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The reduced effectiveness of chemotherapy in many patients undergoing treatment highlights the need for novel drug combinations that target drug resistance mechanisms contributing to tumor survival. Dynamic conditions within the tumor microenvironment influence the response to anti-cancer drugs. Accordingly, identifying effective drug concentrations and interactions (additive, synergistic, or antagonistic) in relevant tumor tissue models will inform new treatment combinations. To address this need for combinatorial chemotherapeutic (CTx) screening assays, we have developed a new assay called CombiCTx, which uses a device with three reservoirs containing gels loaded with anti-cancer drugs. The drug-loaded device is inverted and placed in a standard culture dish above cancer cells, and both are then enclosed in gel. Drugs diffuse from the reservoirs and expose cancer cells to overlapping dynamic drug gradients. We imaged diffusion of the anti-cancer drug doxorubicin in the assay using time-lapse microscopy, and established an imaging protocol for quantifying MDA-MB-231 breast cancer cell survival responses along drug gradients. Finally, evaluating combination effects of navitoclax and gemcitabine with CombiCTx revealed localized effects of navitoclax, attributed to limited diffusion, while gemcitabine seemed to diffuse readily throughout the assay and revealed a mild synergy in navitoclax affected regions. These data demonstrate the capacity of CombiCTx to evaluate the cytotoxic effects of anti-cancer drug combinations while accounting for drug diffusion differences, which is relevant in the context of the 3D tumor environment and may thereby help inform clinical treatment strategies.

Bioprinting is increasingly used to create complex tissue constructs for an array of research applications, and there are also increasing efforts to print tissues for transplantation. Bioprinting may also prove valuable in the context of drug screening for personalized medicine for treatment of diseases such as cancer. However, the rapidly expanding bioprinting research field is currently limited by access to bioprinters. To increase the availability of bioprinting technologies we present here an open source extrusion bioprinter based on the E3D motion system and tool changer to enable high-resolution multimaterial bioprinting. As proof of concept, the bioprinter is used to create collagen constructs using freeform reversible embedding of suspended hydrogels (FRESH) methodology, as well as multimaterial constructs composed of distinct sections of laminin and collagen. Data is presented demonstrating that the bioprinted constructs support growth of cells either seeded onto printed constructs or included in the bioink prior to bioprinting. This open source bioprinter is easily adapted for different bioprinting applications, and additional tools can be incorporated to increase the capabilities of the system.

A new class of materials, bone adhesives, could revolutionise the treatment of highly fragmented fractures. We present the first biological safety investigation of a bio-inspired bone adhesive. The formulation was based upon a modified calcium phosphate cement that included the amino acid phosphoserine. This material has recently been described as substantially stronger than other bioresorbable calcium phosphate cements. Four adhesive groups with the active substance (phosphoserine) and two control groups without phosphoserine were selected for in vitro and in vivo biocompatibility testing. The test groups were subject for cell viability assay and subcutaneous implantation in rats that was followed by gene expression analysis and histology assessment after 6 and 12 weeks. All adhesive groups supported the same rate of cell proliferation compared to the alpha-TCP control and had viability between 45-64% when compared to cell control. There was no evidence of an increased immune response or ectopic bone formation in vivo. To conclude, this bio-inspired bone adhesive has been proven to be safe, in the present study, without any harmful effects on the surrounding soft tissue. 

Phosphoserine (pSER) is a phosphorylated amino acid commonly found in non-collagenous bone proteins and is implicated in the regulation of calcium phosphate (CaP) mineral formation and interfacial interactions in native bone. Here, we soaked 3D printed collagen scaffolds, with or without prior mineralisation, in pSER and investigated the early cellular response by assessing pre-osteoblast viability through ATP content measurements at day 3. The scaffolds were further evaluated in a rat uni-cortical femoral defect model and analysed by micro-computed tomography (µCT) and histology after 6 weeks. In parallel, standard 2D cultures of MC3T3-E1 cells and rat mesenchymal stromal cells (rMSCs) were exposed to pSER, nanohydroxyapatite (nHA), or a combination of both to assess dose-dependent effects in the absence of a scaffold environment. Using a dose-dependent screening approach, we identified 0.05% pSER on mineralised collagen scaffolds as the concentration that resulted in the highest cell viability, whereas higher concentrations were cytotoxic. This concentration was further evaluated in a time-dependent setup over 7 days and again showed increased cell viability compared to untreated collagen scaffolds. Similar trends were observed in vivo where CaP-containing groups demonstrated a higher bone volume fraction than CaP-free collagen groups, and pSER-associated effects were detectable but less pronounced. Overall, the results indicate that collagen scaffolds combining pSER and CaP lead to a dose-dependent enhanced pre-osteoblast viability in vitro. Future work will explore how these pSER-modified collagen bioinks influence bone cell differentiation and aim to elucidate the mechanisms through which pSER supports bone regeneration in vivo.