The treatment of critical-sized bone defects still remains today a challenge, especially when the surrounding soft, vascularized and innervated tissues have been damaged - a lack of revascularization within the injured site leading to physiological disorders, from delayed healing to osteonecrosis. The axial insertion of a vascular bundle (e.g. arterio-venous loop, AVL) within a synthetic bone filler to initiate and promote its revascularization has been foreseen as a promising alternative to the current strategies (e.g., vascularized free flaps) for the regeneration of large bone defects. In a previous work, we showed that the insertion of a vein in a 3D-printed monetite scaffold induced its higher revascularization than AVL, thus a possible simplification of the surgical procedures (no microsurgery required). Going further, we investigate in this study whether or not the presence of a vein could stimulate the formation of mineralized tissue insides a synthetic scaffold filled with bone marrow and implanted in ectopic site.

Monetite scaffolds were produced by additive manufacturing according to a reactive 3D-printing technique co-developed by the authors then thoroughly characterized. Animal study was performed on 14 male Wistar rats. After anesthesia and analgesia, a skin medial incision in rat thigh allowed the site on implantation to be exposed. Bone marrow was collected on the opposite femur through a minimally invasive procedure and the implant was soaked with it. For the control group (N=7), the implant was inserted in the incision and the wound was closed whereas the femoral bundle was dissected and the vein inserted in the implant for the experimental group (N=7). After 8 weeks animals were sacrificed, the implant collected and fixed in a 4% paraformaldehyde solution. Explants were characterized by µCT then embedded in poly-methyl methacrylate prior SEM, histology and immunohistochemistry. Images were analyzed with CT-Analyzer (Bruker) and ImageJ (NIH) and statistical analyses were carried out using SPSS (IBM).

Implants were successfully 3D-printed with a +150 µm deviation from the initial CAD. As expected, implants were composed of 63%wt monetite and 37%wt unreacted TCP, with a total porosity of 44%. Data suggested that scaffold biodegradation was significantly higher when perfused by a vein. Moreover, the latter allowed for the development of a dense vascular network within the implant, which is far more advanced than for the control group. Finally, although mineralized tissues were observed both inside and outside the implant for both groups, bone formation appeared to be much more important in the experimental one.

The ectopic formation of a new mineralized tissue within a monetite implant soaked with bone marrow seems to be highly stimulated by the simple presence of a vein alone. Although AVL have been studied extensively, little is known about the couple angiogenesis/osteogenesis which appears to be a key factor for the regeneration of critical-sized bone defects. Even less is known about the mechanisms that lead to the formation of a new bone tissue, induced by the presence of a vein only. With this in mind, this study could be considered as a proof of concept for further investigations.

Bone metastases are the most common cause of cancer-related pain and often lead to other complications such as pathological fractures and spinal cord compression. Bisphosphonates (BP) are a class of potent anti-resorptive agents commonly prescribed to retard osteoporosis progression. Interestingly, BP may have indirect anti-tumour properties through negative effects on macrophages, osteoclasts, endothelial cells and their ability to suppress matrix metalloproteinase (MMP) activity. Currently, the use of bisphosphonates for cancer therapy is generally restricted to high dose systemic delivery. The purpose of this study was to investigate the effects of direct local delivery of Zoledronate at the metastatic site in a mouse model of breast cancer metastasis to bone.

Seven days following intra-tibial inoculation with MDA-MB-231 (N = 1× 105) breast cancer cells in athymic mice, the experimental group (N = 11) was treated by direct infusion of 2µg of Zoledronate into the tibial lesion (three times/week for three weeks) and compared to vehicle-treated mice (N = 5). The formation of bone metastases and growth of the lesions were followed up by weekly bioluminescence imaging. In a subsequent experiment, a comparison of the effects of local versus systemic delivery of Zoledronate on the formation of osteolytic bone metastases was carried in athymic mice (N = 15). Seven days following intra-tibial inoculation with MDA-MB-231 breast cancer cells, the systemic group (N = 5) was treated with Zoledronate (0.025mg/kg) once per week for four weeks and compared to systemic delivery of vehicle (N = 4). Following treatment, the mice were sacrificed, and micro-CT images of the right tibia were obtained. Bone volume to tissue volume ratio (BV/TV%) was determined using µ-CT biomarkers.

The first experiment showed a statistically significant increase in mean bone volume/tissue volume ratio% (BV/TV%) in the treated group (7.0±1.54%) as compared to the control group (3.8±0.48%) (P <0.001, 95%CI=1.9–4.3). This corresponded to a net increase of 84.21% in response to Zoledronate treatment. Comparison between the local and systemic effects of Zoledronate also revealed a significant increase in the BV/TV% in the locally treated group (6.69±0.62%) when compared to the cohort administered systemic bisphosphonate treatment (4.03±0.44%) (P<0.001, 95%CI=1.24–3.20), corresponding to a net increase of 66.0%.

These preliminary results suggest that high dose sustained release of Zoledronate can lead to a significant inhibition of tumor-induced osteolysis. Moreover, comparison between local and systemic delivery revealed that the effect of local bisphosphonate administration exceeds the benefits of systemic delivery in terms of osteolysis inhibition. Lastly, the noted effects of Zoledronate local delivery triggers the need for further assessment of its anti-tumour activity.