Isabel B. Leonor

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.

Surface controlled biomimetic coating of polycaprolactone nanofiber meshes to be used as bone extracellular matrix analogues

The aim of this work was to develop novel electrospun nanofiber meshes coated with a biomimetic calcium phosphate (BCP) layer that mimics the extracellular microenvironment found in the human bone structure. Poly (ε-caprolactone) (PCL) was selected because of its well-known medical applications, its biodegradability, biocompatibility and its susceptibility to partial hydrolysis by a straightforward alkaline treatment. The deposition of a calcium phosphate layer, similar to the inorganic phase of bone, on PCL nanofiber meshes was achieved by means of a surface modification. This initial surface modification was followed by treatment with solutions containing calcium and phosphate ions. The process was finished by a posterior immersion in a simulated body fluid (SBF) with nearly 1.5 × the inorganic concentration of the human blood plasma ions. After some optimization work, the best conditions were chosen to perform the biological assays. The influence of the bone-like BCP layer on the viability and adhesion, as well as on the proliferation of human osteoblast-like cells, was assessed. It was shown that PCL nanofiber meshes coated with a BCP layer support and enhance the proliferation of osteoblasts for long culture periods. The attractive properties of the coated structures produced in the present work demonstrated that those materials have potential to be used for applications in bone tissue engineering. This is the first time that nanofiber meshes could be coated with a biomimetic bone-like calcium phosphate layer produced in a way that the original mesh architecture can be fully maintained.

In vitro bioactivity of starch thermoplastic/hydroxyapatite composite biomaterials: an in situ study using atomic force microscopy

The in vitro bioactivity of a composite composed by a biodegradable starch-based polymeric matrix and hydroxyapatite fillers was investigated, in situ, as a function of immersion time in a simulated body fluid (SBF) using atomic force microscopy (AFM). The surface roughness of the composite started to increase after the initial 8h because of both the degradation of the polymer matrix and the nucleation of calcium phosphate. After 24h of immersion the surface of the composite was fully covered with calcium phosphate nuclei with diameters around 126 nm. As the immersion time increased, the nuclei increased both in number and size, and coalesced leading to the formation of a dense and uniform calcium phosphate layer on the surface of the composite only after 126 h of SBF immersion. The results of in situ AFM observation agreed with those of standard in vitro bioactivity tests in combination with scanning electron microscopy observations. Thin-film X-ray diffraction demonstrated that the ratio of apatite to the polymer matrix was higher within the surface layer (40 microm deep from the surface) than that in the bulk after the immersion for 7 days. The water-uptake capability of the polymer contributes to the nucleation and growth of the calcium phosphate layer. These results suggest the great potential of the composite for a range of temporary applications in which bone-bonding ability is a desired property.

Antimicrobial functionalized genetically engineered spider silk

Genetically engineered fusion proteins offer potential as multifunctional biomaterials for medical use. Fusion or chimeric proteins can be formed using recombinant DNA technology by combining nucleotide sequences encoding different peptides or proteins that are otherwise not found together in nature. In the present study, three new fusion proteins were designed, cloned and expressed and assessed for function, by combining the consensus sequence of dragline spider silk with three different antimicrobial peptides. The human antimicrobial peptides human neutrophil defensin 2 (HNP-2), human neutrophil defensins 4 (HNP-4) and hepcidin were fused to spider silk through bioengineering. The spider silk domain maintained its self-assembly features, a key aspect of these new polymeric protein biomaterials, allowing the formation of β-sheets to lock in structures via physical interactions without the need for chemical cross-linking. These new functional silk proteins were assessed for antimicrobial activity against Gram - Escherichia coli and Gram + Staphylococcus aureus and microbicidal activity was demonstrated. Dynamic light scattering was used to assess protein aggregation to clarify the antimicrobial patterns observed. Attenuated-total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and circular dichroism (CD) were used to assess the secondary structure of the new recombinant proteins. In vitro cell studies with a human osteosarcoma cell line (SaOs-2) demonstrated the compatibility of these new proteins with mammalian cells.

Growth of a bonelike apatite on chitosan microparticles after a calcium silicate treatment

Bioactive chitosan microparticles can be prepared successfully by treating them with a calcium silicate solution and then subsequently soaking them in simulated body fluid (SBF). Such a combination enables the development of bioactive microparticles that can be used for several applications in the medical field, including injectable biomaterial systems and tissue engineering carrier systems. Chitosan microparticles, 0.6microm in average size, were soaked either for 12h in fresh calcium silicate solution (condition I) or for 1h in calcium silicate solution that had been aged for 24h before use (condition II). Afterwards, they were dried in air at 60 degrees C for 24h. The samples were then soaked in SBF for 1, 3 and 7 days. After the condition I calcium silicate treatment and the subsequent soaking in SBF, the microparticles formed a dense apatite layer after only 7 days of immersion, which is believed to be due to the formation of silanol (Si-OH) groups effective for apatite formation. For condition II, the microparticles successfully formed an apatite layer on their surfaces in SBF within only 1 day of immersion.

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 polymeric 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 silicate 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 α-MEM or osteogenic medium under static and dynamic conditions (provided by a flow perfusion bioreactor). 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 materials for future applications in bone tissue engineering. Additionally, these results highlight the simplicity, economic and reliable production process of those materials.

