Spinal fusion surgery utilising interbody cages with bone autograft is a common treatment for chronic lower back pain when conservative therapy proves ineffective. However, autograft harvesting poses risks, including donor site morbidity, while fusion outcomes are often suboptimal in compromised patients. This study aimed to develop a customisable, osteoconductive bone graft substitute by incorporating magnesium and strontium-substituted hydroxyapatite (sHAP) into polycaprolactone (PCL) composites, intended to replace autografts when contraindicated. To ensure the reproducibility of in vitro testing, a standard operating procedure (SOP) was developed to address cell sensitivity to environmental changes and serum variability. The SOP used a human mesenchymal stem cell (MSC) line under serum-free conditions throughout the project.
Various cell culture parameters were evaluated for their effect on MSC proliferation, including media compositions (serum-free, bovine serum-supplemented, and human serum-supplemented), surface coatings (gelatine and fibronectin), and media change frequencies (partial on day 3, full on day 3, no change in 1 week). Cell proliferation was quantified using a metabolic resazurin assay. For the synthesis of sHAP, multiple formulations with substitution degrees of 5%, 10%, and 20% for Mg and Sr, respectively, were evaluated. Chemical composition, structure, and crystallinity were analysed using ICP-OES, FTIR, and XRD. sHAP was incorporated into 4-arm methacrylated PCL and fabricated into scaffolds via 3D printing. The wettability of different sHAP-PCL composites was analysed via drop-shape analysis, and surface characteristics were examined using SEM. Cytotoxicity of sHAP powders and sHAP-PCL scaffolds was evaluated via a metabolic resazurin assay. Visualisation of cell spreading on scaffolds was performed through actin staining.
Serum-free medium demonstrated comparable cell proliferation to serum-supplemented media, with no medium change necessary over a 1-week culture period. Surface coatings did not significantly impact proliferation. Higher substitution levels increased secondary phase formation, while 20% Mg substitution completely hindered HAP formation. sHAP formulations with 5% Mg, 5% Sr and 10% Mg, 5% Sr exhibited high substituent incorporation with characteristic chemical HAP structure and minimal to no secondary phases. These formulations showed no cytotoxic effects in vitro. The incorporation of sHAP influenced scaffold printability by altering ink rheology, while silica addition potentially reduced hydrophilicity. Scaffolds containing 30% sHAP (5% Mg and Sr) allowed for cell attachment and spreading, demonstrating sustained cell viability and proliferation.
An SOP for serum-free in vitro analysis was established, sHAP was successfully synthesised, and fabrication of sHAP-PCL scaffolds was achieved, advancing the development of customisable fusion cage inserts as potential substitutes for bone autografts.
