Bone tissue engineering is a promising strategy to treat the huge number of bone fractures caused by progressive population ageing and diseases i.e., osteoporosis. The bioactive and biomimetic materials design modulating cell behaviour can support healthy bone tissue regeneration. In this frame, type I collagen and hydroxyapatite (HA) have been often combined to produce biomimetic scaffolds. In addition, mesoporous bioactive glasses (MBGs) are known for their ability to promote the deposition of HA nanocrystals and their potential to incorporate and release therapeutic ions. Furthermore, the use of 3D printing technologies enables the effective design of scaffolds reproducing the natural bone architecture.

This study aims to design biomimetic and bioactive 3D printed scaffolds that mimic healthy bone tissue natural features in terms of chemical composition, topography and biochemical cues. Optimised collagenous hybrid systems will be processed by means of extrusion 3D printing technologies to obtain high resolution bone-like structures. Protocols of human co-cultures of osteoblasts and osteoclasts will be developed and used to test the 3D scaffolds.

Type I collagen has been combined with rod-like nano-HA and strontium containing MBGs (micro- and nano-sized particles) in order to obtain hybrid systems resembling the composition of native bone tissue. A comprehensive rheological study has been performed to investigate the potential use of the hybrid systems as biomaterial inks. Mesh-like structures have been obtained by means of extrusion-based technologies exploiting the freeform reversible embedding of suspended hydrogels (FRESH) approach. Different crosslinking methods have been tested to improve final constructs mechanical properties. Both crosslinked and non-crosslinked biomaterials were cultured with human osteoblasts and osteoclasts to assay the hybrid matrix biocompatibility as well as its influence on cell behaviour.

Homogeneous hybrid systems have been successfully developed and characterised, proving their suitability as biomaterial inks for 3D printing technologies. Mesh-like structures have been extruded in a thermo-reversible gelatine slurry, exploiting the sol-gel transition of the systems under physiological conditions. Covalent bonds between collagen molecules have been promoted by genipin treatment, leading to a significant increase in matrix strength and stability. The collagen methacrylation and the further UV-crosslinking are under investigation as alternative promising method to reinforce the 3D structure during the printing process. Biological tests showed the potential of the developed systems especially for genipin treated samples, with a significant adhesion of primary cells.

Collagenous hybrid systems proved their suitability for bioactive 3D printed structures design for bone tissue engineering. The multiple stimuli provided by the scaffold composition and structure will be investigated on both direct and indirect human osteoblasts and osteoclasts co-culture, according to the developed protocols.

Bone healing especially in elderly patients is a complex process with limited therapeutic options. In recent years the use of BMP2 for fracture healing is investigated extensively. However, for many applications superficial amounts of BMP2 were required for efficacy due to the absence of sustained release carriers and severe side effects have reported thereby limiting the use of BMP2. Here we present an alternative method based on the use of a combination of low molecular weight compounds, testosterone and alendronate, with established safety profiles in men. Moreover, in contrast to BMP2 which activates both osteoblasts and osteoclasts, this combination of drugs enhances osteoblast activity but simultaneously inhibits osteoclast activity resulting in a net effect of bone growth.

Human primary osteoblasts were obtained from bone of patients requiring knee prostheses and cultured in the presence of various concentrations testosterone with and without alendronate. Optimal concentrations were selected and used to stimulate 5×8 mm porcine bone biopsies for 4 weeks. Medium was exchanged regularly and ALP activity was determined. At endpoint biopsies were analyzed in a MicroCT (Bruker Skyscan 1076) to analyze bone volume (BV), trabecular thickness (Tb.Th) and tissue volume (TV). Bone strength was measured using Hounsfield (H10KT) test equipment.

The data obtained showed a significant and dose dependent increase in ALP activity of primary osteoblasts (day 7–10) indicating robust activation of osteoblast activity. Optimal and synergistic ALP activation was observed when treating cells with 15–375 nM testosterone in combination with 2 μM alendronate. Significant inhibition (75%) of osteoclast activity was observed by alendronate (2–10 μM) which was further enhanced by high testosterone levels. This concept was further tested in bovine bone biopsies cultured for 4 weeks in the presence of 75 nM testosterone and 2 μM alendronate. MicroCT analysis of the biopsies revealed a ± 40% increase in both bone volume (trabecular and cortical bone) and bone strength. Moreover bone mineral density was increased by 20% indicating increased mineralization of bone tissue.

Treatment of human primary osteoblasts or human or bovine bone explants with a combination of an androgen (testosterone) and a bisphosphonate (alendronate) significantly enhance bone growth and bone mineral density. Moreover, bone strength was increased indicating the formation of high quality bone tissue. These findings are the basis for the development of sustained release materials to be applied locally at the bone fracture site, which would allow for low amounts of the drugs and no systemic exposure. By encapsulating testosterone and alendronate in a biodegradable polymer coating, a sustained release up to 5 weeks can be achieved, and the loaded coating can be applied in combination with collagen membranes to improve bone healing or as a coating onto implants to improve osseo-integration.