Class I Biocompatible DLP-Printed Acrylate Impairs Adhesion and Proliferation of Human Mesenchymal Stromal Cells in Indirect Cytotoxicity Assay.

The popular method of digital light processing 3D printing (DLP) for complex and individual laboratory equipment requires materials that are as inert as possible for use in contact with cells for subsequent investigations. However, the per se incomplete curing of acrylate resins by UV light leaves residuals that are not suitable for cell culture application. Therefore, we evaluated the cytotoxicity of four commercially available acrylate resins with bone marrow-derived human mesenchymal stromal cells (BM-hMSC) in an indirect cytotoxicity test. This involved incubating the printed cylinders in Transwell™ inserts for 7 days. While the degree of crosslinking did not increase significantly between freshly printed and stored samples (3 weeks in ambient conditions), the storage improved the material's performance in terms of cytocompatibility. The DNA amount and LDH activity showed a direct influence of the resin residuals on cell adhesion. The class I acrylate Surgical Guide™ left no adherent cells after 7 days, regardless of previous storage. In comparison, the Basic Ivory™ resin after storage allowed same amount of adherent cells after 7 days as the polystyrene reference. We conclude that resin residuals of certain materials are released, which allows the use of the resins in indirect contact with cells thereafter.

Osteogenic stimulation of osteoprogenitors by putamen ovi peptides and hyaluronic acid.

Eggshell peptides (EP) majorly contribute to rapid bone building in chicks, wherefore this paper investigated their potential for stimulating osteogenesis in vitro. In this study, the effects of EP, also called putamen ovi peptides and a combination of hyaluronic acid with EP in cell culture medium were tested towards proliferation, differentiation, gene expression and mineralization of bovine osteoprogenitors and primary human osteoblasts. The influence of EP at concentrations of 0.005 g/L, 0.5 g/L and 0.5 g/L with 0.25% hyaluronic acid was analyzed using immunocytochemical staining of bone-specific matrix proteins, namely collagen type I, osteonectin, osteopontin and osteocalcin, to prove osteoblastic differentiation. Additionally, Richardson-staining was performed. All tests revealed a superior osteoblastic differentiation with EP at 0.5 g/L after 5 days of cultivation. Hyaluronic acid alone showed controversial results and partially constrained osteoblastic differentiation in combination with EP to a level as low as for pure EP at 0.005 g/L. Of particular interest is the osteoblast-typical mineralization, as an important indicator of bone formation, which was measured indirectly via the calcium concentration after cultivation over 4 weeks. The mineralization showed an increase by a factor of 286 during the cultivation of primary human osteoblasts with hyaluronic acid and EP. Meanwhile, cell cultures treated with EP (0.5 g/L) only showed an 80-fold increase in calcium concentration.The influence of EP (0.5 g/L) on primary human osteoblasts was investigated by gene expression after 2 weeks of cultivation. Microarray and qRT-PCR analysis showed a strongly increased expression of main important genes in bone formation, bone regeneration and the physiological bone remodelling processes. Namely, BMP 2, osteopontin and the matrix metalloproteinases 1 and 9, were present during in vitro osteoprogenitor culture with EP. By explicitly underlining the potential of eggshell peptides for stimulating osteogenesis, as well as emphasizing complex and controversial interaction with hyaluronan, this manuscript is relevant for developing new functionalized biomaterials for bone regeneration.

The interplay of collagen/bioactive glass nanoparticle coatings and electrical stimulation regimes distinctly enhanced osteogenic differentiation of human mesenchymal stem cells.

Increasing research has incorporated bioactive glass nanoparticles (BGN) and electric field (EF) stimulation for bone tissue engineering and regeneration applications. However, their interplay and the effects of different EF stimulation regimes on osteogenic differentiation of human mesenchymal stem cells (hMSC) are less investigated. In this study, we introduced EF with negligible magnetic field strength through a well-characterized transformer-like coupling (TLC) system, and applied EF disrupted (4/4) or consecutive (12/12) regime on type I collagen (Col) coatings with/without BGN over 28 days. Additionally, dexamethasone was excluded to enable an accurate interpretation of BGN and EF in supporting osteogenic differentiation. Here, we demonstrated the influences of BGN and EF on collagen topography and maintaining coating stability. Coupled with the release profile of Si ions from the BGN, cell proliferation and calcium deposition were enhanced in the Col-BGN samples after 28 days. Further, osteogenic differentiation was initiated as early as d 7, and each EF regime was shown to activate distinct pathways. The disrupted (4/4) regime was associated with the BMP/Smad4 pathways that up-regulate Runx2/OCN gene expression on d 7, with a lesser effect on ALP activity. In contrast, the canonical Wnt/ß-Catenin signaling pathway activated through mechanotransduction cues is associated with the consecutive (12/12) regime, with significantly elevated ALP activity and Sp7 gene expression reported on d 7. In summary, our results illustrated the synergistic effects of BGN and EF in different stimulation regimes on osteogenic differentiation that can be further exploited to enhance current bone tissue engineering and regeneration approaches. STATEMENT OF SIGNIFICANCE: The unique release mechanisms of silica from bioactive glass nanoparticles (BGN) were coupled with pulsatile electric field (EF) stimulation to support hMSC osteogenic differentiation, in the absence of dexamethasone. Furthermore, the interplay with consecutive (12/12) and disrupted (4/4) stimulation regimes was investigated. The reported physical, mechanical and topographical effects of BGN and EF on the collagen coating, hMSC and the distinct progression of osteogenic differentiation (canonical Wnt/ß-Catenin and BMP/Smad) triggered by respective stimulation regime were not explicitly reported previously. These results provide the fundamentals for further exploitations on BGN composites with metal ions and rotation of EF regimes to enhance osteogenic differentiation. The goal is sustaining continual osteogenic differentiation and achieving a more physiologically-relevant state and bone constructs in vitro.