Metabolic switching and cell wall remodelling of Mycobacterium tuberculosis during bone tuberculosis

Bone tuberculosis is widely characterized by irreversible bone destruction caused by Mycobacterium tuberculosis . Mycobacterium has the ability to adapt to various environmental stresses by altering its transcriptome in order to establish infection in the host. Thus, it is of critical importance to understand the transcriptional profile of M. tuberculosis during infection in the bone environment compared to axenic cultures of exponentially growing M.tb. In the current study, we characterized the in vivo transcriptome of M. tuberculosis within abscesses or necrotic specimens obtained from patients with bone TB using whole genome microarrays in order to gain insight into the M. tuberculosis adaptive response within this host microenvironment. A total of 914 mycobacterial genes were found to be significantly over-expressed and 1688 were repressed (fold change>2; p-value ≤ 0.05) in human bone TB specimens. Overall, the mycobacteria displayed a hypometabolic state with significant (p ≤ 0.05) downregulation of major pathways involved in translational machinery, cellular and protein metabolism and response to hypoxia. However, significant enrichment (p ≤ 0.05) of amino-sugar metabolic processes, membrane glycolipid biosynthesis, amino acid biosynthesis (serine, glycine, arginine and cysteine) and accumulation of mycolyl-arabinogalactan-peptidoglycan complex suggests possible mycobacterial survival strategies within the bone lesions by strengthening its cell wall and cellular integrity. Data were also screened for M.tb virulence proteins using Virulent-Pred and VICM-Pred tools, which revealed five genes (Rv1046c, Rv1230c, DppD, PE_PGRS26 and PE_PGRS43) with a possible role in the pathogenesis of bone TB. Next, an osteoblast cell line model for bone TB was developed allowing for significant intracellular multiplication of M.tb. Interestingly, three virulence genes (Rv1046c, DppD and PE_PGRS26) identified from human bone TB microarray data were also found to be overexpressed by intracellular M. tuberculosis in osteoblast cell lines. Overall, these data demonstrate that M. tuberculosis alters its transcriptome as an adaptive strategy to survive in the host and establish infection in bone. Additionally, the in vitro osteoblast model we describe may facilitate our understanding of the pathogenesis of bone TB.

The Effects of Polyphenol, Tannic Acid, or Tannic Acid in Combination with Pamidronate on Human Osteoblast Cell Line Metabolism

Background: This study investigates the effect of tannic acid (TA) combined with pamidronate (PAM) on a human osteoblast cell line. Methods: EC50 for TA, PAM, and different combination ratios of TA and PAM (25:75, 50:50, 75:25) were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The combination index value was utilized to analyze the degree of drug interaction, while trypan blue assay was applied to analyze the cells proliferation effect. The mineralization and detection of bone BSP and Osx genes were determined via histochemical staining and PCR test, respectively. Results: The EC50 of osteoblasts treated with TA and a 75:25 ratio of TA and PAM were more potent with lower EC50 at 0.56 µg/mL and 0.48 µg/mL, respectively. The combination of TA and PAM (75:25) was shown to have synergistic interaction. On Day 7, both TA and PAM groups showed significantly increased proliferation compared with control and combination groups. On Day 7, both the TA and combination-treated groups demonstrated a higher production of calcium deposits than the control and PAM-treated groups. Moreover, on Day 7, the combination-treated group showed a significantly higher expression of BSP and Osx genes than both the TA and PAM groups. Conclusion: Combination treatment of TA and PAM at 75:25 ameliorated the highest enhancement of osteoblast proliferation and mineralization as well as caused a high expression of BSP and Osx genes.

PEEK as a Potential Material for Dental Implants and its Biomechanical Properties and Osteoblast Cell Response

Proper osseointegration is crucial for the success of dental and orthopedic implants. Titanium-6Aluminum-4Vanadium (TAV) is one of the most popular implant materials; however, polyetheretherketone (PEEK) has gained the interest of implant researchers and manufacturers over the past several years due to its lower modulus of elasticity compared to metallic implant materials. Porosity and patterned surface morphologies are thought to improve mechanical interlocking and play an important role in the differentiation of pre-osteoblasts into mature osteoblasts. This study aimed to determine the effects a macro patterned PEEK surface has on the material’s mechanical properties and the proliferation, differentiation, and maturation of pre-osteoblasts. Mechanical testing data indicated that the macro patterning improved the mechanical interlocking and has no detrimental effect on compression strength. DNA data and live/dead imaging showed that pre-osteoblasts on solid PEEK specimens did not readily differentiate but instead encouraged proliferation only. However, ALP data in comparison to the DNA data showed that cells on patterned PEEK specimens more readily entered the differentiation pathway to mineralization. This is further confirmed by the patterned PEEK specimens showing an overall higher amount of cell mineralization. Clinical significance: This study concludes that surface macro patterning of PEEK material increases the mechanical interlocking and enhances the osseointegration capability without diminishing mechanical properties.

Bone Formation Ability and Cell Viability Enhancement of MC3T3-E1 Cells by Ferrostatin-1 a Ferroptosis Inhibitor of Cancer Cells

Recently, ferroptosis has gained scientists’ attention as an iron-related regulated necrosis. However, not many reports have investigated the effect of ferroptosis on bone. Therefore, with the present study, we assessed the effect of ferroptosis inhibition using ferrostatin-1 on the MC3T3-E1 pre-osteoblast cell. Cell images, cell viability, alkaline phosphatase activity test, alizarin red staining, and RUNX2 gene expression using real-time PCR were applied to investigate the effects of ferrostatin and erastin on MC3T3-E1 osteoblast cells. Erastin was used as a well-known ferroptosis inducer reagent. Erastin with different concentrations ranging from 0 to 50 µmol/L was used for inducing cell death. The 25 µmol/L erastin led to controllable partial cell death on osteoblast cells. Ferrostatin-1 with 0 to 40 µmol/L was used for cell doping and cell death inhibition effect. Ferrostatin-1 also displayed a recovery effect on the samples, which had already received the partially artificial cell death by erastin. Cell differentiation, alizarin red staining, and RUNX2 gene expression confirmed the promotion of the bone formation ability effect of ferrostatin-1 on osteoblast cells. The objective of this study was to assess ferrostatin-1’s effect on the MC3T3-E1 osteoblast cell line based on its ferroptosis inhibitory property.

The Effects of Titanium Topography and Chemical Composition on Human Osteoblast Cell

The objective of this study was to evaluate and compare titanium surfaces: machined (MA); sintered ceramic-blasted (HAS); sintered ceramic-blasted and acid-etched (HAS DE) and to determine the effects of surface topography, roughness and chemical composition on human osteoblast cell reaction. Titanium surface samples were analyzed with respect to surface chemical composition, topography, and roughness. The effects of material surface characteristics on osteoblasts was examined by analyzing osteoblast morphology, viability and differentiation. Osteoblasts cultured on these materials had attached, spread and proliferated on every sample. The viability of osteoblasts cultured on HAS and HAS DE samples increased more intensively in time comparing to MA sample. The viability of osteoblast cultured on HAS samples increased more intensively in the early phases of culture while for cells cultured on HAS DE the cells viability increased later in time. Alkaline phosphate activity was the highest for the cells cultured on HAS sample and statistically higher than for the MA sample. The least activity occurred on the smooth MA sample along with the rougher HAS DE samples. All the examined samples were found to be biocompatible, as indicated by cell attachment, proliferation, and differentiation. Titanium surfaces modification improved the dynamics of osteoblast viability increase. Osteoblast differentiation was found to be affected by the etching procedure and presence of Ca and P on the surface.