Preparation of bioactive coatings on the surface of bioinert polymers through an innovative auto-catalytic electroless route

The aim of this research was to develop a new methodology to obtain bioactive coatings on bioinert and biodegradable polymers that are not intrinsically bioactive. In this study, three types of materials were used as substrates: (i) high molecular weight polyethylene (HMWPE) and two different types of starch based blends (ii) starch/ethylene vinyl alcohol blends, SEVA-C, and (iii) starch/cellulose acetate blends, SCA. These materials were obtained by injection moulding and by extrusion with blowing agents in order to obtain compact/porous 3D architectures. Three types of baths were developed in order to produce the newly proposed auto-catalytic Ca-P coatings: (i) alkaline, (ii) acid, and (iii) oxidant bath. The obtained results indicated that it was possible to coat the materials surfaces with calcium phosphate (Ca-P) layer with only 60 min of immersion in the different types of auto-catalytic solutions. These innovative auto-catalytic electroless route allows for the production of an adherent bioactive film on the polymeric surfaces. Furthermore, it was possible observe by SEM/EDS the clear bioactive nature of the Ca-P coatings after different immersion periods, in a simulated body fluid (SBF)

Bioactive starch-based scaffolds and human adipose stem cells are a good combination for bone tissue engineering

Silicon is known to have an influence on calcium phosphate deposition and on the differentiation of bone precursor cells. This study explores the effect of the incorporation of silanol (Si–OH) groups into poly- meric scaffolds on the osteogenic differentiation of human adipose stem cells (hASC) cultured under dynamic and static conditions. A blend of corn starch with polycaprolactone (30/70 wt.%, SPCL) was used to produce three-dimensional fibre meshes scaffolds by the wet-spinning technique, and a calcium sili- cate solution was used as a non-solvent to develop an in situ functionalization with Si–OH groups. In vitro assessment, using hASC, of functionalized and non-functionalized scaffolds was evaluated in either a- MEM or osteogenic medium under static and dynamic conditions (provided by a flow perfusion bioreac- tor). The functionalized materials, SPCL–Si, exhibit the capacity to sustain cell proliferation and induce their differentiation into the osteogenic lineage. The formation of mineralization nodules was observed in cells cultured on the SPCL–Si materials. Culturing under dynamic conditions using a flow perfusion bioreactor was shown to enhance the hASC proliferation and differentiation and a better distribution of cells within the material. The present work demonstrates the potential of these functionalized mate- rials for future applications in bone tissue engineering. Additionally, these results highlight the simplicity, economic and reliable production process of those materials.Fundação para a Ciência e a Tecnologia (FCT) - (Grant SFRH/BD/69962/2010, SFRH/BPD/26648/2006, PTDC/CTM/67560/2006European NoE EXPERTISSUES - (NMP3-CT-2004- 50028

