Recombinant Unique Cartilage Matrix-associated Protein Potentiates Osteogenic Differentiation and Mineralization of MC3T3-E1 Cells

Objective: The relative balance of osteoblasts in bone formation and osteoclasts in bone resorption is crucial for maintaining bone health. With age, this balance between osteoblasts and osteoclasts is broken, resulting in bone loss. Anabolic drugs are continuously being developed to counteract this low bone mass. Recombinant proteins are used as biotherapeutics due to being relatively easy to produce on a large scale and are cost-effective through various expression systems. This study aimed to develop a recombinant protein that would positively impact osteoblast differentiation and mineralized nodule formation using unique cartilage matrix-associated protein (UCMA). Methods: A recombinant glutathione-S-transferase (GST)-UCMA fusion protein was generated in an E.coli system, and purified by affinity chromatography. MC3T3-E1 osteoblast cells and Osterix (Osx)-knockdown stable cells were cultured for 14 days to investigate osteoblast differentiation and nodule formation in the presence of the recombinant GST-UCMA protein. The differentiated cells were assessed by alizarin red S staining and quantitative PCR of the osteoblast differentiation marker osteocalcin. In addition, cell viability in the presence of the recombinant GST-UCMA protein was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell adhesion assay. Results: The isolation of both purified recombinant GST-only and GST-UCMA proteins were confirmed at 26 kDa and 34 kDa, respectively, by Coomassie staining and western blot analysis. Neither dose-dependent nor time-dependent presence of recombinant GSTUCMA affected MC3T3-E1 cell viability. However, MC3T3-E1 cell adhesion to the recombinant GST-UCMA protein increased dose-dependently. Osteoblast differentiation and nodule formation were promoted in both MC3T3-E1 osteoblast cells and Osxknockdown stable cells when cultured in the presence of recombinant GST-UCMA protein. Conclusion: A recombinant GST-UCMA protein induces osteogenic differentiation and mineralization, suggesting its potential use as an anabolic drug to increase low bone mass in osteoporotic patients.

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.

Treatment of Bone Marrow Cancer Based on Model Predictive Control

Bone marrow is a spongy tissue that contains stem cells that are found inside some bones, including the hip and femur. Bone marrow cancer is a type of cancer that is caused by stem cells that make up the blood cells in the bone marrow. Sometimes these cells grow too fast or abnormally, which is called bone marrow cancer. Bone tissue cells are mainly composed of osteoblasts and osteoclasts. Osteoblast cells constantly build new bone throughout the life of each bone, and other cells called osteoclasts constantly absorb pieces of bone, so the bone is constantly being renewed. In this paper, mathematical models of tumors, the effect of the body on the drug, and the drug on the body are introduced, and then the appropriate dose of the drug to reduce tumor density is calculated using the model predictive control (MPC) algorithm. To obtain an adaptive MPC strategy, the extended least squares (ELS) method developed to learn the parameters of the tumor growth model is used. Finally, the simulation in MATLAB, assuming the model is correct, shows that the tumor is gone, and the bone mass improves over a period of time. The results demonstrate that the proposed method is effective for the treatment of bone marrow cancer.

Herbal Extracts from Lycii Radicis Corex and Achyranthes japonica Prevent Multiple Myeloma

