Bone tissue engineering has emerged as a promising strategy to overcome the limitations of current treatments for bone-related disorders, but the trade-off between mechanical properties and bioactivity remains a concern for many polymeric materials. To address this need, novel polymeric blends of poly-L-lactic acid (PLLA), polycaprolactone (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have been explored. Blend filaments comprising PLLA/PCL/PHBV at a ratio of 90/5/5 wt% have been prepared using twin-screw extrusion. The PLLA/PCL/PHBV blends were enriched with nano-hydroxyapatite (nano-HA) and strontium-substituted nano-HA (Sr-nano-HA) to produce composite filaments. Three-dimensional scaffolds were printed by fused deposition modelling from PLLA/PCL/PHBV blend and composite filaments and evaluated mechanically and biologically for their capacity to support bone formation in vitro. The composite scaffolds had a mean porosity of 40%, mean pores of 800 µm, and an average compressive modulus of 32 MPa. Polymer blend and enriched scaffolds supported cell attachment and proliferation. The alkaline phosphatase activity and calcium production were significantly higher in composite scaffolds compared to the blends. These findings demonstrate that thermoplastic polyesters (PLLA and PCL) can be combined with polymers produced via a bacterial route (PHBV) to produce polymer blends with excellent biocompatibility, providing additional options for polymer blend optimization. The enrichment of the blend with nano-HA and Sr-nano-HA powders enhanced the osteogenic potential in vitro.

Nanohydroxyapatite (nanoHA) is the major mineral component of bone. It is highly biocompatible, osteoconductive, and forms strong bonds with native bone, making it an excellent material for bone regeneration. However, enhanced mechanical properties and biological activity for nanoHA can be achieved through enrichment with strontium ions. Here, nanoHA and nanoHA with a substitution degree of 50 and 100% of calcium with strontium ions (Sr-nanoHA_50 and Sr-nanoHA_100, respectively) were produced via wet chemical precipitation using calcium, strontium, and phosphorous salts as starting materials. The materials were evaluated for their cytotoxicity and osteogenic potential in direct contact with MC3T3-E1 pre-osteoblastic cells. All three nanoHA-based materials were cytocompatible, featured needle-shaped nanocrystals, and had enhanced osteogenic activity in vitro. The Sr-nanoHA_100 indicated a significant increase in the alkaline phosphatase activity at day 14 compared to the control. All three compositions revealed significantly higher calcium and collagen production up to 21 days in culture compared to the control. Gene expression analysis exhibited, for all three nanoHA compositions, a significant upregulation of osteonectin and osteocalcin on day 14 and of osteopontin on day 7 compared to the control. The highest osteocalcin levels were found for both Sr-substituted compounds on day 14. These results demonstrate the great osteoinductive potential of the produced compounds, which can be exploited to treat bone disease.

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 μm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.

The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.