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Bioactive cations, including calcium, copper and magnesium, have shown the potential to become the alternative to protein growth factor-based therapeutics for bone healing. Ion substitutions are less costly, more stable, and more effective at low concentrations. Although they have been shown to be effective in providing bone grafts with more biological functions, the precise control of ion release kinetics is still a challenge. Moreover, the synergistic effect of three or more metal ions on bone regeneration has rarely been studied. In this study, vaterite-calcite CaCO3 particles were loaded with copper (Cu2+) and magnesium (Mg2+). The polyelectrolyte multilayer (PEM) was deposited on CaCuMg-CO3 particles via layer-by-layer technique to further improve the stability and biocompatibility of the particles and to enable controlled release of multiple metal ions. The PEM coated microcapsules were successfully combined with collagen at the outmost layer, providing a further stimulating microenvironment for bone regeneration. The in vitro release studies showed remarkably stable release of Cu2+ in 2 months without initial burst release. Mg2+ was released in relatively low concentration in the first 7 days. Cell culture studies showed that CaCuMg-PEM-Col microcapsules stimulated cell proliferation, extracellular maturation and mineralization more effectively than blank control and other microcapsules without collagen adsorption (Ca-PEM, CaCu-PEM, CaMg-PEM, CaCuMg-PEM). In addition, the CaCuMg-PEM-Col microcapsules showed positive effects on osteogenesis and angiogenesis in gene expression studies. The results indicate that such a functional and controllable delivery system of multiple bioactive ions might be a safer, simpler and more efficient alternative of protein growth factor-based therapeutics for bone regeneration. It also provides an effective method for functionalizing bone grafts for bone tissue engineering.
Continuous manufacturing is becoming more important in the biopharmaceutical industry. This processing strategy is favorable, as it is more efficient, flexible, and has the potential to produce higher and more consistent product quality. At the same time, it faces some challenges, especially in cell culture. As a steady state has to be maintained over a prolonged time, it is unavoidable to implement advanced process analytical technologies to control the relevant process parameters in a fast and precise manner. One such analytical technology is Raman spectroscopy, which has proven its advantages for process monitoring and control mostly in (fed-) batch cultivations. In this study, an in-line flow cell for Raman spectroscopy is included in the cell-free harvest stream of a perfusion process. Quantitative models for glucose and lactate were generated based on five cultivations originating from varying bioreactor scales. After successfully validating the glucose model (Root Mean Square Error of Prediction (RMSEP) of ∼0.2 g/L), it was employed for control of an external glucose feed in cultivation with a glucose-free perfusion medium. The generated model was successfully applied to perform process control at 4 g/L and 1.5 g/L glucose over several days, respectively, with variability of ±0.4 g/L. The results demonstrate the high potential of Raman spectroscopy for advanced process monitoring and control of a perfusion process with a bioreactor and scale-independent measurement method.