The administration of bovine hydroxyapatite-alendronate implant accelerates bone defect healing in an osteoporotic rat.

BACKGROUND: Bone fracture is the main consequence of osteoporosis, which may become a neglected disease. OBJECTIVE: This study aims to fabricate bovine hydroxyapatite-gelatine (BHA-GEL) based bone-implant with alendronate (ALE) in vivo. METHODS: Wistar rats were used for an osteoporotic animal model induced by ovariectomy. There were three groups: negative control, BHA-GEL implant, and BHA-GEL-ALE implant. Each group performed a defect by drilling the femur (diameter of 2.2 mm and depth of 2 mm). Observations on the closure of bone defects were performed by X-ray radiography at the second and sixth week after surgery. The mechanism of bone healing was observed by using hematoxylin-eosin (HE) staining and immunohistochemical technique with anti-vascular endothelial growth factor (VEGF) and anti-alkaline phosphatase (ALP) antibodies. RESULTS: The radiograph examination showed the implanted group had accelerated bone growth. In addition, the osteoblast, osteoclast and osteocyte had accelerated migration to the defect area. Moreover, the immunoreactive score (IRS) of VEGF at the sixth week in the BHA-GEL-ALE group was lower than the other groups. Meanwhile, the IRS of ALP in BHA-GEL-ALE was higher compared to other groups. CONCLUSION: The BHA-GEL-ALE implant accelerates the healing of bone defect in the osteoporotic rat by increasing the ALP expression and the total number of cells.

Computational approach in searching for dual action multitarget inhibitors for osteosarcoma.

Osteosarcoma is a common primary malignant bone tumor that typically manifests in the second decade of life. This study aimed to identify osteogenic compounds that potentially serve as multitarget inhibitors for osteosarcoma. The study was a molecular docking study of nine Food and Drug Administration-approved compounds with osteogenic properties to the key membrane proteins of osteosarcoma. The ligands used were raloxifene, simvastatin, dexamethasone, risedronate, ibandronate, zoledronic acid, ascorbic acid, alendronate, and ß-glycerophosphate, whereas the target proteins used were RET, fibroblast growth factor receptor 1, KIT, PDGFRA, VEGFR1, and VEGFR2. Chem3D version 15.0.0.106 was used for ligand preparation, and AutoDockTools version 1.5.6 was used for protein preparation, whereas molecular docking was conducted using AutoDock Vina. Raloxifene, simvastatin, and dexamethasone had the lowest binding activity to the target proteins. The binding affinity of raloxifene was from -8.4 to -10.0 kcal mol-1, that of simvastatin was -8.3 to -9.2 kcal mol-1, whereas dexamethasone ranged from -6.9 to -9.1 kcal mol-1. Most types of interactions were hydrophobically followed by hydrogen bonding. The current study suggests that raloxifene, simvastatin, and dexamethasone have the potential to act as multitarget inhibitors for osteosarcoma with the ability to induce bone remodeling.

Bovine hydroxyapatite-based scaffold accelerated the inflammatory phase and bone growth in rats with bone defect.

Hydroxyapatite (HA) is a biomaterial widely used to treat bone defect, such as due to traffic accident. The HA scaffold is obtained from synthetic HA or natural sources, such as bovine hydroxyapatite (BHA). This study aims to compare the characteristics and in vivo performance of BHA-based and HA-based scaffolds. For this purpose, the scaffold was formulated with gelatin (GEL) and characterised by SEM-EDX, FTIR and mini autograph. The defect model was carried out on the femur area of Wistar rats classified into three animal groups: defect, HA-GEL and BHA-GEL. Postoperatively (7, 14 and 28 days), the bone was radiologically evaluated, and stained with haematoxylin-eosin, anti-CD80 and anti-CD163. The BHA-GEL scaffold showed a regular surface and spherical particle shape, whereas the HA-GEL scaffold exhibited irregular surface. The BHA-GEL scaffold had higher pore size and compressive strength and lower calcium-to-phosphorus ratio than the HA-GEL scaffold. In vivo study showed that the expression of CD80 in the three experimental groups was not significantly different. However, the expression of CD163 differed significantly between the groups. The BHA-GEL group showed robust expression of CD163 on day 7, which rapidly decreased over time. It also showed increased osteoclasts, osteoblasts and osteocytes cell count that contributed to the integrity of the defect area. In conclusion, the BHA-based scaffold exhibited the desired physical and chemical characteristics that benefit in vivo performance versus the HA-based scaffold. Thus, the BHA-based scaffold may be used as a bone graft.

