pure polymer.

Electrospinning has been recognized as an efficient technique for the forming of polymer nanofibers. Silk fibroin (SF) nanofibers were electrospun from SF solution using trifluoroacetic acid solution as a solvent. In the present work, we have systematically evaluated the effects of instrument parameters, including applied voltage, tip-target distance, solution flow rate, solution parameters; such as polymer concentration and solution viscosity on the morphology of electrospun SF fibers. The applied voltage and flow rate was monitored at fixed tip target distance during the electrospinning process and it was correlated with the characteristics of the fibers obtained. The number of deposited fibers also increases with the applied voltage. Also, viscosity, flow rate and applied voltage strongly affect the shape and morphology of the fibers. A particular interest, we demonstrated that by monitoring the applied voltage and flow rate it is possible to control the fibers morphology and bead concentration. Rheological study showed a strong dependence of spinnability and fiber morphology on solution viscosity. Solution concentrations has been found to most strongly affect fiber size, with fiber diameter increasing with increasing solution concentration and the morphology of the deposition on the collector changed from spherical beads to interconnected fibrous networks. FTIR analysis clearly shows that there are no spectral differences between fibers and which suggests that there was no chemical modification developed during the process. Under optimized conditions, homogenous (not interconnected) SF fibers with a mean diameter of 234 nm were prepared.