Abstract
Objectives: This study evaluated the cellular response of primary osteoblasts exposed to two different presentations of a low-temperature non-sintered deproteinized bovine bone matrix (DBBM). Materials and methods: Six different baths of a commercially available DBBM block (Bonefill® Porous Block) and one of DBBM granule (Bonefill® Porous) were evaluated to identify the mineral structure and organic or cellular remnants. Samples of the same baths were processed in TRIZOL for RNA extraction and quantification. For the immunologic cell reaction assay, primary human osteoblasts (pOB) were exposed to DBMM block (pOB + B) or granules (pOB + G), or none (control) for 1, 3, or 7 days of cell cultivation. Expression of proinflammatory cytokines by pOB was evaluated by crosslinked ELISA assay. In addition, total DNA amount, as well as cell viability via LDH evaluation, was assessed. Results: Organic remnants were present in DBBM blocks; 45.55% (±7.12) of osteocytes lacunae presented cellular remnants in blocks compared to 17.31% (±1.31) in granules. In three of five batches of blocks, it was possible to isolate bovine RNA. The highest concentration of TGF-β1 was found in supernatants of pOB + G on day 7 (218.85 ± 234.62 pg/mL) (p < 0.05), whereas pOB + B presented the lowest amount of TGF-β1 secretion at the end of evaluation (30.22 ± 14.94 pg/mL, p < 0.05). For IL-6 and OPG, there was no statistical difference between groups, while pOB + G induced more IL-8 secretion than the control (3.03 ± 3.38 ng/mL, p < 0.05). Considering the kinetics of cytokine release during the study period, all groups presented a similar pattern of cytokines, estimated as an increasing concentration for IL-6, IL-8, and OPG during cultivation. Adherent cells were observed on both material surfaces on day 7, according to H&E and OPN staining. Conclusion: Neither tested material induced a pronounced inflammatory response upon osteoblast cultivation. However, further studies are needed to elucidate the potential influence of organic remnants in bone substitute materials on the regeneration process.
Highlights
The use of bone substitute material to support bone regeneration is a common purpose in regenerative medicine and oral surgery
The purification process is essential to remove the organic remnants of the appropriate donor, which potentially carry pathogens, proteins, or foreign genetic material which can lead to disease transmission or exacerbate an inflammatory reaction [7,8]
In H&E staining, organic remnants were present inside the Haversian canals and in the margin of trabecula, indicating possible fragments of periosteum not removed by the chemical baths (Figure 1A,B)
Summary
The use of bone substitute material to support bone regeneration is a common purpose in regenerative medicine and oral surgery. There are numerous commercially available materials which are indicated for bone regeneration procedures [1,2]. They exhibit different chemical composition, physicochemical structure, and mechanical properties, and they undergo various producing or purification processes [3,4]. Regarding their origin, the bone materials can be autologous, allogeneic, xenogeneic, synthetic, or alloplastic [5,6]. Bone substitute materials should accomplish a series of requirements to be suitable for clinical use. In addition to advantageous properties such as osteoconductivity or osteoinductivity of a material, biocompatibility is one of the most important requirements [9,10,11,12]
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