Novel starch thermoplastic/Bioglass® composites: Mechanical properties, degradation behavior and in-vitro bioactivity

The present research aims to evaluate the possibility of creating new degradable, stiff and highly bioactive composites based on a biodegradable thermoplastic starch-based polymeric blend and a Bioglass® filler. Such combination should allow for the development of bioactive and degradable composites with a great potential for a range of temporary applications. A blend of starch with ethylene–vinyl alcohol copolymer (SEVA-C) was reinforced with a 45S5 Bioglass® powder presenting a granulometric distribution between 38 and 53 μm. Composites with 10 and 40 wt % of 45S5 Bioglass® were compounded by twin-screw extrusion (TSE) and subsequently injection molded under optimized conditions. The mechanical properties of the composites were evaluated by tensile testing, and their bioactivity assessed by immersion in a simulated body fluid (SBF) for different periods of time. The biodegradability of these composites was also monitored after several immersion periods in an isotonic saline solution. The tensile tests results obtained indicated that SEVA-C/Bioglass® composites present a slightly higher stiffness and strength (a modulus of 3.8 GPa and UTS of 38.6 MPa) than previously developed SEVA-C/Hydroxylapatite (HA) composites. The bioactivity of SEVA-C composites becomes relevant for 45S5 amounts of only 10 wt %. This was observed by scanning electron microscopy (SEM) and confirmed for immersion periods up to 30 days by both thin-film X-ray diffraction (TF-XRD) (where HA typical peaks are clearly observed) and induced coupled plasma emission (ICP) spectroscopy used to follow the elemental composition of the SBF as function of time. Additionally, it was observed that the composites are biodegradable being the results correlated with the correspondent materials composition.

An innovative auto-catalytic deposition route to produce calcium-phosphate coatings on polymeric biomaterials

The aim of this research is 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. Two types of baths were originally proposed and studied to produce novel auto-catalytic calcium–phosphate (Ca–P) coatings. Then, the coated surfaces were analyzed by scanning electron microscopy and energy dispersive spectroscopy (SEM/EDS), as produced, and after different immersion periods in SBF. The evolution of Ca and P concentrations was determined by induced-coupled plasma emission (ICP) spectroscopy. The crystalline phases present on the films formed on the different material surfaces, after a certain soaking time, were identified by thin-film X-ray diffraction (TF-XRD). The obtained results indicated that it was possible to coat the materials surfaces with a Ca–P layer with only 60 min of immersion in both types of auto-catalytic solutions. Furthermore, it was possible to observe the clear bioactive nature of the Ca–P coatings after different immersion periods in a simulated body fluid (SBF). The results from TF-XRD confirmed the presence of partially amorphous Ca–P films with clearly noticeable hydroxylapatite peaks. These new methodologies allow for the production of an adherent bioactive film on the polymeric surfaces prior to implantation, which may allow for the development of bone-bonding, bioabsorbable implants and fixation devices. ©2003 Kluwer Academic Publisher

In situ functionalization of wet-spun fibre meshes for bone tissue engineering.

Bone tissue engineering success strongly depends on our ability to develop new materials combining osteoconductive, osteoinductive and osteogenic properties. Recent studies suggest that biomaterials incorporating silanol (SiOH) groups promote and maintain osteogenesis. The purpose of the present research work was to provide evidence that using wet‐spinning technologies and a calcium silicate solution as a coagulation bath, it was possible to develop an in situ functionalization methodology to obtain 3D wet‐spun fibre meshes with SiOH groups, through a simple, economic and reliable process. SPCL (blend of starch with polycaprolactone) fibre meshes were produced by wet‐spinning, using a calcium silicate solution as a non‐solvent and functionalized in situ with SiOH groups. In vitro tests, using goat bone marrow stromal cells (GBMSCs), showed that SPCL–Si scaffolds sustained cell viability and proliferation. Furthermore, high ALP activity and matrix production indicated that SiOH groups improve cellular functionality towards the osteoblastic phenotype. Using this methodology, and assembling several wet‐spun fibre meshes, 3D meshes can be developed, aiming at designing osteoconductive/osteoinductive 3D structures capable of stimulating bone ingrowth in vivo. Copyright

Undifferentiated human adipose-derived stromal/stem cells loaded onto wet-spun starch–polycaprolactone scaffolds enhance bone regeneration: Nude mice calvarial defect in vivo study

The repair of large bony defects remains challenging in the clinical setting. Human adipose-derived stromal/stem cells (hASCs) have been reported to differentiate along different cell lineages, including the osteogenic. The objective of the present study was to assess the bone regeneration potential of undifferentiated hASCs loaded in starch-polycaprolactone (SPCL) scaffolds, in a critical-sized nude mice calvarial defect. Human ASCs were isolated from lipoaspirate of five female donors, cryopreserved, and pooled together. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an SPCL scaffold alone, or SPCL scaffold with human ASCs. Histological analysis and Micro-CT imaging of the retrieved implants were performed. Improved new bone deposition and osseointegration was observed in SPCL loaded with hASC engrafted calvarial defects as compared to control groups that showed little healing. Nondifferentiated human ASCs enhance ossification of nonhealing nude mice calvarial defects, and wet-spun SPCL confirmed its suitability for bone tissue engineering. This study supports the potential translation for ASC use in the treatment of human skeletal defects.