Collagen barrier membranes do not adsorb hypoxia mimetic activity‐Activity of gingival fibroblasts cultured directly on collagen barrier membranes loaded with hypoxia mimetic agents

Abstract

Hypoxia-based strategies for applications in oral surgery and periodontology have been proposed where collagen barrier membranes (CBM) are loaded with hypoxia mimetic agents (HMA) to induce a pro-angiogenic response. While it was found that CBM release HMA, it remained unclear if CBM adsorb HMA activity. Here we evaluated the response of oral cells cultured on CBM, supplemented with the HMA dimethyloxalylglycine (DMOG), desferrioxamine (DFO), and l-mimosine (l-MIM). Gingival fibroblasts (GF) were cultured on unwashed CBM as well as on CBM that had been washed with serum-free medium for 48 hours. The pro-angiogenic response was measured based on vascular endothelial growth factor (VEGF) production. Viability and proliferation were assessed based on MTT and BrdU assays. We found that GF seeded onto CBM loaded with DFO and l-MIM, but not DMOG, showed an increase in VEGF to 6.1-fold and 7.7-fold compared to unloaded CBM, respectively. Cells remained vital, but a trend for decreased proliferation was observed on DMOG and DFO-loaded CBM which did not reach the level of significance. Evaluation of washed CBM revealed no difference between the unloaded CBM and CBM supplemented with DMOG, DFO, or l-MIM. In conclusion, our results suggest that CBM do not adsorb hypoxia mimetic activity but release HMA within the first hours. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 874-879, 2018.

Effect of prolyl hydroxylase inhibitor-loaded collagen barrier membranes on osteoclastogenesis and osteoblastogenesis

“…This is in line with bioassays using RAW 264.7 and MC3T3-E1 cells showing that supernatants of collagen barrier membranes in the first 6 h initiated changes in metabolic activity, cell proliferation, and VEGF production in monolayer cell cultures. Furthermore, these findings are in line with the burst release, which was observed by Hamid et al 17 Our data together with the observation that collagen barrier membranes do not adsorb biologic activity of PHD inhibitors 22 highlight the difference to growth and differentiation factors, which can adsorb the collagen barrier membranes. 26,27 In this study, collagen barrier membranes were loaded with one dose of a PHD inhibitor.…”

“…Recent studies revealed that collagen barrier membranes can serve as carrier for PHD inhibitors and showed release kinetics that differ from bone substitute materials, 17,21 whereas no PHD inhibitor activity remained after 48 h on loaded collagen membranes. 22 PHD inhibitors have been advocated as tools for oral surgery and periodontology as they can support wound healing and bone regeneration especially when healing is compromised. [12][13][14][23][24][25] Here we evaluated the impact of released PHD inhibitors of loaded collagen barrier membranes on differentiation and activity of osteoclasts and osteoblasts as an extension to Hamid et al 17 Our findings show that DMOG and DFO are released from collagen barrier membranes.…”

Deferoxamine but Not Dimethyloxalylglycine, L-Mimosine, or Cobalt Dichloride Can Interfere with the MTT Assay

“…Thus, potential misinterpreted results as is possible with DFO can have a major impact on follow-up studies. Especially studies on DFO-loaded materials such as bone substitutes, collagen matrices, or hydrogels need to be performed with a feasible test method [14, 21]. The impact on cell viability in the presence of high dosage of DFO might be underestimated based on the MTT results.…”

“…The wells with and without cells were incubated with 1 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 2 h as previously described [14, 22–24]. MTT solution was discarded and 100 μ L dimethyl sulfoxide (DMSO) was added per well.…”

“…Various biomaterials are under development and evaluation for the release capacity of HMAs. These include collagen barrier membranes and bone substitute materials [12, 14–16]. The success of new scaffolds depends on the biocompatibility and release kinetics of these materials [17].…”