Human Platelet Lysate Can Replace Fetal Calf Serum as a Protein Source to Promote Expansion and Osteogenic Differentiation of Human Bone-Marrow-Derived Mesenchymal Stromal Cells.

Fetal calf serum (FCS) is frequently used as a growth factor and protein source in bone-marrow-derived mesenchymal stromal cell (BMSC) culture media, although it is a xenogenic product presenting multiple disadvantages including but not limited to ethical concerns. A promising alternative for FCS is human platelet lysate (hPL), which is produced out of human platelet concentrates and happens to be a stable and reliable protein source. In this study, we investigated the influence of hPL in an expansion medium (ESM) and an osteogenic differentiation medium (ODM) on the proliferation and osteogenic differentiation capacity of human BMSC. Therefore, we assessed population doublings during cell expansion, performed alizarin red staining to evaluate the calcium content in the extracellular matrix and determined the activity of alkaline phosphatase (ALP) as osteogenic differentiation correlates. The proliferation rate of BMSC cultured in ESM supplemented with hPL exceeded the proliferation rate of BMSC cultured in the presence of FCS. Furthermore, the calcium content and ALP activity was significantly higher in samples incubated in hPL-supplemented ODM, especially in the early phases of differentiation. Our results show that hPL can replace FCS as a protein supplier in cell culture media and does not negatively affect the osteogenic differentiation capacity of BMSC.

Supplementation with 45S5 Bioactive Glass Reduces In Vivo Resorption of the ß-Tricalcium-Phosphate-Based Bone Substitute Material Vitoss.

Compared to other materials such as 45S5 bioactive glass (BG), ß-tricalcium phosphate (ß-TCP)-based bone substitutes such as Vitoss show limited material-driven stimulation of osteogenesis and/or angiogenesis. The unfavorable degradation kinetics of ß-TCP-based bone substitutes may result in an imbalance between resorption and osseous regeneration. Composite materials like Vitoss BA (Vitoss supplemented with 20 wt % 45S5-BG particles) might help to overcome these limitations. However, the influence of BG particles in Vitoss BA compared to unsupplemented Vitoss on osteogenesis, resorption behavior, and angiogenesis is not yet described. In this study, Vitoss and Vitoss BA scaffolds were seeded with human mesenchymal stromal cells before subcutaneous implantation in immunodeficient mice for 10 weeks. Scaffold resorption was monitored by micro-computed tomography, while osteoid formation and vascularization were assessed by histomorphometry and gene expression analysis. Whilst slightly more osteoid and improved angiogenesis were found in Vitoss BA, maturation of the osteoid was more advanced in Vitoss scaffolds. The volume of Vitoss implants decreased significantly, combined with a significantly increased presence of resorbing cells, whilst the volume remained stable in Vitoss BA scaffolds. Future studies should evaluate the interaction of 45S5-BG with resorbing cells and bone precursor cells in greater detail to improve the understanding and application of ß-TCP/45S5-BG composite bone substitute materials.

Osteogenic differentiation of mesenchymal stem cells is enhanced in a 45S5-supplemented ß-TCP composite scaffold: an in-vitro comparison of Vitoss and Vitoss BA.

Since the amount of autologous bone for the treatment of bone defects is limited and harvesting might cause complications, synthetic bone substitutes such as the popular ß-tricalcium phosphate (ß-TCP) based Vitoss have been developed as an alternative grafting material. ß-TCPs exhibit osteoconductive properties, however material-initiated stimulation of osteogenic differentiation is limited. These limitations might be overcome by addition of 45S5 bioactive glass (BG) particles. This study aims to analyze the influence of BG particles in Vitoss BA (20 wt% BG particles with a size of 90-150 µm) on osteogenic properties, cell vitality and cell proliferation in direct comparison to Vitoss by evaluation of the underlying cellular mechanisms. For that purpose, Vitoss and Vitoss BA scaffolds were seeded with human mesenchymal stem cells (MSC) and underwent osteogenic differentiation in-vitro for up to 42 days. Cell vitality, proliferation, and osteogenic differentiation were monitored by quantitative gene expression analysis, determination of alkaline phosphatase activity, PrestoBlue cell viability assay, dsDNA quantification, and a fluorescence-microscopy-based live/dead-assay. It was demonstrated that BG particles decrease cell proliferation but do not have a negative impact on cell vitality. Especially the early stages of osteogenic differentiation were significantly improved in the presence of BG particles, resulting in earlier maturation of the MSC towards osteoblasts. Since most of the stimulatory effects induced by BG particles took place initially, particles exhibiting another surface-area-to-volume ratio should be considered in order to provide long-lasting stimulation.