Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and demonstrated ability to support bone formation, in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASC), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol - HFIP), pore sizes (250–500μm vs. 500–1000μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes with decellularized trabecular bone (DCB) included as a “gold standard”. The scaffolds were seeded with hASC and cultured for 7 weeks in osteogenic media. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2, and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation.

To facilitate optimal application of appropriate scaffold architectures for clinical trials, there is a need to compare different scaffold modifications under similar experimental conditions. In this study was assessed the effectiveness of poly-e-caprolactone (PCL) scaffolds fabricated by fused deposition modelling (FDM), with varying material modifications, for the purposes of bone tissue engineering. The incorporation of hydroxyapatite (HA) in PCL scaffolds, as well as precalcification through immersion in a simulated body fluid (SBF) to produce a biomimetic apatite coating on the scaffolds, was assessed. A series of in vitro studies spanning 3 weeks as well as in vivo studies utilizing a subcutaneous nude mouse model were carried out. PCL and HA-PCL scaffolds demonstrated increasing tissue growth extending throughout the implants, as well as superior mechanical strength and mineralization, as evidenced by X-ray imaging after 14 weeks in vivo. No significant difference was found between PCL and HA-PCL scaffolds. Precalcification with SBF did not result in increased osteoconductivity and cell proliferation as previously reported. Conversely, tensile forces exerted by tissue sheets bridging adjacent struts of the PCL scaffold caused flaking of the apatite coating that resulted in impaired cell attachment, growth and mineralization. The results suggest that scaffolds fabricated by FDM may have load-bearing applications.

In the present work, a new methodology to produce bioactive coatings on the surface of starch-based biodegradable polymers or other polymeric biomaterials is proposed. A sodium silicate gel is employed as an alternative nucleating agent to the more typical bioactive glasses for inducing the formation of a calcium-phosphate (Ca-P) layer. The method has the advantage of being able to coat efficiently both compact materials and porous 3D architectures aimed at being used on tissue replacement applications and as tissue engineering scaffolds. By means of this treatment, it is possible to observe the formation of an apatite-like layer, only after 6 hours of simulated body fluid immersion. For the porous materials, this layer could also be observed inside the pores, clearly covering the cell walls. Furthermore, an increase of the surface hydrophilicity (higher amount of polar groups in the surface) might contribute to the formation of silanol groups that also act as apatite inductors. After 30 days of SBF immersion, the apatite-like films exhibit a partially amorphous nature and the Ca/P ratios became much closer to the value attributed to hydroxyapatite (1.67). The obtained results are very promising for the development of cancellous bone replacement materials and for pre-calcifying bone tissue engineering scaffolds.

During the last few years Strontium has been shown to have beneficial effects when incorporated at certain doses in bone by stimulating bone formation. It is believed that its presence locally at the interface between an implant and bone will enhance osteointegration and therefore, ensure the longevity of a joint prosthesis. In this study we explore the possibility of incorporating Sr into nano-apatite coatings prepared by a solution-derived process according to an established biomimetic methodology for coating titanium based implants. The way this element is incorporated in the apatite structure and its effects on the stereochemistry and morphology of the resulting apatite layers was investigated, as well as its effect in the mineralization kinetics. By using the present methodology it was possible to incorporate increasing amounts of Sr in the apatite layers. Sr was found to incorporate in the apatite layer through a substitution mechanism by replacing Ca in the apatite lattice. The presence of Sr in solution induced an inhibitory effect on mineralization, leading to a decrease in the thickness of the mineral layers. The obtained Sr-substituted biomimetic coatings presented a bone-like structure similar to the one found in the human bone and therefore, are expected to enhance bone formation and osteointegration.

An important challenge in the biomaterials field is to mimic the structure of functional tissues via cell and extracellular matrix (ECM) alignment and anisotropy. Toward this goal, silk-based scaffolds resembling bone lamellar structure were developed using a freeze-drying technique. The structure could be controlled directly by solute concentration and freezing parameters, resulting in lamellar scaffolds with regular morphology. Different post-treatments were investigated to induce water stability, such as methanol, water annealing and steam sterilization. The resulting structures exhibited significant differences in terms of morphological integrity, structure and mechanical properties. the lamellar thicknesses were around ~2,6 μm for the methanol treated scaffolds and ~5,8 μm for water-annealed. These values are in the range of the reported for human lamellar bone. Human bone marrow-derived mesenchymal stem cells (hMSCs) were seeded on these silk fibroin lamellar scaffolds and grown under osteogenic conditions to assess the effect of the microstructure on cell behaviour. Collagen in the newly deposited ECM, was found aligned along the lamellar architectures. In the case of methanol treated lamellar structures the hMSCs were able to migrate into the interior of the scaffolds producing a multilamellar hybrid construct. The present morphology constitutes a useful pattern onto which hMSCs cells attach and proliferate for guided formation of a highly oriented extracellular matrix.