In vivo study of bovine hydroxyapatite-gelatin-hydroxypropyl methylcellulose with alendronate as injectable bone substitute composite in osteoporotic animal model.

The injectable bone substitute (IBS) is a self-setting local drug delivery system that adjusts the shape of the bone gap in the fracture. This study aimed to examine the effectiveness of IBS composites of bovine hydroxyapatite (BHA) and alendronate (Ale) in accelerating bone growth in osteoporotic rats. IBS was made by mixing BHA with gelatin 5%, hydroxypropyl methylcellulose (HPMC) 2%, and Ale 10%. The physical properties of IBS were viscosity, injectability, and density tests. Twenty-four female Wistar rats were divided into four groups. After 8 weeks, 2 mm gap was made in the right femur of all rats and filled with IBS. The healing process was observed after 6 weeks with X-ray imaging and H and E staining. The obtained results showed viscosity, injectability, and density value of IBS from 30.4 to 39.4 dPa.s, 98.22%-98.64%, and 0.6325-0.8409 g/cm3, respectively. X-ray imaging and histology results proved the condition of osteoporosis in rats with ovariectomy. The addition of BHA-Gel-HPMC-Ale significantly affected the number of osteoblasts, osteocytes, and osteoclasts (P < 0.05). After 45 days of observation, the addition of BHA-Gel-HPMC-Ale showed the highest mean number of osteoblasts, osteocytes, and osteoclasts, which were 25.00 ± 3.00, 64.33 ± 11.15, and 5.67 ± 0.58 compared to BHA-Gel-HPMC and positive control groups. The BHA-Gel-HPMC-Ale IBS has the potential to reverse osteoporosis. Nevertheless, the underlying potential of these biomaterials to reverse osteoporosis needs further research.

Acceleration of Bone Fracture Healing through the Use of Bovine Hydroxyapatite or Calcium Lactate Oral and Implant Bovine Hydroxyapatite-Gelatin on Bone Defect Animal Model.

Bone grafts a commonly used therapeutic technique for the reconstruction and facilitation of bone regeneration due to fractures. BHA-GEL (bovine hydroxyapatite-gelatin) pellet implants have been shown to be able accelerate the process of bone repair by looking at the percentage of new bone, and the contact between the composite and bone. Based on these results, a study was conducted by placing BHA-GEL (9:1) pellet implants in rabbit femoral bone defects, accompanied by 500 mg oral supplement of BHA or calcium lactate to determine the effectiveness of addition supplements. The research model used was a burr hole defect model with a diameter of 4.2 mm in the cortical part of the rabbit femur. On the 7th, 14th and 28th days after treatment, a total of 48 New Zealand rabbits were divided into four groups, namely defect (control), implant, implant + oral BHA, and implant + oral calcium lactate. Animal tests were terminated and evaluated based on X-ray radiology results, Hematoxylin-Eosin staining, vascular endothelial growth Factor (VEGF), osteocalcin, and enzyme-linked immunosorbent assay (ELISA) for bone alkaline phosphatase (BALP) and calcium levels. From this research can be concluded that Oral BHA supplementation with BHA-GEL pellet implants showed faster healing of bone defects compared to oral calcium lactate with BHA-GEL pellet implants.

Nigella sativa toothpaste promotes anti-inflammatory and anti-destructive effects in a rat model of periodontitis.

OBJECTIVE: Periodontitis is an infectious disease that results in gingiva tissue damage. This study aimed to evaluate the effects of Nigella sativa (N. sativa) toothpaste in a periodontitis tissue repair based on inflammation and periodontal extracellular matrix in vivo. DESIGN: The periodontitis disease model was developed using Wistar rats infected with Porphyromonas gingivalis (P. gingivalis). The rats were divided into three main groups as follows: those that did not receive any toothpaste treatment; those that were treated with N. sativa toothpaste twice a day (simultaneously with P. gingivalis induction); and normal healthy rats. The rats were sacrificed after 1 and 7 days of animal modeling. The number of inflammatory cells, matrix metalloproteinase (MMP)1 + and MMP8 + cells, levels of cytokines (interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2)) and density of collagen type 1 were determined in the gingival tissues of the rats. RESULTS: The rats treated with N. sativa toothpaste had significantly lower numbers of neutrophils, macrophages and lymphocytes than the non-treated rats after 1 and 7 days of treatment; likewise, the levels of IL-1ß and PGE2 were lower in the treated experimental rats. In addition, the group treated with N. sativa toothpaste had fewer numbers of MMP1 + and MMP8 + cells and higher collagen density after 1 and 7 days of administration. CONCLUSIONS: N. sativa toothpaste exhibited anti-inflammatory effects by reducing both inflammatory cell count and activity. Additionally, N. sativa toothpaste demonstrated anti-destructive effects on the periodontal extracellular matrix. Thus, N. sativa toothpaste might be potentially used for the management of periodontitis.

