Breast cancer is the most frequent malignancy in women with an estimation of 2.1 million new diagnoses in 2018. Even though primary tumours are usually efficiently removed by surgery, 20–40% of patients will develop metastases in distant organs. Bone is one of the most frequent site of metastases from advanced breast cancer, accounting from 55 to 58% of all metastases. Currently, none of the therapeutic strategies used to manage breast cancer bone metastasis are really curative. Tailoring a suitable model to study and evaluate the disease pathophysiology and novel advanced therapies is one of the major challenges that will predict more effectively and efficiently the clinical response. Preclinical traditional models have been largely used as they can provide standardization and simplicity, moreover, further advancements have been made with 3D cultures, by spheroids and artificial matrices, patient derived xenografts and microfluidics. Despite these models recapitulate numerous aspects of tumour complexity, they do not completely mimic the clinical native microenvironment. Thus, to fulfil this need, in our study we developed a new, advanced and alternative model of human breast cancer bone metastasis as potential biologic assay for cancer research. The study involved breast cancer bone metastasis samples obtained from three female patients undergoing wide spinal decompression and stabilization through a posterior approach. Samples were cultured in a TubeSpin Bioreactor on a rolling apparatus under hypoxic conditions at time 0 and for up to 40 days and evaluated for viability by the Alamar Blue test, gene expression profile, histology and immunohistochemistry. Results showed the maintenance and preservation, at time 0 and after 40 days of culture, of the tissue viability, biological activity, as well as molecular markers, i.e. several key genes involved in the complex interactions between the tumour cells and bone able to drive cancer progression, cancer aggressiveness and metastasis to bone. A good tis sue morphological and microarchitectural preservation with the presence of lacunar osteolysis, fragmented trabeculae locally surrounded by osteoclast cells and malignant cells and an intense infiltration by tumour cells in bone marrow compartment in all examined samples. Histomorphometrical data on the levels of bone resorption and bone apposition parameters remained constant between T0 and T40 for all analysed patients. Additionally, immunohistochemistry showed homogeneous expression and location of CDH1, CDH2, KRT8, KRT18, Ki67, CASP3, ESR1, CD8 and CD68 between T0 and T40, thus further confirming the invasive behaviour of breast cancer cells and indicating the maintaining of the metastatic microenvironment. The novel tissue culture, set-up in this study, has significant advantages in comparison to the pre-existent 3D models: the tumour environment is the same of the clinical scenario, including all cell types as well as the native extracellular matrix; it can be quickly set-up employing only small samples of breast cancer bone metastasis tissue in a simple, ethically correct and cost-effective manner; it bypasses and/or decreases the necessity to use more complex preclinical model, thus reducing the ethical burden following the guiding principles aimed at replacing/reducing/refining (3R) animal use and their suffering for scientific purposes; it can allow the study of the interactions within the breast cancer bone metastasis tissue over a relatively long period of up to 40 days, preserving the tumour morphology and architecture and allowing also the evaluation of different biological factors, parameters and activities. Therefore, the study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumour subtypes so predicting patient specific responses.

Autologous bone grafting is a standard procedure for the clinical repair of skeletal defects, and good results have been obtained. Autologous vascularized bone grafting is currently the procedure of choice because of high osteogenic potential and resistance against reabsorption. Disadvantages of this procedure include limited availability of donor sites, clinical difficulty in handling, and a failure rate exceeding 10%. Allografts are often used for massive bone loss, but since only the marginal portion is newly vascularized after the implantation non healing fractures are often reported, along with a graft reabsorption. To overcome these problems, some studies in literature tried to conjugate bone graft and vascular supply, with encouraging results. On the other side, several studies in literature reported the ability of bone marrow derived cells to promote neo-vascularization. In fact, bone marrow contains not only hematopoietic stem cells (HSCs) and MSCs as a source for regenerating tissues but also accessory cells that support angiogenesis and vasculogenesis by producing several growth factors. In this scenario a new procedure was developed, consisting in an allogenic bone graft transplantation in a critical size defect in rabbit radius, plus a deviation at its inside of the median artery and vein with a supplement of autologous bone marrow concentrate on a collagen scaffold.

Twenty-four New Zealand male white rabbits (2500–3000 g) were divided into 2 groups, each consisting of 12 animals. Surgeries were performed as follow:

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Group 1 (#12): allogenic bone graft (left radius) / allogenic bone graft + vascular pedicle + autologous bone marrow concentrate (right radius)

For each group, 3 experimental time: 8, 4 and 2 weeks (4 animals for each time).

