# Anti-Inflammatory Activity of a Demineralized Bone Matrix: An In Vitro Pilot Study
## 1 Introduction  
Demineralized bone matrix (DBM) is used to reconstruct bony defects [1], including in dental [2, 3], trauma [4] and spinal surgery [5]. DBM provides an osteophilic surface where new bone can be laid, thereby allowing the ingrowth of new bone into the defect site, ideally bridging the defect [6]. The conglomerate of the DBM, the new bone, and the concomitant bone marrow undergoes remodeling and creeping substitution of the DBM, thus allowing a restitution ad integrum of the bone and a reshaping of the anatomical contours of the former defect site [6]. Demineralized bone is derived from cadaver bone that undergoes multiple steps of processing until it is available as a ready-to-use allograft [1]. This processing pays particular attention to maintain the activity of the growth factors stored in the extracellular matrix, most of all the members of the bone morphogenetic protein (BMP) family with their unique osteoinductive properties [7, 8]. DBM is thus equipped with the ability to initiate de novo bone formation at ectopic sites, which is usually tested in rodent models [9, 10]. Hence, there is evidence that DBM allografts are a source of growth factors that can potentially support bone regeneration, resulting in graft consolidation in a patient.  
Bone is a rich source of TGF-\(\beta\)[11, 12]. We have recently identified TGF-\(\beta\) to be released into a liquid fraction from bone allografts, e.g., demineralized ground cortical (DGC) andmoldable demineralized fibers (MDF) [13]. Apart from the growth factor activity, TGF-\(\beta\) is thought to have anti-inflammatory functions [14]. It is therefore reasonable to assume that DGC and MDF exert an anti-inflammatory activity. Surprisingly, however, allografts are usually not tested for their potential anti-inflammatory activity [1]. It would be interesting to know if allografts exert such activity. This is because a transient local inflammation occurs during early bone regeneration [15] and, if not resolved, the chronic inflammation causes the pathological event of inflammatory osteolysis [16]. Theoretically, upon creeping substitution, DBM could potentially exert inflammation-modulating activity.  
To determine the possible anti-inflammatory activity of DGC and MDF, we took the advantage of our previously established protocol to prepare acid lysates of bone [12] and dentin [17]. We then applied the lysates of DGC and MDF to our established in vitro inflammation assay. Our bioassays include murine RAW 264.7 macrophages and primary bone marrow-derived macrophages being exposed to bacterial endotoxins [18], as well as murine ST2 bone marrow-derived mesenchymal cells [19] and human gingival fibroblasts being exposed to inflammatory cytokines interleukin 1\(\beta\) and TNF\(\alpha\)[20]. The modulation of the inflammatory response is measured by changes in gene expression, including IL6, and by the intensity and nuclear translocation of phosphorylated p65 [21]. IL1 as well as IL6 are lead cytokines that represent a large panel of inflammatory mediators, all of which initiate and enhance an inflammatory process that is resolved in a physiologic situation of wound healing [22] and fracture repair [15]--but, if not being resolved, becomes the main cause of tissue destruction [23]. Inducible nitric oxide synthases (iNOS) is also involved in fracture healing [24] and mesenchymal cells expressing chemokine ligand 5 (CCL5; also known as RANTES) support revascularization [25]. In this pilot study, we show in vitro data suggesting that acid lysates prepared from DGC and MDF lower the expression of inflammatory mediators.