Fabrication and Characterization of Submicron-Scale Bovine Hydroxyapatite: A Top-Down Approach for a Natural Biomaterial.

Submicron hydroxyapatite has been reported to have beneficial effects in bone tissue engineering. This study aimed to fabricate submicron-scale bovine hydroxyapatite (BHA) using the high-energy dry ball milling method. Bovine cortical bone was pretreated and calcined to produce BHA powder scaled in microns. BHA was used to fabricate submicron BHA with milling treatment for 3, 6, and 9 h and was characterized by using dynamic light scattering, scanning electron microscope connected with energy dispersive X-Ray spectroscopy, Fourier-transform infrared spectroscopy, and X-ray diffractometry to obtain its particle size, calcium-to-phosphorus (Ca/P) ratio, functional chemical group, and XRD peaks and crystallinity. Results showed that the particle size of BHA had a wide distribution range, with peaks from ~5 to ~10 µm. Milling treatment for 3, 6, and 9 h successfully gradually reduced the particle size of BHA to a submicron scale. The milled BHA's hydrodynamic size was significantly smaller compared to unmilled BHA. Milling treatment reduced the crystallinity of BHA. However, the treatment did not affect other characteristics; unmilled and milled BHA was shaped hexagonally, had carbonate and phosphate substitution groups, and the Ca/P ratio ranged from 1.48 to 1.68. In conclusion, the fabrication of submicron-scale BHA was successfully conducted using a high-energy dry ball milling method. The milling treatment did not affect the natural characteristics of BHA. Thus, the submicron-scale BHA may be potentially useful as a biomaterial for bone grafts.

Signaling Pathway and Transcriptional Regulation in Osteoblasts during Bone Healing: Direct Involvement of Hydroxyapatite as a Biomaterial.

Bone defects and periodontal disease are pathological conditions that may become neglected diseases if not treated properly. Hydroxyapatite (HA), along with tricalcium phosphate and bioglass ceramic, is a biomaterial widely applied to orthopedic and dental uses. The in vivo performance of HA is determined by the interaction between HA particles with bone cells, particularly the bone mineralizing cells osteoblasts. It has been reported that HA-induced osteoblastic differentiation by increasing the expression of osteogenic transcription factors. However, the pathway involved and the events that occur in the cell membrane have not been well understood and remain controversial. Advances in gene editing and the discovery of pharmacologic inhibitors assist researchers to better understand osteoblastic differentiation. This review summarizes the involvement of extracellular signal-regulated kinase (ERK), p38, Wnt, and bone morphogenetic protein 2 (BMP2) in osteoblastic cellular regulation induced by HA. These advances enhance the current understanding of the molecular mechanism of HA as a biomaterial. Moreover, they provide a better strategy for the design of HA to be utilized in bone engineering.

Predicting the molecular mechanism of glucosamine in accelerating bone defect repair by stimulating osteogenic proteins.

OBJECTIVES: Bone defect is serious condition that is usually caused by traffic accident. Chitosan is a polymer developed as a scaffold to treat bone defect. However, the mechanism by which chitosan can accelerate bone growth in defect area is still unclear. This study aims to identify proteins which are crucial to the osteogenic properties of chitosan monomer using an in silico study. METHODS: Molecular docking was carried out on chitosan monomer, which are d-glucosamine and glucosamine 6-phosphate units against bone morphogenetic protein 2 (BMP-2), fibronectin, fibroblast growth factor (Fgf), and phosphate transporter (PiT) using AutoDock Vina. Ligand preparation was carried out using Chem3D version 15.0.0.106, while protein preparation was performed using AutoDockTools version 1.5.6. RESULTS: The results showed that glucosamine 6-phosphate had the best binding affinity with fibronectin and PiT, which was -5.7 kcal mol-1 on both proteins, while d-glucosamine had the best binding affinity with PiT (-5.2 kcal mol-1). CONCLUSIONS: This study suggests that the osteogenic properties of chitosan may be due to the presence of bonds between glucosamine units and fibronectin and/or PiT. However, in vitro studies need to be done to prove this.