Introduction Multiple Myeloma (MM) is a plasma cell malignancy that, despite advances in treatment, remains incurable. In over 90% of MM patients, aberrant bone remodeling occurs and results in osteolytic lesions. It severely reduces the patient's quality of life and increases mortality. Korean traditional medicine has a long-standing interest in healthy foods to enhance the immune system, energy-boost, and Yin-and-Yang balance. A healthy diet in Tradition medicine is based on accumulated observations from countless cases over multi-centuries. Traditional medicine has used herbal extracts (HE) from natural plants with fewer side effects and long-term treatment tolerance. In earlier studies, lycii radices cortex (LRC) and achyranthes japonica (AJ) containing herbal extracts (HE) have demonstrated the ability to enhance cell growth and mineralization of osteoblast cells while also inhibiting osteoclast differentiation. Furthermore, a sesquiterpene glucoside, (1'R,3'S,5'R,8'S,2 Z,4 E)-dihydrophaseic acid 3'-O-β-d-glucopyranoside (DPA3G) were isolated from the LRC ethanol extract and shown to be a bioactive compound for enhancement of cell growth and bone anabolic activity (Park et al). The current study investigated the effects of LRC+AJ containing HE on murine 5TGM1 MM-bearing mice. Methods We transplanted 1x10 6 cells of the enforced luciferase-expressing 5TGM1 (5TGM1-Luc) into 8~12-week-old Nod-Scid-IL2Rg null (NSG) mice via tail vein (representing an equal number of each sex). For LRC+AJ containing HE treatment, mice were divided into three groups; i) PBS group; ii) treatment group at 300 mg/kg, gavage, TIW started two weeks before MM transplant (TbT); and iii) treatment group at 300 mg/kg, gavage, TIW two weeks after MM transplant (TaT). We assessed tumor burden by weekly bioluminescence imaging (IVIS 200 Imager, Perkin Elmer). The spine was extracted from carcass and scanned at postmortem by Dual Energy X-ray Absorptiometry using PIXImus Densitometer (G.E. Lunar, Madison, USA) and micro-computed tomography (microCT; Scanco Medical AG, Switzerland). HE effects in cell growth were tested in the myeloma cells (5TGM1 and U266) and preosteoblast cells (MC3T3-E1). The cells were grown in various concentrations (8, 80, 800 ug/ml) of HE up to 96 hours. In addition, Chromatogram and Mass Spectrometry were conducted to identify the DPA3G. Results As shown in Figure 1A, this HE significantly increased the mouse survivals of both TbT and TaT groups with median survivals of undetermined and 52.5 days, respectively, while MM control had a median survival of 42 days. The Mantel-Cox test found that TbT and TaT mice were significantly different from the control group (P=0.0014 and 0.0182, respectively). Furthermore, DEXA scans at postmortem showed a significant increase in bone mineral density (BMD) and bone mineral content (BMC) in TbT and TaT groups than control MM mice. For a histomorphometry analysis, the spines were scanned by micro-CT and revealed that TbT and TaT groups had significantly increased bone volume over total volume (BV/TV) than control. To see if HE affects cell growth of myeloma cells and osteoblast cells, we further investigated the HE on preosteoblast cell line MC3T3-E1, mouse MM cell line 5TGM1-Luc, and human MM cell line U-266. Cells were treated with HE in various concentrations, and viability was assessed at 48 and 96 hours post-treatment. Remarkably, LRC+AJ containing HE increased MC3T3-E1 cell growth while it decreased 5TGM1 and U-266 cell viability. We identified the DAP3G and many other compounds in HE used in this study. Conclusions Our results demonstrated that LRC+AJ HE prevents MM and promotes bone formation in 5TGM1 engrafted NSG mice. We also found that LRC+AJ HE suppresses myeloma cell growth and enhances osteoblast cell survival. Although it is yet to be defined, a correlation of osteoblast activity and myeloma cell inhibition suggests a potential mechanism of the HE action to prevent MM progression. 1. Park E, Kim J, Yeo S, Lim E, Choi CW, Choi S, Li WY, Lee JW, Park JH, Huh D, Jeong SY. Anti-Osteoporotic Effects of Combined Extract of Lycii Radicis Cortexand Achyranthes japonica in Osteoblast and Osteoclast Cells and Ovariectomized Mice. Nutrients. 2019 Nov 9;11(11):2716. doi: 10.3390/nu11112716. PMID: 31717518; PMCID: PMC6893723.' Figure 1 Figure 1. Disclosures Huh: Dongwoodang Pharmacy Company: Current Employment. OffLabel Disclosure: Herbal Extracts from Lycii Radicis Corex and Achyranthes Japonica

Osteogenic Activity of Lupeol Isolated from Clinacanthus nutans Lindau: Activity and Mode of Action

Clinacanthus nutans Lindau has been traditionally used for healing of bone fragility, but the mechanism of actions has not been clarified yet. In this study, the bone regeneration activity of lupeol derived from C. nutans was assessed using an in vitro model of osteoblast cells MC3T3-E1. The finding revealed that the compound was not significantly toxic to osteoblast cells at concentration of ≤40 μg/mL. Lupeol demonstrated the osteogenic activity through enhancement of alkaline phosphatase (ALP) of osteoblast cells up to 31.2%, 21%, and 12% at concentrations of 5, 10, and 20 µg/mL, respectively ( p < 0.05). Besides, the mineralization activity was increased up to 170, 230, 185, and 117% at concentration of 5, 10, 20, and 40 μg/mL, respectively ( p < 0.05). The marker genes related to osteoblast differentiation evaluated on the expression level in the presence of lupeol, including collagen I (col 1), osteopontin (opn), osterix (osx), and runx2, showed upregulated expression in all the test genes ( p < 0.05). The Western blot analysis demonstrated a clear effect of lupeol on expression of p38/p-p38, and ERK/p-ERK proteins involved in the MAPK signaling pathway. Thus, lupeol isolated from C. nutans exhibited the osteogenic activity by enhancing expression of important markers of osteogenesis, as well as affected the MAPK signaling pathway relating to osteoblast differentiation. This is the first report on the detailed mechanism of action of lupeol on bone regeneration and also explains for the traditional use of this medicinal plant for bone healing.