Stimulatory effects of inorganic ions on osteogenesis in vitro

Introduction: Several studies demonstrated the effect of silicate ions (Si) on differentiation of bone precursor cells1,2, although its exact role in processes related to bone formation and remodeling is still incompletely understood. The focus of this work is to explore the effect of calcium and silicate ions on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs). This strategy may reduce the need for growth factors required to stimulate bone formation in regenerative approaches, decreasing the associated costs and overcoming stability issues. Materials and Methods In order to define the range of Si concentrations that are not toxic to cells, we performed a preliminary study varying Si concentrations from 0.00357mM to 4mM. The concentration of the Ca ions was selected based on the earlier study by Barradas et. al3. Cell culture media were supplemented by using sodium silicate (Na2SiO3) and/or calcium chloride dehydrate (CaCl2*2H2O) as Si and Ca precursors, respectively. hMSCs derived from bone marrow were seeded at a seeding density of 2.000 cells/cm2 and allowed to adhere overnight. Then, the medium was replaced by the appropriate supplemented medium and cells were cultured for 3, 7, 14 and 18 days. Basic and osteogenic media were used as negative and positive controls. Cell proliferation was evaluated by DNA quantification. hMSCs osteogenic gene expression was evaluated by Q-PCR. Results DNA quantification indicated an increase in cell number during the culture time for all the conditions. Results obtained by Q-PCR revealed a significantly higher expression of osteocalcin (OC) and bone morphogenetic protein-2 (BMP2) in cells cultured in media supplemented by both ions, as compared to media containing either Ca or Si alone. Discussion and Conclusions DNA quantification studies indicated that none of the selected concentrations had a negative influence on cell proliferation. The increase in osteogenic gene expression for cells cultured with both Ca and Si suggested a synergistic effect of the two ions on osteogenic differentiation of hMSCs. We further showed that cells cultured in the medium with the highest concentration of Ca (7.8mM) revealed a higher expression of the selected genes, which is in accordance with the earlier results by Barradas et al3. The obtained results suggest the importance of combining both ions, Ca and Si, for promoting the osteogenic differentiation of hMSCs. References 1. Hoppe A, Biomaterials 32: 2757-2774, 2011. 2. Beck Jr GR, Nanomedicine: Nanotechnology, Biology, and Medicine,1-11, 2011 3. Barradas AMC et al., Biomaterials 3205-3215, 2012. Acknowledgments The author thanks the Portuguese Foundation for Science and Technology (FCT) for the grant (SFRH/BD/69962/2010). Disclosures The authors have nothing to disclose

Incorporation of proteins and enzymes at different stages of the preparation of calcium phosphate coatings on a degradable substrate by a biomimetic methodology

In this work, the possibility of incorporating proteins into calcium phosphate (Ca-P) coatings, prepared on the surface of starch polymeric biomaterials by means of a biomimetic route, was investigated. The morphology, chemical composition and crystallinity of Ca-P coatings was assessed and related to the incorporation of the studied biomolecules. For that, bovine serum albumin (BSA) and aamylase were added in concentrations of 1 mg/ml to simulated body fluid (SBF) solutions, being both added at the nucleation or growth stages of the biomimetic coating process. A biodegradable blend of corn starch/ethylene vinyl alcohol (SEVA-C) was used as substrate and bioactive glass (45S5 BioglassR) was used as the nucleating agent. The obtained Ca-P coatings were characterised by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy using an attenuated reflectance device (FTIR-ATR) and thin-film X-ray diffraction (TF-XRD). Additionally, to evaluate the activity of the incorporated enzyme and the stability of the Ca-P films, coated samples were immersed in an SBF solution for different periods of time. The enzyme activity was measured and the morphology of the coating examined by SEM. The results obtained showed that the presence of protein molecules, at the nucleation or growth stages, lead to the formation of a dense Ca-P film presenting different morphologies that were different of the selected coating conditions. FTIR-ATR analysis detected the presence of carbonate and phosphate groups on the Ca-P layer, indicating the formation of a coating similar to the mineral component of vertebrates bone tissue. When proteins were added, amide I and amide II bands, characteristic groups of protein molecules, were also detected, revealing the efficient incorporation of these biomolecules into the Ca-P coatings. Ca-P coatings, with a-amylase incorporated at the nucleation stage, showed no degradation of the film after incubation in SBF for 28 days. The release of increasing concentration of reducing sugars with degradation time revealed that a-amylase was efficiently incorporated in the coating remaining active throughout the coating preparation. This can be a strategy that will allow, in addition of conferring osteoconductive properties to biodegradable polymers, also simultaneously tailoring their degradation kinetics.Fundação para a Ciência e a Tecnologia (FCT

Silk fibroin-spider silk-like protein biomaterials for preventing microbial infections

