Critical size bone defects pose a serious clinical problem, as the intrinsic healing capacity of bone fails due to the size of the defect. Bone healing might be aided by addition of 1,25(OH)2 vitamin D3 (vitD3) to bone tissue engineering scaffolds. VitD3can promote osteogenic differentiation of human stem cells such as adipose stem cells (hASCs), which is a clinical-relevant source of mesenchymal stem cells. However, it is unknown which release kinetics of vitD3, i.e. short or sustained release from scaffolds, leads to the most optimal osteogenic differentiation of hASCs. We hypothesized that sustained release of vitD3 leads to more osteogenic differentiation of hASCs than shorter applications. hASCs (1×105, passage 3–4) were seeded on 20 ± 1 mg of calcium phosphate particles (day 0), cultured for 20 days, and treated with a total amount of 124 ng vitD3. This treatment was provided either during 30 min before seeding (pre-incubation, short stimulation: [200 nM]), after seeding, over the first 2 days (burst- release high: [100 nM]), or over the total culture period of 20 days (sustained-release: [10 nM]). In the extra condition: burst-release low the hASCs were treated for 2 days after seeding with 6.2 ng vitD3 ([10 nM]) per day. Live/dead staining followed by fluorescent microscopy showed that hASCs attached to the calcium phosphate particles and were mostly viable (±75 %) at day 2. VitD3 applied for any duration did not affect the proliferation of hASCs at day 7 and day 20, measured with an alamar blue assay. At day 7, sustained-release increased the release of active alkaline phosphatase on average by 3.5-fold, compared to all the other conditions. At day 20, this was increased 4.3-fold. At both day 7 and day 20 total protein levels were similar in all conditions. Our results suggest that sustained release of VitD3 from bone tissue engineered scaffolds may be beneficial for the osteogenic differentiation of human stem cells for the treatment of critical bone size defects

In England and Wales in 2012 over 160,000 primary total hip and knee replacements were performed with 57% of hip replacements utilising uncemented prostheses. The main cause of failure, affecting approximately 10% of patients, is aseptic loosening. Previous research has found that functionalising titanium with lysophosphatidic acid (LPA) induces an increase in human osteoblast maturation on the implant surface through co-operation with active metabolites of vitamin D3. This feature, the small size of the LPS molecule and its affinity to readily bind to titanium and hydroxylapatite makes it an especially desirable molecule for bone biomaterials. Nevertheless biomaterials that also demonstrate anti-microbial properties are highly desirable. To test the antimicrobial efficacy of the LPA-functionalised titanium, a clinical isolate of Staphylococcus aureus, obtained from an infected revision surgery, was cultured on the surface of titanium discs functionalised with 0, 0.1. 0.5, 1, 2 and 5μM LPA. Bacterial adhesion was quantified at 1, 2, 6, 12 and 24 hours by live/dead counts and biofilm mass quantified by crystal violet staining after 24, 48, 72 and 96 hours culture. To elucidate the mechanisms of action of LPA, proteomic analysis of adhered bacteria was performed using SDS-PAGE and Western blots. 500nM to 1μM LPA were the optimum concentrations to significantly inhibit bacterial adhesion (ANOVA, p<0.001). These concentrations also reduced biofilm mass on the surface of the titanium. Proteomic analysis highlighted an increase in low molecular weight proteins as a result of optimal LPA surface concentrations. Fatty acid chains as found in LPA have previously been associated with causing leakage of low molecular weight proteins through increased cell membrane permeability. LPA coatings have the potential to enhance implant osseointegration whilst simultaneously reducing bacterial attachment. This technology may reduce both septic and aseptic failure of cementless joint prostheses, ultimately prolonging implant longevity and patient quality of life