Human Osteoblastic Cells Research Articles

Calcium silicate-based cements cause environmental stiffness and show diverse potential to induce osteogenesis in human osteoblastic cells

Calcium silicate-based cements differ markedly in their radiopacifiers and the presence of calcium sulfate, aluminates, carbonates and other components that can affect their biological properties. This study aimed to compare the biological properties of six calcium silicate cements in human osteoblastic cell culture (Saos-2 cells): Bio-C Repair (Bio-C), PBS HP (PBS-HP), Biodentine (Biodentine), MTA Repair HP (MTA-HP), NeoMTA Plus (NeoMTA-P), and ProRoot MTA (ProRoot). After exposure to these materials, the cells were analyzed by MTT, wound healing, cell migration, and alkaline phosphatase activity (ALP) assays, real-time PCR (qPCR) analysis of the osteogenesis markers (osteocalcin or bone gamma-carboxyglutamate protein, BGLAP; alkaline phosphatase, ALPL; bone sialoprotein or secreted phosphoprotein 1, BNSP), and alizarin red staining (ARS). Curiously, the migration rates were low 24–48 h after exposure to the materials, despite the cells showing ideal rates of viability. The advanced and intermediate cell differentiation markers BGLAP and BNSP were overexpressed in the Bio-C, MTA-HP, and ProRoot groups. Only the Biodentine group showed ALPL overexpression, a marker of initial differentiation. However, the enzymatic activity was high in all groups except Biodentine. The mineralization area was significantly large in the NeoMTA-P, ProRoot, PBS-HP, MTA-HP, and Bio-C groups. The results showed that cellular environmental stiffness, which impairs cell mobility and diverse patterns of osteogenesis marker expression, is a consequence of cement exposure. Environmental stiffness indicates chemical and physical stimuli in the microenvironment; for instance, the release of cement compounds contributes to calcium phosphate matrix formation with diverse stiffnesses, which could be essential or detrimental for the migration and differentiation of osteoblastic cells. Cells exposed to Bio-C, PBS-HP, ProRoot, NeoMTA-P, and MTA-HP seemed to enter the advanced or intermediate differentiation phases early, which is indicative of the diverse potential of cements to induce osteogenesis. Cements that quickly stimulate osteoblast differentiation may be ideal for reparative and regenerative purposes since they promptly lead to dentin or bone deposition.

Characterization of porous fluorohydroxyapatite bone-scaffolds fabricated using freeze casting

With the increasing demand for orthopedic and dental reconstruction surgeries, there comes a shortage of viable bone substitutes. This study was therefore designed to assess the efficacy of porous fluorohydroxyapatite (FHA) as a potential bone substitute. For this, porous FHA scaffolds were fabricated using the freeze casting technique. They were then sintered at 1250, 1350 and, 1450°C, and microstructural, mechanical, and in vitro properties were analyzed. The microstructure analyses revealed the porosity remained constant within the temperature range. However, the pore size decreased with increasing sintering temperature. The greatest compressive strength and elastic modulus were obtained at 1450°C, which were 13.5±4.0MPa and 379±182MPa, respectively. These are comparable values to human trabecular bone and other porous scaffolds made using hydroxyapatite. This analysis has thus helped to attain an understanding of the mechanical and material properties of freeze-cast FHA scaffolds that have not been presented before. In vitro studies revealed an increasing rate of human osteoblast cell proliferation on freeze-cast FHA scaffolds with increasing sintering temperature, suggesting improved osteogenic properties. Additionally, osteoblasts cells were also shown to proliferate into the interior pores of all freeze-cast FHA scaffolds. These results indicate the potential of porous FHA scaffolds fabricated using the freeze-casting technique to be utilized clinically as bone substitutes.

Glycogen synthase kinase 3β promotes osteosarcoma invasion and migration via regulating PTEN and phosphorylation of focal adhesion kinase

