(52) Inflammatory Response Attenuated by Extracellular Matrix Proteins in Demineralized Bone Matrix

Abstract: While successful tissue regeneration of new bone requires osteoconductive, osteoinductive and osteogenic components, bone healing after injury necessitates additional elements for consideration. Current natural scaffolds, such as demineralized bone matrix (DBM), contain an osteoconductive collagenous network and numerous osteoinductive growth factors such as TGF-β and BMP-2 to promote new bony deposition.1 But the immediate stages after tissue damage such as inflammation, angiogenesis, and cell recruitment are still regulated by a myriad of growth factors, hormones, and cytokines that influence the local cellular environment prior to stimulating new bone tissue formation.1 Therefore, in this study, the effect of released proteins from DBM on select cytokines pertinent to the inflammation process were evaluated. Both proinflammatory (TNFα, IL-1β, IL-6, IL-8) and anti-inflammatory (IL-4, IL-10, IL-13) cytokine release by cells in skeletal (macrophages) or dental (gingival fibroblasts) models exposed to demineralized bone matrix proteins were studied.Experiments were performed with commercially available human leukemia monocytic cells (THP-1) and primary human gingival fibroblasts (HGF) cells. Demineralized bone matrix fibers (DBF) and demineralized ground cortical (DGC) were provided by LifeNet Health. For acid lysates of DBF and DGC, demineralized bone was submerged to reach 0.1g/ml in 1N HCl and samples were left shaking overnight at room temperature. Lysates were then collected, the pH neutralized using NaOH, and run through a 0.2 µm filter. Acid extraction was also done on a deactivated DBM powder control. For cell differentiation and treatment, THP-1 cells were plated at 100,000 cells per well and differentiated for 3 days, then washed twice with PBS and allowed to recover for 2 days with fresh DMEM containing 10% FBS and 1% pen/strep. HGF cells were plated at 100,000 cells per well with fresh DMEM containing 10% FBS and 1% pen/strep. Both cell lines were pre-exposed to 25% acid lysate from DGC and DBF for 30 minutes, then treated with either IL-1β and TNFα, both at 10ng/ml, or 100ng/mL of LPS, in serum-free media for 24 hours. For a control, cells were treated with 25% lysate from the deactivated DBM, and then treated with either IL-1β and TNFα, or LPS. For cytokine quantification, the resulting supernatants (100µL) were used in corresponding human Quantikine ELISA kits.Pre-treating differentiated monocytes with acid lysates from either DBF or DGC, followed by LPS treatment, led to a reduction in the expression of pro-inflammatory cytokines IL-6 and IL-8 (Figure 1a,b). Similarly, in fibroblasts exposed to IL-1β and TNFα, the acid lysates again reduced the expression of both IL-6 and IL-8 (Figure 1c,d).The results of this study suggest that DBM granules and fibers can influence cytokine release by cells involved in the immune process. This repression arises whether the cells are stimulated by growth factors or cytokines (IL-1β/TNFα) produced by host immune cells or the presence of foreign material (LPS). The immunosuppressive response can ensure that acute inflammation remains transient, and that chronic inflammation is prevented. However, inflammation in general is necessary for bone healing to initiate and proceed without incident, as macrophages, neutrophils, T cells, B cells play a role in removing cell and tissue debris, recruiting cells for callus formation, and preconditioning mesenchymal stem cells to prime osteogenic functionality and mitigate the proinflammatory response (shifting macrophages to the M2 phenotype). Therefore, complete elimination of inflammation is not considered an ideal situation and may negatively impact the bone healing process.[4].Understanding the effect of released proteins from DBM on the overall profile of pro-inflammatory and anti-inflammatory cytokines may provide a better understanding of its impact on the bone repair process, whether in long or flat bones of the skeletal system or in dental applications.

Figure 1: Attenuated IL-6 release in stimulated THP-1 cells (a) and fibroblasts (c) in response to DBM lysates. Additionally, similar IL-8 release in stimulated THP-1 cells (b) and fibroblasts (d) in response to DBM lysates.