# Anti-Inflammatory Activity of a Demineralized Bone Matrix: An In Vitro Pilot Study
## 4 Discussion  
Demineralized bone matrix [1] may have biological activities that remain overshadowed by the impressive osteoinductive properties when transplanted ectopically in a rodent model [9,10]. In a clinical scenario, however, demineralized freeze-dried bone allografts failed to exert the hallmark signs of osteoinductivity [28]. Moreover, demineralized teeth are proposed as grafts for bone reconstruction [17,29]. DBM research is closely related to the discovery of the BMPs [1,7,30], yet attempts to identify other growth factors that survive the demineralization process are minor [13,31,32]. Surprisingly, however, even though the early stages of graft consolidation occur in a catabolic inflammatory environment [15], we lack research on whether DBM might influence the process of inflammation. We therefore tested the potential anti-inflammatory properties of acid lysates prepared from DGC and MDF based on our established bioassays. The main finding of the present research was that acid lysates from granules and fibers reduced the inflammatory response of RAW 264.7 macrophages, particularly IL1 and IL6 expression. This anti-inflammatory activity was not restricted to RAW 264.7 macrophages as the LPS-induced expression of inflammatory cytokines in the primary macrophages was also diminished by DGC and MDF. Care should be taken when interpreting these findings because NaCl generated by the neutralization of HCl might have anti-inflammatory properties; however, it needs around 200 mM NaCl to cause an M1 to M2 shift of primary human macrophages [27]. We confirmed that adding 300 mM NaCl to the culture medium is toxic to RAW 264.7 macrophages and observed an anti-inflammatory activity by adding 100 mM  
Figure 6: Desalted DGC and MDF reduced the expression of IL6, iNOS and CCL5 in ST2 cells. NaCl to the culture medium. To rule out that the anti-inflammatory activity of DGC and MDF is an artefact caused by the disbalanced salt concentration of the culture medium, the lysates were subjected to desalting and buffer exchange for serum-free medium. In support of the overall conclusion, desalted and buffer-exchanged DGC and MDF greatly reduced the LPS-induced expression of IL1 and IL6 in RAW 264.7 macrophages. To rule out that DGC and MDF bind to LPS and thereby neutralize its activity, we first exposed the RAW 264.7 macrophages to LPS before adding the lysates. In this setting, the desalted lysates of both allografts had an anti-inflammatory activity with respect to the expression of IL1 and IL6 in the RAW 264.7 macrophages. To further rule out this possibility, we implemented the ST2 murine mesenchymal cell line where IL1\(\beta\) and TNF\(\alpha\) can provoke an inflammatory response [19]. We show here that with ST2 cells, desalted DGC and MDF significantly attenuated the expression of IL6, iNOS and CCL5. Together, these findings suggest that the anti-inflammatory activity of the allograft lysates cannot be simply blamed on the neutralization of LPS. Moreover, these data extend our observation obtained with macrophages towards mesenchymal cells. Surprisingly, however, the inflammatory response of human gingival fibroblast to IL1\(\beta\) and TNF\(\alpha\) was only moderately reduced by DGC and MDF. These findings suggest that the murine cells are potentially more sensitive than human cells to lysates of DGC and MDF in their activity in terms of reducing the expression of inflammatory mediators. The present study has limitations. Even though we describe here an anti-inflammatory effect of acid lysates of DGC and MDF in macrophages and ST2 cells, many questions remain to be answered. First, we have not yet identified a specific molecule in DGC and MDF that is responsible for the anti-inflammatory activity. TGF-\(\beta\) signaling can be ruled out because blocking of the TGF-\(\beta\) receptor type I kinase with SB431542 failed to reverse the anti-inflammatory activity.