Publicado em "Frontiers in Bioengineering and Biotechnology. Conference Abstract: 10th World Biomaterials Congress"Introduction: Microbial contamination of medical devices, such as sutures, are one of the major causes of hospital infections despite improvements in medical healthcare[1]. Thus, it is important to explore new biomaterials with antimicrobial properties in order to overcome microbial colonisation and biofilm formation. Spider silk has been considered an excellent biomaterial because of its toughness, strength and outstanding elasticity[2]. Also, through recombinant DNA technology, we can bioengineer and functionalize spider silk-based materials with antimicrobial peptides (AMP), thus evolving a new type of biomaterials[3]. Herein, the aim of this study was to develop silk-based fibers with antimicrobial properties by combining silk fibroin (SF) with recombinant spider silk proteins functionalized with AMP. Materials and Methods: Silk-based sutures were produced by combining spider silk chimeric proteins SSP (6mer and 6mer-HNP1) with different ratios of SF extracted from Bombyx mori through wet-spinning. The structure and topography of the sutures were characterized by scanning electron microscopy (SEM) and the tensile properties and knot strength of the sutures was assessed using an INSTRON 5540 Universal Machine. The formation of biofilm on the fibers was assessed and hemolytic effects of the materials were evaluated. Results and Discussion: Silk fibers containing spider silk 6mer or 6mer-HNP1 showed improved tensile behaviour when compared to silk fibers without SPP (Figure 1). The results suggest that the combination of spider silk chimeric proteins with SF increased the tensile stress of the fibers, when compared to those without spider silk chimeric proteins. No bacterial biofilm was observed on the fibers containing SSP functionalized with AMP, suggesting that the presence of the 6mer-HNP1 prevented the formation of biofilm. Conclusions: The outcomes suggest that silk-based fibers functionalized with AMP showed better mechanical properties when compared to silk fibers alone. The data also demonstrate the positive effect of the presence of AMP in preventing biofilm formation on the fibers, suggesting that the functionalized silk fibers could be used as a new strategy to produce sutures capable of preventing microbial proliferation, while retaining useful mechanical properties. Further investigation is still need in order to understand the in vivo performance of sutures and their physical changes during wound healing.Portuguese Foundation for Science and Technology under the scope of the project PTDC/BBB­BIO/0827/201

Effects of protein incorporation on calcium phosphate coating

The incorporation of proteins into calcium phosphate (Ca–P) coatings is expected to alter their properties. The aim of this work is, therefore, to study the effect of protein concentration on the formation of Ca–P film. A biodegradable blend of corn starch/ethylene vinyl alcohol (SEVA-C) was used as substrate and bioactive glass (45S5 Bioglass®) was used as a nucleating agent. Bovine serum albumin (BSA) and α-amylase were added, separately, at a concentration of 0.5, 1, and 5 mg/mLto simulated body fluid (SBF) solutions, at the nucleation stage. The incorporation of protein molecules was shown to affect the properties of Ca–P coatings in terms of morphology, composition and crystallinity. Both proteins seem to inhibit in some extent and/or retard the growth of Ca–P nuclei at 0.5 and 5 mg/mL concentrations. FTIR analyses revealed the presence of phosphate and carbonate groups, confiming the formation of a Ca–P layer. The characteristic groups of protein molecules were also detected on the IR spectra, which indicate the efficient incorporation of the proteins into the coatings. When α-amylase was added to the SBF solution the production of reducing sugars was detected, proving the retention of enzyme activity. These results suggest the carrier potential of Ca–P coatings for the sustained delivery of other biologically active proteins and consequently with a strong potential for inducing bone tissue regeneration.This work was partially supported by Portuguese Foundation for Science and Technology (FCT) and was performed within the framework of the project BIOLEARN (POCTI/CTM/38803/2001) through funds from the POCH and/or FEDER Programmes. 1. B. Leonor thanks FCT for providing her a PhD scholarship (SFRH/BD/9031/2002)

Redox activity of melanin from the ink sac of Sepia officinalis by means of colorimetric oxidative assay

The redox properties of natural extract from cuttlefish ink sac (Sepia officinalis) and synthetic melanin used as a biomimetic in melanin structural investigation were determined by comparison of this phenol-based heterogeneous pigment with gallic acid used as a standard in Folinâ Ciocalteu colorimetric assay widely employed for characterisation of oxidative properties of biomaterials. Reactivity of sepia melanin reported here is much higher than previously indicated and this protocol should allow the redox characterisation of all melanins irrespective of their origin and composition.European Union’s Seventh Framework Programme (FP7/2007-2013) [grant agreement number REGPOT-CT2012-316331-POLARIS