Molecular docking studies of Nigella sativa L and Curcuma xanthorrhiza Roxb secondary metabolites against histamine N-methyltransferase with their ADMET prediction.

OBJECTIVES: Histamine N-methyltransferase (HNMT) is an enzyme that plays a crucial role in the inactivation of histamine in central nervous system, kidneys and bronchi. Inhibition of HNMT is known to have a potential role in treating attention-deficit hyperactivity disorder, memory impairment, mental illness and neurodegenerative illnesses. Therefore, to find potential compounds that could be developed as novel HNMT inhibitors, this study conducted an in silico study of the secondary metabolites of Nigella sativa L and Curcuma xanthorrhiza Roxb. METHODS: In this study, we conducted a molecular docking study of 36 secondary metabolites of N. sativa L and 26 secondary metabolites of C. xanthorrhiza Roxb using an in silico approach targeting HNMT protein (PDB ID: 2AOT) using AutoDockVina software. The prediction of ADMET characteristics was done using the pkCSM Online Tool. RESULTS: This study obtained one metabolite from N. sativa L (longifolene) and seven metabolites from C. xanthorrhiza Roxb {(+)-beta-atlantone, humulene epoxide, (-)-beta-curcumene, (E)-caryophyllene, germacrone, (R)-(-)-xanthorrhizol, and (-)-beta-caryophyllene epoxide} which were predicted to have potential to be developed as HNMT inhibitors. CONCLUSIONS: This study found several secondary metabolites of N. sativa L and C. xanthorrhiza Roxb which had activity as HNMT inhibitors. This research can likewise be utilized as a basis for further research, both in vitro, in vivo, and clinical trials related to the development of secondary metabolites from N. sativa L and C. xanthorrhiza Roxb as novel HNMT inhibitor compounds.

The impact of glutaraldehyde on the characteristics of bovine hydroxyapatite-gelatin based bone scaffold as gentamicin delivery system.

OBJECTIVES: Biomaterials are widely used as drug delivery systems targeting bone tissue, such as to treat bone infectious disease. However, the addition of drugs to biomaterials weakens their mechanical properties. Crosslinkers are compounds that improve the mechanical properties of biomaterials. This study aims to determine the effect of glutaraldehyde (GTA) as a crosslinker on the characteristics of bovine hydroxyapatite-gelatin-based bone scaffold with gentamicin as antibiotics (BHA-GEL-GEN-GTA). METHODS: BHA-GEL-GEN-GTA scaffold with GTA solid content ranging from 0.1 to 1.4 wt% was made by direct compression. The compressive strength test was carried out using autograph. Scaffold degradation test was carried out by dissolving the scaffolds in PBS. Scaffold toxicity was performed by MTT assay using BHK-21 fibroblast cells. RESULTS: There was a significant difference in the scaffolds' compressive strength due to differences in GTA volume. Scaffold crosslinked using GTA with solid content 0.1 and 0.2 wt% in 2 mL solution had higher compressive strength than those in 1 mL solution. Furthermore, GTA with solid content 0.6, 1, 1.2, and 1.4 wt% showed higher compressive strength than those without GTA. Degradation test results showed that GTA increased the percentage of weight loss and swelling of the scaffold. The scaffold exhibited a nontoxic profile in MTT assay. CONCLUSIONS: GTA with optimum solid content shows great compressive strength, stable swelling profile with low percentage of scaffold's weight loss, and is considered as nontoxic.

