A Preliminary Direct Comparison of the Inflammatory Reduction and Growth Factor Production Capabilities of Three Commercially Available Wound Products: Collagen Sheet,Manuka Honey Sheet,and a Novel Bioengineered Collagen Derivative + Manuka Honey + Hydroxyapatite Sheet

Many commercially available wound products focus on improving one stage of the wound healing cascade. While this targeted approach works for specific wounds, there is a need for products that can reliably and comprehensively progress a wound through multiple stages. This preliminary in vitro study was performed to directly compare the inflammatory reduction and growth factor production effects of three commercially available wound care products: a collagen sheet (COL), a Manuka Honey Calcium Alginate sheet (MH), and a novel bioengineered sheet comprised of a collagen derivative (gelatin), Manuka honey, and hydroxyapatite (BCMH). Macrophages and human dermal fibroblasts were directly seeded on all three commercial products, and supernatants were analyzed for inflammatory markers and growth factors, respectively. Comparing the MMP-9/TIMP-1 ratio, BCMH resulted in 11× lower levels of this inflammation biomarker compared to COL, and 3× lower levels compared to MH. Both the COL and BCMH products created an environment conducive to expression and release of relevant growth factors, while the MH product showed the lowest levels of growth factor expression of all three commercially available products tested. The favorable 11× lower MMP-9/TIMP-1 ratio observed with the BCMH product compared to the COL product suggests that the BCMH products provided a superior comprehensive approach to healthy progression of the wounds by providing an additional benefit of reducing the inflammatory response in vitro.     

Effects of the Novel Compound DK223 ([1E,2E-1,2-Bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) on Migration and Proliferation of Human Keratinocytes and Primary Dermal Fibroblasts

Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM) secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS) increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-β1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-β1 induction.     

Dexamethasone Induces Changes in Osteogenic Differentiation of Human Mesenchymal Stromal Cells via SOX9 and PPARG,but Not RUNX2

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field.