The bone used as graft was previously collected from an uncorrelated study. An in vitro evaluation of bone marrow concentrate was performed in all cases, and at the time of sacrifice histological and histomorphometrical assessment were performed with immunohistochemical assays for VEGF, CD31 e CD146 to highlight the presence of vessels and endothelial cells. Micro-CT Analysis with quantitative bone evaluation was performed in all cases.

The bone marrow concentrate showed a marked capability to differentiate into osteogenic, chondrogenic and agipogenic lineages. No complications such as infection or intolerance to the procedure were reported. The bone grafts showed only a partial integration, mainly at the extremities in the group with vascular and bone marrow concentrate supplement, with a good and healthy residual bone. immunohistochemistry showed an interesting higher VEGF expression in the same group. Micro CT analysis showed a higher remodeling activities in the groups treated with vascular supplement, with an area of integration at the extremities increasing with the extension of the sacrifice time.

The present study suggests that the vascular and marrow cells supplement may positively influence the neoangiogenesis and the neovascularization of the homologous bone graft. A longer time of follow up and improvement of the surgical technique are required to validate the procedure.

Aims: Surface modification of biomaterials to be used as scaffolds in tissue engineering is a promising method to improve device multi-functionality and biological properties. Biomimetic surface treatments, such as changes in nano-structure and attachment of biomolecular signals, enhance material bioactivity and affinity for specific cells. In this study the functionalization of a titanium surface with vitronectin-derived nonapeptide(HVP) and RGD peptides was investigated. Bone forming cells were used to analyse the role of each surface modification in the initial steps of cell adhesion process and then proliferation and differentiation.

Method: Smooth titanium samples were functionalized by different chemical treatments in order to obtain varying amount of peptide adhesion. Human marrow stro-mal cells (MSC) were seeded and cultured in osteogenic medium. Cell adhesion and morphology were assessed by fluorescence microscopy after 6 hours. Viability of MSC was quantified at 7 and 14 days from plating, proliferation was measured using DNA and total protein content, and osteoblast phenotype expression was assayed using alkaline phosphatase (ALP) and calcium content.

Results: The results showed that presence of HVP and RGD peptides improves cell morphology in early adhesion on surface, compared to control (titanium without peptides). Activity of ALP and Ca²⁺ content of (1:1000)HVP and RGD samples were higher than the other experimental surfaces with or without peptides, even if they did not reach the values of control cells on tissue culture polystyrene.

Discussion: Properties acquired with chemical treatments can improve titanium surfaces. These data provide information useful to develop biomimetic cell-friendly surfaces for bone engineering.

Aims: Different polyurethane foam matrices (PUF) loaded with hydroxyapatite (HA) or α-tricalcium phosphate were proposed as scaffold for bone regeneration [1,2]. In this work new PUFs were developed and loaded with HA or α-TCP.

Methods: PUFs were synthesized by a one-step polymerisation from a hydrophilic polyol mixture (LF 2946, Elastogran, Italy) and polymeric MDI (B141, BASF), using Fe acetyl-acetonate as catalyst and 2% water as expanding agent. The composites were prepared in the same way, by adding HA or α-TCP to the reacting mixture.

In vitro cell interactions were evaluated with human osteoblasts (HOB, 2nd passage) isolated from the trabecular bone of the femoral head of patients undergoing total hip replacement and cultured following the usual procedure. HOB cells (1x105 cells/sample) were kept in contact with the scaffolds for 7 and 14 days. At each time endpoint HOB metabolic activity, intracellular and released ALP were evaluated.

Results: By water adsorption test, newly synthesized PUFs showed a higher hydrophilicity compared to that of the previous matrices (600% vs 110% water uptake after 100h). Due to the presence of inorganic salts, composite scaffolds showed density values higher (0.131B80.200g/cm3) than those of unloaded PUFs (0.071B80.093g/cm3). Yet, open cell percentage (57–75%) and average pore size (350B8520mm) resulted similar to those of the PUFs.

HOB cells grown on scaffold samples showed an increase of metabolic activity from 7 to 14 days. The amount of intracellular ALP increased too, whereas the amount of ALP in the medium was quite low. HOB cells, after 14 days, appeared closely adherent to the scaffolds, with an elongated and flattened shape.

Conclusions: These preliminary results showed that, even if slow, the growth of HOB onto PUF scaffolds was quite good. After 14 days, PUF composites showed higher cells growth than PUF-matrices, confirming the role of the bone-like inorganic particles in improving osteoblasts functions. Long-term in vitro tests are now in progress.