Aim: Typical features of human osteosarcoma are highly invasive and migratory capacities. Our study aimed to investigate the roles of glycogen synthase kinase 3β (GSK3β) in human osteosarcoma metastasis.Methods: GSK3β expressions in clinical osteosarcoma tissues with or without metastasis were examined by immunohistochemical staining. The expressions of GSK3β, p-GSK3βSer9, and p-GSK3βTyr216 in human osteoblast cells (hFOB1.19) and human osteosarcoma cells (MG63, SaOS-2, and U2-OS) were detected by Western blotting. The GSK3β activity was measured by non-radio isotopic in vitro kinase assay. Migration and invasion abilities of MG-63 cells treated with small-molecular GSK3β inhibitors were respectively examined by monolayer-based wound-healing assay and transwell assay. The mRNA expressions of GSK3β, matrix metalloproteinase-2 (MMP-2), MMP-9, phosphatase with tensin homology (PTEN), and focal adhesion kinase (FAK) were detected after siRNA transfection for 72 h. Meanwhile, protein expressions of GSK3β, FAK, p-FAKY397, PTEN, MMP-2, and MMP-9 were measured by Western blotting.Results: Clinical osteosarcoma tissues with metastasis showed higher GSK3β expressions. MG63 and U2-OS cells that were easy to occur metastasis showed significantly higher expressions and activities of GSK3β than SaOS-2 cells. Inhibition of GSK3β with small-molecular GSK3β inhibitors in MG63 cells significantly attenuated cell migration and invasion. These effects were associated with reduced expressions of MMP-2 and MMP-9. Moreover, increased PTEN and decreased p-FAKY397 expressions were observed following GSK3β knockdown by siRNA transfection. Conclusion: GSK3β might promote osteosarcoma invasion and migration via pathways associated with PTEN and phosphorylation of FAK.

Upregulation of 15 Antisense Long Non-Coding RNAs in Osteosarcoma.

The human genome encodes thousands of natural antisense long noncoding RNAs (lncRNAs); they play the essential role in regulation of gene expression at multiple levels, including replication, transcription and translation. Dysregulation of antisense lncRNAs plays indispensable roles in numerous biological progress, such as tumour progression, metastasis and resistance to therapeutic agents. To date, there have been several studies analysing antisense lncRNAs expression profiles in cancer, but not enough to highlight the complexity of the disease. In this study, we investigated the expression patterns of antisense lncRNAs from osteosarcoma and healthy bone samples (24 tumour-16 bone samples) using RNA sequencing. We identified 15 antisense lncRNAs (RUSC1-AS1, TBX2-AS1, PTOV1-AS1, UBE2D3-AS1, ERCC8-AS1, ZMIZ1-AS1, RNF144A-AS1, RDH10-AS1, TRG-AS1, GSN-AS1, HMGA2-AS1, ZNF528-AS1, OTUD6B-AS1, COX10-AS1 and SLC16A1-AS1) that were upregulated in tumour samples compared to bone sample controls. Further, we performed real-time polymerase chain reaction (RT-qPCR) to validate the expressions of the antisense lncRNAs in 8 different osteosarcoma cell lines (SaOS-2, G-292, HOS, U2-OS, 143B, SJSA-1, MG-63, and MNNG/HOS) compared to hFOB (human osteoblast cell line). These differentially expressed IncRNAs can be considered biomarkers and potential therapeutic targets for osteosarcoma.

3D printing of PCL/nano-hydroxyapatite scaffolds derived from biogenic sources for bone tissue engineering

Bioactive composites made of ∽85 wt% poly(ε-caprolactone) (PCL) and ∽15 wt% nanometric hydroxyapatite (HA) produced from biogenic sources were 3D printed by an extrusion-based process to obtain porous scaffolds suitable for bone regeneration. Three different composite formulations were considered by using HA synthesized from three distinct natural sources, which were collected as food wastes: cuttlefish bones, mussel shells and chicken eggshells. Composition and thermal properties of the materials were analysed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and x-ray spectroscopy (XRD), while the morphological and mechanical properties of the 3D scaffolds were studied by means of electron microscopy (SEM) and compression tests. Bioactivity was tested by seeding human osteoblast cell line (MG63) onto the scaffolds which were analysed by confocal microscopy and Alamar Blue and PicoGreen® tests after 1 to 7 culture days. The elastic modulus (177–316 MPa) is found to be within the range reported for typical trabecular bones being increased by the presence of the bio-HA particles. Moreover, cells adhesion, viability and proliferation are largely promoted in the scaffolds containing nanometric HA with respect to pure PCL, the best results being revealed when mussel shell-derived HA is used. Indeed, different biological sources result in different cell proliferation rates, pointing that the biological origin has an impact on the cells-scaffold interaction. In general, the results show that PCL/bio-HA scaffolds possess improved mechanical properties and enhanced bioactivity when compared with pure PCL ones.