Spider silk-bone sialoprotein fusion proteins for bone tissue engineering

The remarkable mechanical characteristics of the spider silk protein major ampullate spidroin protein suggest this polymer as a promising biomaterial to consider for the fabrication of scaffolds for bone regeneration. Herein, a new functionalized spider silk-bone sialoprotein fusion protein was designed, cloned, expressed, purified and the osteogenic activity studied. Bone sialoprotein (BSP) is a multidomain protein with the ability to induce cell attachment and differentiation and the deposition of calcium phosphates (CaP). Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) was used to assess the secondary structure of the fusion protein. In vitro mineralization studies demonstrated that this new fusion protein with BSP retained the ability to induce the deposition of CaP. Studies in vitro indicated that human mesenchymal stem cells had significant improvement towards osteogenic outcomes when cultivated in the presence of the new fusion protein vs. silk alone. The present work demonstrates the potential of this new fusion protein for future applications in bone regenerationPhD grant SFRH/BD/28603/2006; Chimera project, PTDC/EBB-EBI/109093/2008; NIH, P41 EB002520, EB003210 and DE017207.Foundation for Science and Technolog

Biomimetic apatite formation on different polymeric microspheres modified with calcium silicate solutions

Proceedings of the 18th International Symposium on Ceramics in Medicine, The Annual Meeting of the International Society for Ceramics in Medicine (ISCM), Kyoto, Japan, 5-8 December 2005. Published in : Key Enggineering Materials, vol. 309 - 311Bioactive polymeric microspheres can be produced by pre-coating them with a calcium silicate solution and the subsequent soaking in a simulated body fluid (SBF). Such combination should allow for the development of bioactive microspheres for several applications in the medical field including tissue engineering. In this work, three types of polymeric microspheres with different sizes were used: (i) ethylene-vinyl alcohol co-polymer (20-30 'm), (ii) polyamide 12 (10-30 'm) and (iii) polyamide 12 (300 'm). These microspheres were soaked in a calcium silicate solution at 36.5ºC for different periods of time under several conditions. Afterwards, they were dried in air at 100ºC for 24 hrs. Then, the samples were soaked in SBF for 1, 3 and 7 days. Fourier transformed infrared spectroscopy, thin-film X-ray diffraction, and scanning electron microscopy showed that after the calcium silicate treatment and the subsequent soaking in SBF, the microspheres successfully formed a bonelike apatite layer on their surfaces in SBF within 7 days due to the formation of silanol (Si-OH) groups that are quite effective for apatite formation.I. B. Leonor thanks the Portuguese Foundation for Science and Technology (FCT) for providing her a PhD scholarship (SFRH/BD/9031/2002) and the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Designing biomaterials based on biomineralization of bone

In nature, organisms control crystal nucleation and growth using organic interfaces as templates. Scientists, in the last decades, have tried to learn from nature how to design biomimetic biomaterials inspired by the hierarchical complex structure of bone and other natural mineralised tissues or to control the biomineralization process onto biomaterials substrates to promote the osteoconductive properties of implantable devices. The design of synthetic bone analogues, i.e., with a structure and properties similar to bone, would certainly constitute a major breakthrough in bone tissue engineering. Moreover, many strategies have been proposed in the literature to develop bioactive bone-like materials, for instance using bioactive glasses. Fundamental aspects of biomineralization may be also important in order to propose new methodologies to improve calcification onto the surface of biomaterials or to develop bioactive tridimensional templates that could be used in regenerative medicine. In particular, it has been shown that some chemical groups and proteins, as well as the tridimensional matrix in which calcification would occur, play a fundamental role on the nucleation and growth of hydroxyapatite. All these distinct aspects will be reviewed and discussed in this paper.I. B. Leonor thanks the Portuguese Foundation for Science and Technology (FCT) for providing her a post-doctoral scholarship (SFRH/BPD/26648/2006). This work was supported by the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and by the Portuguese Foundation for Science and Technology, FCT, through the projects PTDC/CTM/68804/2006, PTDC/CTM/67560/2006 and PTDC/FIS/68209/2006