Glucocorticoid promotes osteoblast viability by activating autophagy via the SGK1/FOXO3a signal pathway

Background: Autophagy is important for cellular survival under numerous stimuli. Previous studies have demonstrated that endogenous glucocorticoids (GCs) could induce autophagy, contribute to bone metabolism and development under physiological condition, but the precise mechanisms involved are unknown. Methods: Mouse embryonic osteoblastic precursor cells, MC3T3-E1 cells, were treated with low dose of GCs (10-8M dexamethasone, Dex) and/or autophagy inhibitor, 3-methyladenine (3-MA) for different time periods. Gene interference with serum- and glucocorticoid induced kinase-1 (SGK1) was performed before above stimulations. After that, CCK-8 assay was performed to determine cell viability and the flow cytometry was performed to detect the apoptosis rates of the osteoblast cells. The expressions of SGK1, phosphorylated-FOXO3a (p-FOXO3a)/FOXO3 and autophagy-related proteins were detected with the western blotting. Results: A low dose (10-8M) of Dex induced a significant time-dependent increase in the expression and activation of SGK1 in MC3T3-E1 cells，accompanied by an increase in cell viability and decrease in cell apoptosis. 3-MA significantly inhibited the viability-promoting effect of Dex. Moreover, we found that Dex increased LC3II and Beclin-1 levels and decreased SQSTM/p62 levelin a time-dependent manner, which could be attenuated by pretreatment with 3-MA. Transfection of MC3T3-E1 cells exposed to Dex with shRNA-SGK1 resulted in a significant reduction in cell viability, and an increase in apoptosis. 3-MA further aggravated the effects of SGK1 inhibition on cell viability and apoptosis. Knocking down SGK1 before Dex exposure significantly downregulated p-FOXO3a/FOXO3, suppressed LC3II and Beclin-1 levels and increased SQSTM/p62 levels in MC3T3-E1 cells, which could be amplifies by 3-MA. Conclusions: A low dose of GCs increased osteoblast viability by activating autophagy via SGK1/FOXO3a pathway.

Surface Properties and Biocompatibility of Anodized Titanium with a Potential Pretreatment for Biomedical Applications

The effects of anodized titanium (Ti) with a potential hydrogen fluoride (HF) acid pretreatment through cathodization on the formation of nano-porous Ti dioxide (TiO2) layer were characterized using field-emission scanning electron microscopy, grazing incidence X-ray diffractometer, and contact angle goniometer. The biocompatibility was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test. Analytical results found that a well-aligned nano-porous structure was formed on the anodized Ti surface with HF pretreatment concentration above 0.5%. Microstructure of the nano-porous Ti dioxide surface generated by anodization with HF pretreatment was composed of anatase and rutile phases, while the anodized Ti sample with HF pretreatment concentration of 0.5% presented excellent hydrophilicity surface. An in-vitro biocompatibility also indicated that osteoblast cells grown on the surface of the anodized Ti sample with HF pretreatment increased with the increase of culture time. The filopodia of osteoblast cells not only adhered flat, but also tightly grabbed the nano-porous structure for promoting cell adhesion and proliferation. Therefore, the anodized Ti with HF pretreatment can form a functionalized surface with great biocompatibility for biomedical applications, particularly for dental implants.

Bioactivity of Ti6Al4V alloy with bioglass and corrosion protection by silane coating

The Ti6Al4V alloy is usually employed as a biomaterial, however, when in use, exhibits a few drawbacks such as corrosion, caused by the release of aluminum and vanadium ions besides the bioinert behavior. Bioactive coatings offer a barrier effect and bioactivity, promoting biocompatibility and osseointegration processes. The present work aims to study the biocompatibility behavior of a bioglass-containing silane film deposited on a titanium alloy (Ti6Al4V) substrate. The effect of the surface roughness of the metallic substrate was also evaluated. Film/substrate systems were characterized as their morphological, chemical, physical, electrochemical behavior, and cell cytotoxicity and cell viability. The main results pointed out that silane films augment corrosion resistance of titanium alloy substrates. The biological results indicated a growth of osteoblast cells (MG-63), for all the test conditions. The bioglass film deposited on the ground substrate exhibits the highest cell density.