Silver diamine fluoride (SDF) is commonly used to arrest caries lesions, especially in early childhood caries. Recently, it was suggested that SDF can be combined with potassium iodide (KI) to minimize the discoloration of demineralized dentine associated with SDF application. However, the antibacterial efficacy of SDF alone or combined with KI on in-situ biofilm is unknown. Hence, we compared the anti-plaque biofilm efficacy of two different commercially available SDF solutions, with or without KI, using an in-situ biofilm, analysed using viability real-time PCR with propidium monoazide (PMA). Appliance-borne in-situ biofilm samples (n = 90) were grown for a period of 6 h in five healthy subjects who repeated the experiment on three separate occasions, using a validated, novel, intraoral device. The relative anti-biofilm efficacy of two SDF formulations; 38.0% Topamine (SDFT) and 31.3%, Riva Star (SDFR), KI alone, and KI in combination with SDFR (SDFR+KI) was compared. The experiments were performed by applying an optimized volume of the agents onto the biofilm for 1min, mimicking the standard clinical procedure. Afterwards the viability of the residual biofilm bacteria was quantified using viability real-time PCR with PMA, then the percentage of viable from total bacteria was calculated. Both SDF formulations (SDFT and SDFR) exhibited potent antibacterial activities against the in-situ biofilm; however, there was non-significant difference in their efficacy. KI alone did not demonstrate any antibacterial effect, and there was non-significant difference in the antibacterial efficacy of SDF alone compared to SDF with KI, (SDFT v SDFR/KI). Thus, we conclude that the antibacterial efficacy of SDF against plaque biofilms is not modulated by KI supplements. Viability real-time PCR with PMA was successfully used to analyze the viability of naturally grown oral biofilm; thus, the same method can be used to test the antimicrobial effect of other agents on oral biofilms in future research.
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.
The aim of this study was to compare the ability of demineralized (DMB) and decellularized (DCC) bovine bone granules to support bone regeneration in rat calvaria critical-size defects. DMB and DCC were prepared using a previously published method. The granule size used ranged between 500 and 750 μm. A total of forty-eight Sprague-Dawley rats were divided into two groups (n = 24). A pair of 5 mm diameter defects were created on the calvaria of the rats in the right and left parietal bone in both groups. Group A animals received DMB granules and Group B received DCC granules in the right parietal defect side while the left parietal untreated defect acted as sham surgery for both groups. Four animals per group were euthanized in a CO2 chamber at day 7, 14 and 21 post-surgery and the calvaria implantation site biopsy harvested was subjected to osteogenic gene expression analysis. Another four animals per group were euthanized at days 15, 30 and 60 post surgery and the calvaria implantation site biopsy harvested was subjected to histological, immunohistochemistry, RAMAN spectroscopy and Micro-CT analysis at the mentioned time points. Statistical analysis was conducted using t-tests and ANOVA. Histomorphometry showed significantly higher new bone formation in the DCC sites (p<0.05) compared to DMB. Both DMB and DCC implantation sites showed distinct staining for osteocalcin and osteopontin proteins compared to their respective sham sites. By day 21 after implantation, DCC sites demonstrated significantly elevated mRNA levels of osteonectin (p<0.001), osteopontin (p<0.001), osteocalcin (p<0.0001), ALP (p<0.01), and BMP-2 (p<0.001) compared to DMB. However, VEGF expression showed no significant differences at this time point between the two groups. Micro-CT analysis also showed enhanced defect closure and higher bone density in DCC implanted sites while RAMAN spectra demonstrated increased abundance of collagen and bone minerals, especially, PO43- ions than DMB. In conclusion, both DMB and DCC granules demonstrated favorable osteogenic potential in critical-sized defects, with DCC exhibited superior osteoconductive, osteoinductive and osteogenesis properties.
Hindlimb suspension (HLS) mice exhibit osteoporosis of the hindlimb bones and may be an excellent model to test pharmacological interventions. We investigated the effects of inhibiting endoplasmic reticulum (ER) stress with 4-phenyl butyrate (4-PBA) on the morphology, physicochemical properties, and bone turnover markers of hindlimbs in HLS mice. We randomly divided 21 male C57BL/6J mice into three groups, ground-based controls, untreated HLS group and 4-PBA treated group (HLS+4PBA) (100mg/kg/day, intraperitoneal) for 21 days. We investigated histopathology, micro-CT imaging, Raman spectroscopic analysis, and gene expression. Untreated HLS mice exhibited reduced osteocyte density, multinucleated osteoclast-like cells, adipocyte infiltration, and reduced trabecular striations on micro-CT than the control group. Raman spectroscopy revealed higher levels of ER stress, hydroxyproline, non-collagenous proteins, phenylalanine, tyrosine, and CH2Wag as well as a reduction in proteoglycans and adenine. Furthermore, bone alkaline phosphatase and osteocalcin were downregulated, while Cathepsin K, TRAP, and sclerostin were upregulated. Treatment with 4-PBA partially restored normal bone histology, increased collagen crosslinking, and mineralization, promoted anti-inflammatory markers, and downregulated bone resorption markers. Our findings suggest that mitigating ER stress with 4-PBA could be a therapeutic intervention to offset osteoporosis in conditions mimicking hindlimb suspension.
Increased oxidative stress in bone cells is known to negatively alter favorable bone regeneration. This study aimed to develop a porous polycaprolactone (PCL) membrane incorporated with 25 wt % Vitamin C (PCL-Vit C) and compared it to the PCL membrane to control oxidative stress and enhance biomineralization in vitro. Both membranes were characterized using SEM-EDS, FTIR spectroscopy, and surface hydrophilicity. Vitamin C release was quantified colorimetrically. Assessments of the viability and attachment of human fetal osteoblast (hFOB 1.19) cells were carried out using XTT assay, SEM, and confocal microscopy, respectively. ROS generation and wound healing percentage were measured using flow cytometry and ImageJ software, respectively. Mineralization study using Alizarin Red in the presence or absence of osteogenic media was carried out to measure the calcium content. Alkaline phosphatase assay and gene expression of osteogenic markers (alkaline phosphatase (ALP), collagen Type I (Col1), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN)) were analyzed by real-time PCR. SEM images revealed smooth, fine, bead-free fibers in both membranes. The FTIR spectrum of pure vitamin C was replaced with peaks at 3436.05 and 2322.83 cm-1 in the PCL-Vit C membrane. Vitamin C release was detected at 15 min and 1 h. The PCL-Vit C membrane was hydrophilic, generated lower ROS, and showed significantly higher viability than the PCL membrane. Although both PCL and PCL-Vit C membranes showed similar cellular and cytoskeletal morphology, more cell clusters were evident in the PCL-Vit C membrane. Lower ROS level in the PCL-Vit C membrane displayed improved cell functionality as evidenced by enhanced cellular differentiation with more intense alizarin staining and higher calcium content, supported by upregulation of osteogenic markers ALP, Col1, and OPN even in the absence of osteogenic supplements. The presence of Vitamin C in the PCL-Vit C membrane may have mitigated oxidative stress in hFOB 1.19 cells, resulting in enhanced biomineralization facilitating bone regeneration.