Current immunological issues in bone grafting regarding the transfer of xenogeneic donor bone cells into the recipient are challenging the industry to produce safer acellular natural matrices for bone regeneration. The aim of this study was to investigate the efficacy of a novel decellularization technique for producing bovine cancellous bone scaffold and compare its physicochemical, mechanical, and biological characteristics with demineralized cancellous bone scaffold in an in-vitro study. Cancellous bone blocks were harvested from a bovine femoral head (18–24 months old) subjected to physical cleansing and chemical defatting, and further processed in two ways. Group I was subjected to demineralization, while Group II underwent decellularization through physical, chemical, and enzymatic treatments. Both were then freeze-dried, and gamma radiated, finally producing a demineralized bovine cancellous bone (DMB) scaffold and decellularized bovine cancellous bone (DCC) scaffold. Both DMB and DCC scaffolds were subjected to histological evaluation, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), fourier-transform infrared spectroscopy (FTIR), quantification of lipid, collagen, and residual nucleic acid content, and mechanical testing. The osteogenic potential was investigated through the recellularization of scaffolds with human osteoblast cell seeding and examined for cell attachment, proliferation, and mineralization by Alizarin staining and gene expression. DCC produced a complete acellular extracellular matrix (ECM) with the absence of nucleic acid content, wider pores with extensive interconnectivity and partially retaining collagen fibrils. DCC demonstrated a higher cell proliferation rate, upregulation of osteogenic differentiation markers, and substantial mineralized nodules production. Our findings suggest that the decellularization technique produced an acellular DCC scaffold with minimal damage to ECM and possesses osteogenic potential through the mechanisms of osteoconduction, osteoinduction, and osteogenesis in-vitro.

Introduction: Alveolar bone is critical in supporting natural teeth, dental implants as well as a removable and fixed prosthesis. Alveolar bone volume diminishes when its associated natural tooth is lost. Objective: The aim of this study is to evaluate the effectiveness of bovine bone granules on alveolar bone socket augmentation for ridge preservation following atraumatic tooth extraction. Materials and Methods: Twenty medically fit patients (12 males and 8 females aged between 18 and 40 years) who needed noncomplicated tooth extraction of 1 mandibular premolar tooth were divided randomly and equally into 2 groups. In control group I, the empty extraction socket was left untreated and allowed to heal in a conventional way. In group II, the empty extraction socket wound was filled with lyophilized bovine bone xenograft granules 0.25 to 1 mm of size, 1 mL/vial. A resorbable pericardium membrane was placed to cover the defect. Clinical and 3-dimensional radiological assessments were performed at day 0, 3 months, and 9 months postoperative. Results: There were no clinical differences in general wound healing between the groups. Comparisons within the groups showed a significant difference of bone resorption of 1.49 mm (95% confidence interval, 0.63–2.35) at 3 months, and further resorption of 1.84 mm (P ≤ 0.05) at 9 months in the control group. No significant changes of bone resorption were observed in group II during the same time interval. Comparison between groups showed a significant difference of bone resorption at 3 and 9 months (2.40 and 2.88 mm, respectively). Conclusion: The use of lyophilized demineralized bovine bone granules in socket preservation to fill in the extraction socket seems essential in preserving the alveolar bone dimension as it showed excellent soft and hard tissue healing. This study concludes that the alveolar bone socket exhibited a dynamic process of resorption from the first day of tooth extraction. Evidence shows the possibility of using bovine bone granules routinely in socket volume preservation techniques following tooth extraction.

The aim of this study was to determine the efficacy of immediate implant placement with alveolar bone augmentation on socket preservation following atraumatic tooth extraction and comparing it with a tooth alveolar socket that was allowed to heal in a conventional way.Twenty medically fit patients (8 males and 12 females aged between 18 and 40 years) who needed noncomplicated tooth extraction of mandibular premolar teeth were divided randomly and equally into 2 groups. In Group I, the empty extraction socket was left untreated and allowed to heal in a conventional way. In Group II, the immediate implant was placed and the gap between the implant and the inner buccal plate surface of the socket wall was filled with lyophilized bovine bone granules and the wound was covered with pericardium membrane. The patients were followed up clinically and radiologically for regular reviews at 1 week, 3 months, and 9 months postoperative. Cone beam computerized tomography images of the alveolar ridge and socket were analyzed to determine the structural changes of the alveolar ridge. Resonance frequency analysis was measured at 9 months for Group II to assess the degree of secondary stability of the implants by using Osstell machine.A significant difference of bone resorption of 1.49 mm (confidence interval, CI 95%, 0.63-2.35) was observed within the control group at 3 months, and 1.84 mm (P ≤ 0.05) at 9 months intervals. No significant changes of bone resorption were observed in Group II. Comparison between groups showed a highly significant difference at 3 months; 2.56 mm (CI 95% 4.22-0.90) and at 9 months intervals; 3.2 mm (CI 95%, 4.70-1.62) P ≤ 0.001 between Group I and II. High resonance frequency analysis values were observed at 9 months postoperative in Group II.In conclusion, the insertion of immediate implants in fresh extraction sockets together with grafting the circumferential gap between the bony socket wall and the implant surface with bovine bone granules was able to preserve a greater amount of alveolar ridge volume when compared with an extraction socket that was left to heal in a conventional way.

The performance of apparently biocompatible implanted bovine bone grafts may be compromised by unresolved chronic inflammation, and poor graft incorporation leading to implant failure. Monitoring the intensity and duration of the inflammatory response caused by implanted bone grafts is crucial. In this study, the ability of demineralized (DMB) and decellularized (DCC) bovine bone substitutes in initiating inflammatory responses to peripheral blood monocyte-derived macrophages (PBMMs) was investigated. The response of PBMMs to bone substitutes was evaluated by using both direct and indirect cell culture, reactive oxygen species (ROS) generation, apoptosis, immunophenotyping, and cytokine production. Analysis of DMB and DCC substitutes using scanning electron microscope (SEM) showed a roughened surface with a size ranging between 500 and 750 μm. PBMMs treated with DMB demonstrated cell aggregation and clumping mimicking lipopolysaccharide (LPS) treated PBMMs and a higher proliferation ability (166.93%) compared to control (100%) and DCC treatments (115.64%; p<0.001) at 24h. This was associated with a significantly increased production of intracellular ROS in PBMMs exposed to DMB substitutes than control (3158.5 vs 1715.5; p<0.001) and DCC treatment (2117.5). The bone substitute exposure also caused an increase in percentage apoptosis which was significantly (p<0.0001) higher in both DMB (27.85) and DCC (29.2) treatment than control (19.383). A significant increase in proinflammatory cytokine expression (TNF-α: 3.4 folds; p<0.05) was observed in DMB substitute-treated PBMMs compared to control. Notably, IL-1β mRNA was significantly higher in DMB (21.75 folds; p<0.0001) than control and DCC (5.01 folds). In contrast, DCC substitutes exhibited immunoregulatory effects on PBMMs, as indicated by the expression for CD86, CD206, and HLDR surface markers mimicking IL-4 treatments. In conclusion, DMB excites a higher immunological response compared to DCC suggesting decellularization process of tissues dampen down inflammatory reactions when exposed to PBMM.