Silencing of LINC00707 suppresses cell proliferation, migration, and invasion of osteosarcoma cells by modulating miR-338-3p/AHSA1 axis.

Osteosarcoma is the most common type of primary malignant tumor of the bone, with a high metastatic rate and poor prognosis. Therefore, it is important to further elucidate the molecular mechanisms involved in the development of osteosarcoma and explore new molecular therapeutic targets. Long intergenic nonprotein-coding RNA 707 (LINC00707) is an oncogenic gene in several cancers. In this study, we further clarified its role and regulatory mechanism in osteosarcoma. We found that LINC00707 levels are significantly higher in the osteosarcoma cell lines SW 1353, HOS, U-2 OS, MG-63, and Saos-2 compared to those in human fetal osteoblastic cell line hFOB1.19. LINC00707 silencing suppressed cell proliferation, migration, and invasion of MG-63 and Saos-2 cells. Moreover, LINC00707 can act as a competitive endogenous RNA of miR-338-3p, and miR-338-3p inhibitor and AHSA1 overexpression alleviated the effect of LINC00707 silencing. In conclusion, we demonstrated high expression of LINC00707 in osteosarcoma cell lines and that silencing LINC00707 suppresses cell proliferation, migration, and invasion by targeting the miR-338-3p/AHSA1 axis in MG-63 and Saos-2 cells. These findings suggest that LINC00707 may serve as a potential target for osteosarcoma treatment.

Association of RDM1 with osteosarcoma progression via cell cycle and MEK/ERK signalling pathway regulation.

RAD52 motif‐containing 1 (RDM1), a key regulator of DNA double‐strand break repair and recombination, has been reported to play an important role in the development of various human cancers, such as papillary thyroid carcinoma, neuroblastoma and lung cancer. However, the effect of RDM1 on osteosarcoma (OS) progression remains unclear. Here, this study mainly explored the connection between RDM1 and OS progression, as well as the underlying mechanism. It was found that RDM1 was highly expressed in OS cells compared with human osteoblast cells. Knockdown of RDM1 caused OS cell proliferation inhibition, cell apoptosis promotion and cell cycle arrest at G1 stage, whereas RDM1 overexpression resulted in the opposite phenotypes. Furthermore, RDM1 silencing leads to a significant decrease in tumour growth in xenograft mouse model. RDM1 also increased the protein levels of MEK 1/2 and ERK 1/2. All these findings suggest that RDM1 plays an oncogenic role in OS via stimulating cell cycle transition from G1 to S stage, and regulating MEK/ERK signalling pathway, providing a promising therapeutic factor for the treatment of OS.

Improved Biological Responses of Titanium Coating Using Laser-Aided Direct Metal Fabrication on SUS316L Stainless Steel.

Direct metal fabrication (DMF) coatings have the advantage of a more uniform porous structure and superior mechanical properties compared to coatings provided by other methods. We applied pure titanium metal powders to SUS316L stainless steel using laser-aided DMF coating technology with 3D printing. The purpose of this study was to determine the efficacy of this surface modification of stainless steel. The capacity of cells to adhere to DMF-coated SUS316L stainless steel was compared with machined SUS316L stainless steel in vitro and in vivo. Morphological in vitro response to human osteoblast cell lines was evaluated using scanning electron microscopy. Separate specimens were inserted into the medulla of distal femurs of rabbits for in vivo study. The distal femurs were harvested after 3 months, and were then subjected to push-out test and histomorphometrical analyses. The DMF group exhibited a distinct surface chemical composition, showing higher peaks of titanium compared to the machined stainless steel. The surface of the DMF group had a more distinct porous structure, which showed more extensive coverage with lamellipodia from osteoblasts than the machined surface. In the in vivo test, the DMF group showed better results than the machined group in the push-out test (3.39 vs. 1.35 MPa, respectively, p = 0.001). In the histomorphometric analyses, the mean bone-to-implant contact percentage of the DMF group was about 1.5 times greater than that of the machined group (65.4 ± 7.1% vs. 41.9 ± 5.6%, respectively; p < 0.001). The porous titanium coating on SUS316L stainless steel produced using DMF with 3D printing showed better surface characteristics and biomechanical properties than the machined SUS316L.

Delivery of Bioactive Gene Particles via Gelatin-Collagen-PEG-Based Electrospun Matrices.

