One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues.

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan-PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4-HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4-PEO2-HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4-HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP-IN) and on the fibers (NP-ON). It was shown that incorporation on the CHI4-HAc2 fibers (NP-ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP-IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4-HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP-ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.

This study reports on the production of chitosan fibers and 3-D fiber meshes for the use as tissue engineering scaffolds. Both structures were produced by means of a wet spinning technique. Maximum strain at break and tensile strength of the developed fibers were found to be 8.5% and 204.9 MPa, respectively. After 14 d of immersion in simulated body fluid (SBF), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and inductively coupled plasma emission (ICP) spectroscopy analyses showed that a bioactive Ca-P layer was formed on the surface of the fibers, meaning that they exhibit a bioactive behavior. The samples showed around 120% max. swelling in physiological conditions. The pore sizes of 3-D chitosan fiber mesh scaffolds were observed to be in the range of 100-500 microm by SEM. The equilibrium-swelling ratio of the developed scaffolds was found to be around 170% (w/w) in NaCl solution at 37 degrees C. Besides that, the limit swelling strain was less than 30%, as obtained by mechanical spectroscopy measurements in the same conditions. The viscoelastic properties of the scaffolds were also evaluated by both creep and dynamic mechanical tests. By means of using short-term MEM extraction test, both types of structures (fibers and scaffolds) were found to be non-cytotoxic to fibroblasts. Furthermore, osteoblasts directly cultured over chitosan fiber mesh scaffolds presented good morphology and no inhibition of cell proliferation could be observed.Osteoblast-like cells proliferating over chitosan based fibers after 7 d of culture.

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on post-implantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structural proteins, homotypic interactions and angiogenic potential of human EC cultured on a scaffold made of a blend of starch and poly(caprolactone). The nano-network allowed cells to span between individual micro-fibers and influenced cell morphology. Furthermore, on nano-fibers as well as on micro-fibers ECs maintained the physiological expression pattern of the structural protein vimentin and PECAM-1 between adjacent cells. In addition, ECs growing on the nano/micro-fiber-combined scaffold were sensitive to pro-inflammatory stimulus. Under pro-angiogenic conditions in vitro, the ECM-like nano-network provided the structural and organizational stability for ECs' migration and organization into capillary-like structures. The architecture of nano/micro-fiber-combined scaffolds elicited and guided the 3D distribution of ECs without compromising the structural requirements for bone regeneration.

Tissue engineering offers a promising new approach to create biological alternatives to repair or restore function of damaged or diseased tissues. To obtain three-dimensional tissue constructs, stem or progenitor cells must be combined with a highly porous three-dimensional scaffold, but many of the structures purposed for tissue engineering cannot meet all the criteria required by an adequate scaffold because of lack of mechanical strength and interconnectivity, as well as poor surface characteristics. Fiber-based structures represent a wide range of morphological and geometric possibilities that can be tailored for each specific tissue-engineering application. The present article overviews the research data on tissue-engineering therapies based on the use of biodegradable fiber architectures as a scaffold.

One limitation associated with the delivery of bioactive agents concerns the short half-life of these molecules when administered intravenously, which results in their loss from the desired site. Incorporation of bioactive agents into depot vehicles provides a means to increase their persistence at the disease site. Major issues are involved in the development of a proper carrier system able to deliver the correct drug, at the desired dose, place and time. In this work, starch-poly-e-caprolactone (SPCL) microparticles were developed for use in drug delivery and tissue engineering (TE) applications. SPCL microparticles were prepared by using an emulsion solvent extraction/evaporation technique, which was demonstrated to be a successful procedure to obtain particles with a spherical shape (particle size between 5 and 900 lm) and exhibiting different surface morphologies. Their chemical structure was confirmed by Fourier transform infrared spectroscopy. To evaluate the potential of the developed microparticles as a drug delivery system, dexamethasone (DEX) was used as model drug. DEX, a well-known component of osteogenic differentiation media, was entrapped into SPCL microparticles at different percentages up to 93%. The encapsulation efficiency was found to be dependent on the polymer concentration and drug-to-polymer ratio. The initial DEX release seems to be governed mainly by diffusion, and it is expected that the remaining DEX will be released when the polymeric matrix starts to degrade. In this work it was demonstrated that SPCL microparticles containing DEX can be successfully prepared and that these microparticular systems seem to be quite promising for controlled release applications, namely as carriers of important differentiation agents in TE.

This study proposes a new route for producing fiber mesh scaffolds from a starch-polycaprolactone (SPCL) blend. It was demonstrated that the scaffolds with 77% porosity could be obtained by a simple wet-spinning technique based on solution/precipitation of a polymeric blend. To enhance the cell attachment and proliferation, Ar plasma treatment was applied to the scaffolds. It was observed that the surface morphology and chemical composition were significantly changed because of the etching and functionalization of the fiber surfaces. XPS analyses showed an increase of the oxygen content of the fiber surfaces after plasma treatment (untreated scaffolds O/C:0.32 and plasma-treated scaffolds O/C:0.41). Both untreated and treated scaffolds were examined using a SaOs-2 human osteoblast-like cell line during 2 weeks of culture. The cell seeded on wet-spun SPCL fiber mesh scaffolds showed high viability and alkaline phosphatase enzyme activity, with those values being even higher for the cells seeded on the plasma-treated scaffolds.

Bone-like apatite coating of polymeric substrates by means of biomimetic process is a possible way to enhance the bone bonding ability of the materials. The created apatite layer is believed to have an ability to provide a favorable environment for osteoblasts or osteoprogenitor cells. The purpose of this study is to obtain bone-like apatite layer onto chitosan fiber mesh tissue engineering scaffolds, by means of using a simple biomimetic coating process and to determine the influence of this coating on osteoblastic cell responses. Chitosan fiber mesh scaffolds produced by a previously described wet spinning methodology were initially wet with a Bioglass((R))-water suspension by means of a spraying methodology and then immersed in a simulated body fluid (SBF) mimicking physiological conditions for one week. The formation of apatite layer was observed morphologically by scanning electron microscopy (SEM). As a result of the use of the novel spraying methodology, a fine coating could also be observed penetrating into the pores, that is clearly within the bulk of the scaffolds. Fourier Transform Infrared spectroscopy (FTIR-ATR), Electron Dispersive Spectroscopy (EDS) and X-ray diffraction (XRD) analysis also confirmed the presence of apatite-like layer. A human osteoblast-like cell line (SaOs-2) was used for the direct cell contact assays. After 2 weeks of culture, samples were observed under the SEM. When compared to the control samples (unmodified chitosan fiber mesh scaffolds) the cell population was found to be higher in the Ca-P biomimetic coated scaffolds, which indicates that the levels of cell proliferation on this kind of scaffolds could be enhanced. Furthermore, it was also observed that the cells seeded in the Ca-P coated scaffolds have a more spread and flat morphology, which reveals an improvement on the cell adhesion patterns, phenomena that are always important in processes such as osteoconduction.