TGFb1 by fibroblasts was inhibited. HB-EGF was also reduced in cultures with IL-1a or TGFb1 siRNA-transfected fibroblasts and in co-cultures with antiIL-1a or anti-TGFb1 antibodies, indicating that the keratinocyte proliferation upregulated by IL-1a and TGFb1 in the co-culture was partially mediated by HB-EGF. The effects of the anti-HBEGF antibody on keratinocyte proliferation were significantly greater than those of the anti-IL-1a or anti-TGFb1 antibodies, suggesting that HB-EGF plays a central role in the regulation of kerotinocyte growth in these co-cultures. Since siRNA can only partially or/and Tulathromycin B transiently block target cytokine production and the inhibition antibodies we used in our experiments were all overdosage, the inhibitory effects of siRNA was always weaker than those of inhibition antibodies in our observations. After the early stage of the co-culture, the keratinocytes in all of the culture conditions proliferated at similar rates, with the exception of the EGF control. The EGF culture showed faster late stage growth than the other cultures, suggesting a reduction of cell growth factors in these cultures. However, when the concentrations of the HB-EGF, IL1a, and TGFb1 were examined, it was found that they continued to increase as the cells grew. Further analysis into the ratio of HBEGF levels and cell count revealed that the increase in HB-EGF, IL-1a, and TGFb1 levels failed to match the growth rate, resulting in lower levels of these cytokines per cell during the later stage 
of the culture. Both keratinocytes and fibroblasts have very high motility in culture, which enables them to meet and form cell-to-cell contacts even when co-cultured at very low densities. To confirm the effects of cell contact on keratinocyte proliferation and migration that was promoted by fibroblasts and relevant cytokines, transwell plates were used to separate the two cell types in this study. The two cell types were separated into the upper and lower chambers of the transwell to eliminate Gomisin-D direct contact with each other, and the cell type being observed was placed in the lower chamber. The data clearly showed that without direct contact, the fibroblasts and the IL-1a and TGFb1 cytokines produced by the fibroblasts did not have a significant effect on the proliferation and migration of co-cultured keratinocytes. To further confirm the effects of IL-1a, TGFb1 and HB-EGF on keratinocyte proliferation, these cytokines were added to keratinocyte cultures at different concentrations and compared to the kerotinocye/ fibroblast co-culture. The results showed that all three cytokines were able to stimulate keratinocyte proliferation, but require a 10fold higher concentration of the cytokines, which suggested that since the overall cytokine level of the culture was insufficient to account for the observed effects, direct contact may be required to provide a microenvironment with sufficient cytokine levels. Cell death could be a factor that affects the evaluation of cell proliferation by cell counting. To eliminate the interference of cell death on our experimental results, we did Anexin V binding assay for some samples in addition to our routine Trypan Blue Exclusion test for all samples. Trypan Blue Exclusion was assessed on days 5, 10 and 15, when the cells were harvested. Organ tissues are comprised of groups of cells that coordinate their activities so as to achieve a functional outcome. In bone, the functional unit of cells is called a ‘basic multicellular unit’. BMUs are transient functional groupings of cells that progress through the bone.