The fabrication of fiber mats via electrospinning has been adopted in the last decades to produce high quality scaffolds for tissue engineering. However, an effective combination of electrospinning methods with gene delivery therapies remains a challenge. In this study, we describe how the delivery of gene complexes via electrospun mats that contain different volumes of gelatin (Gel), collagen (Col), and polyethylene glycol (PEG) can affect gene expression by transfected cells. Non-viral complexes were formulated by using lipid modified polyethylenimine (PEI) polymer and plasmid DNAs (pDNA) like the reporter Green Fluorescent Protein (GFP) and the therapeutically relevant Bone Morphogenetic Protein-2 (BMP-2) and electrospuned after being mixed with different volumes of Gel-Col-PEG mats and delivered to human myoblast (C2C12) and mouse osteoblast cells (MC3T3). The entrapment of GFP complexes via different homogeneous electrospun fiber mats revealed that a high fraction of collagen in the mats affected the quality of the fibers and led to reduced transfection efficiency on target cells. On the other hand, the fabrication of double-layered mats that contained collagen without complexes as a first layer and gelatin-collagen-PEG with complexes as a second layer successfully induced GFP expression and ALP activity in C2C12 cells. We conclude that this study has established the advantage of formulating multilayered bioactive collagen-based mats for gene delivery applications.

NPR3, transcriptionally regulated by POU2F1, inhibits osteosarcoma cell growth through blocking the PI3K/AKT pathway

Natriuretic peptide receptor 3 (NPR3), mediates natriuretic peptides degradation, was reported to act as a tumor suppressor or promoter in some types of cancer. Previous studies showed that NPR3 was significantly decreased in osteosarcoma (OS) samples. However, the function and potential regulatory mechanism of NPR3 in OS development are unknown. By analyzing the protein expression of NPR3 in OS cell lines (n = 5) and human osteoblast cell line hFOB 1.19, we found that NPR3 expression was also significantly decreased in OS cells. The loss/gain-of-function analysis indicated that NPR3 overexpression observably decreased OS cell viability, arrested cell cycle, and induced apoptosis. However, NPR3 knockdown further enhanced the malignant phenotype of OS cells. Furthermore, NPR3 downregulation activated the PI3K/AKT pathway in OS cells, and the effects of NPR3 silencing on cell proliferation were reversed by the blockade of PI3K/AKT pathway. Of note, dual-luciferase reported assay and site-directed mutagenesis assay indicated that transcription factor POU domain class 2 transcription factor 1 (POU2F1) was proved to suppress NPR3 promoter activity by mainly binding to the −900 to −800 bp region of NPR3 promoter. Moreover, NPR3 overexpression inversed the promotion effect of POU2F1 on cell proliferation. In vivo experiments confirmed that NPR3 overexpression suppressed the growth of xenograft tumors. Taken together, the present study demonstrates that NPR3 may serve as a novel tumor suppressive factor through blocking the PI3K/AKT pathway and transcriptionally regulated by POU2F1.

Abstract 1350: Regulation of pH by growth factors is dependent on the expression of the calcineurin homologous protein-2 in human bone sarcoma cells

Abstract Bone sarcoma is a malignant tumor that includes multiple subtypes. The osteosarcoma is the sixth most common malignancy in adolescents, while chondrosarcoma is primarily a disease of adults. Current treatments for both osteosarcoma and chondrosarcoma have not demonstrated improvement in patients' survival over the past three decades. Extracellular acidosis affects osteoblast and chondrocyte function by reducing bone mineralization and increasing production of cartilage matrix, respectively. Thereby, it weakens bone matrix and expands cartilage matrix, and may play a role in the development of bone characteristics seen in patients with osteosarcoma or chondrosarcoma. Development of extracellular acidosis in cancer is due to dysregulation of pH dynamics mediated by constitutive activation of Na+/H+ exchanger-1 (NHE1) activity, the main transporter responsible for pH regulation. Current therapeutic agents that target inhibition of NHE1 activity are ineffective due to broad inhibitory effects. The calcineurin homologous protein (CHP) family binds to and regulates NHE1 activity. CHP isoform 1 (CHP1) is expressed in non-cancerous cells and forms a dynamic complex with NHE1. On the other hand, CHP isoform 2 (CHP2) is expressed primarily in cancerous cells and has a stronger binding affinity to NHE1 compared with CHP1. We hypothesize that CHP2 binding to NHE1 in cancer cells induces the over-activity of NHE1, making the extracellular pH of cancer cells more acidic than non-cancerous cells. We measured CHP1 or CHP2 protein expression levels in human osteoblast (hFOB), osteosarcoma (143B), and chondrosarcoma (SW1353) cells and studied whether the levels of CHP2 protein expression affected NHE activity in these cell lines. We determined that: 1. hFOB, 143B, and SW1353 cells expressed mainly NHE1; NHE activity measured using spectrofluorometry was completely blocked by a highly selective NHE1 inhibitor (zoniporide, 10-8 M); 2. NHE1 activity was increased by serum-nutrient deprivation, used to mimic a cancerous microenvironment and NHE1 over-activity was associated with increased levels of CHP2 protein expression; 3. silencing of CHP2 by shRNA inhibited NHE1 activity in response to serum-nutrient deprivation in 143B or SW1353 cells and restored the NHE1 response inducted by serum-deprivation in non-cancerous cells. Regulation of NHE1 through CHP2 is proposed as a promising novel target in sarcoma treatment and to overcome the wide range of side effects induced by inhibitors of NHE1 activity. Citation Format: Tiffany Chang, Serena S. Luong, Adam P. Zobel, Elitsa Ananieva, Victor Babich, Francesca Di Sole. Regulation of pH by growth factors is dependent on the expression of the calcineurin homologous protein-2 in human bone sarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1350.