Structure-based virtual screening of bioactive compounds from Indonesian medical plants against severe acute respiratory syndrome coronavirus-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a virus that causes the infectious disease coronavirus disease-2019. Currently, there is no effective drug for the prevention and treatment of this virus. This study aimed to identify secondary metabolites that potentially inhibit the key proteins of SARS-CoV-2. This was an in silico molecular docking study of several secondary metabolites of Indonesian herbal plant compounds and other metabolites with antiviral testing history. Virtual screening using AutoDock Vina of 216 Lipinski rule-compliant plant metabolites was performed on 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), and spike glycoprotein. Ligand preparation was performed using JChem and Schrödinger's software, and virtual protein elucidation was performed using AutoDockTools version 1.5.6. Virtual screening identified several RdRp, spike, and 3CLpro inhibitors. Justicidin D had binding affinities of -8.7, -8.1, and -7.6 kcal mol-1 on RdRp, 3CLpro, and spike, respectively. 10-methoxycamptothecin had binding affinities of -8.5 and -8.2 kcal mol-1 on RdRp and spike, respectively. Inoxanthone had binding affinities of -8.3 and -8.1 kcal mol-1 on RdRp and spike, respectively, while binding affinities of caribine were -9.0 and -7.5 mol-1 on 3CLpro and spike, respectively. Secondary metabolites of compounds from several plants were identified as potential agents for SARS-CoV-2 therapy.

Comparison of Bacterial Community Structure and Diversity in Traditional Gold Mining Waste Disposal Site and Rice Field by Using a Metabarcoding Approach.

Traditional small-scale gold mining mostly use mercury to extract the gold from ores. However, mercury contamination in the environment can affect the composition and structure of the bacterial community. The purpose of this study was to determine the effect of mercury contamination on the bacterial community in the traditional gold mining waste disposal site and in the rice field. Mercury analysis was carried out using the CVAFS method. Analysis of bacterial communities and structure was carried out based on the results of metabarcoding of the V3-V4 16S rRNA regions obtained from paired-end Illumina MiSeq reads. The results showed that the sample from the mining waste disposal site had a mercury level of 230 mg/kg, while the sample from the rice field had 3.98 mg/kg. The results showed that there were differences in microbial composition and community structure in both locations. With the total reads of 57,031, the most dominant phylum was Firmicutes in the mining disposal site sample. Meanwhile, with the total reads of 33,080, the sample from rice field was dominated by Planctomycetes. The abundant classes of bacteria in the mining waste disposal site, from the highest were Bacilli, Gammaproteobacteria and Planctomycetia, while the sample from the rice field was dominated by the Planctomycetia and Acidobacteria subdivision 6. The families that dominated the sample in disposal site were Bacillaceae and Aeromonadaceae, while the sample from the rice field was dominated by Gemmataceae. The abundant genera in both locations were Bacillus and Gemmata. This study concluded that the high level of mercury in the soil reduced the richness and diversity of bacterial phyla and lower taxa. There was also a shift in the dominance of phyla and lower taxa in both locations. This study provides an understanding of the microbial community structure in the area that is highly contaminated with mercury to open insight into the potential of these bacteria for mercury bioremediation.

The change of proinflammatory cytokine tumor necrosis factor α level in the use of meloxicam in rat model of osteoarthritis.

Background Osteoarthritis (OA) is a chronic disease in the joints. One of the proinflammatory cytokines that is thought to have a major role in the inflammatory process, the emergence of pain, and cartilage damage in OA is tumor necrosis factor α (TNF-α). Meloxicam is a nonsteroidal anti-inflammatory drug class of drugs that is relatively selective in inhibiting the activity of cyclooxygenase 2 (COX-2) formation. This study is conducted to prove the change in TNF-α level in the use of meloxicam with model in animals suffering from OA. Methods The OA rat model was induced with sodium monoiodoacetate intra-articularly. Rats were divided into 5 groups: negative control group, positive control group, and treatment groups with various doses of meloxicam. Hyperalgesia effect was evaluated using a warm plate test, and TNF-α level was determined using enzyme-linked immunosorbent assay. Results The treatment groups that received meloxicam at a dose of 1.0, 3.0, or 10.0 mg/kg body weight (BW) did not show significant differences in rat knee joint diameter (p = 0.99), but showed a significant difference in sensitivity to heat stimulation (p = 0.02) compared to the control group. Osteoarthritis rats experienced a significant reduction in TNF-α level after being given meloxicam at a dose of 10 mg/kg BW compared with the control group. This shows that the 10 mg/kg BW of meloxicam is a potential dose in reducing the TNF-α level in OA rat models. Conclusions Based on these data, it can be concluded that the inhibition of pain and the development of OA by meloxicam in animal models may be assigned to a decreased level of TNF-α.