Influence of surface termination of ultrananocrystalline diamond films coated on titanium on response of human osteoblast cells: A proteome study

Successful osseointegration, i.e. the fully functional connection of patient's bone and artificial implant depends on the response of the cells to the direct contact with the surface of the implant. The surface properties of the implant which trigger cell responses leading to its integration into the surrounding bone can be tailored by surface modifications or coating with thin layers. One potential material for such applications is ultrananocrystalline diamond (UNCD). It combines the exceptional mechanical properties of diamond with good biocompatibility and possibility of coating as thin uniform films on different substrates of biological interest. In the current work we firstly deposited UNCD films on titanium-coated substrates and applied oxygen or ammonia plasma to modify their surface properties. The as-grown and modified UNCD exhibited relatively smooth surfaces with topography dominated by rounded features. The modifications induced oxygen- or amino-terminated surfaces with increased hydrophilicity. In addition, the UNCD coatings exhibited very low coefficient of friction when diamond was used as a counterpart. As-grown and modified UNCD samples were applied to study the responses of human osteoblast MG63 cells triggered by surfaces with various terminations assessed by proteomic analysis. The results revealed that the coating of Ti with UNCD as well as the plasma modifications resulting in O- or NH2-terminated UNCD induced upregulation of proteins specific for cytoskeleton, cell membrane, and extracellular matrix (ECM) involved in the cell-ECM-surface interactions. Proteins from each of these groups, namely, vimentin, cadherin and fibronectin were further studied immunocytochemically and the results confirmed their increased abundance leading to improved cell-to-surface adhesion and cell-to-cell interactions. These findings demonstrate the potential of implant coating with UNCD and its surface modifications for better osseointegration and bone formation.

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.

Protective Effects of Collagen Tripeptides in Human Aortic Endothelial Cells by Restoring ROS-Induced Transcriptional Repression.

Collagen tripeptide (CTP) is defined as a functional food material derived from collagenase digests of type I collagen and contains a high concentration of tripeptides with a Gly-X-Y sequence. CTP has several biological effects, including the acceleration of fracture healing, ameliorating osteoarthritis, and improving dryness and photoaging of the skin. Recently, an antiatherosclerotic effect of CTP has been reported, although its molecular mechanism is yet to be determined. In this study, we examined the effects of CTP on primary cultured human aortic endothelial cells (HAECs) under oxidative stress, because oxidative endothelial dysfunction is a trigger of atherosclerosis. DNA microarray and RT-qPCR analyses showed that CTP treatment recovered the downregulated expression of several genes, including the interleukin-3 receptor subunit alpha (IL3RA), which were suppressed by reactive oxygen species (ROS) treatment in HAECs. Furthermore, IL3RA knockdown significantly decreased the viability of HAECs compared with control cells. RT-qPCR analysis also showed that solute carrier 15 family peptide transporters, which are involved in CTP absorption into cells, were expressed in HAECs at levels more than comparable to those of a CTP-responsive human osteoblastic cell line. These results indicated that CTP exerts a protective effect for HAECs, at least in part, by regulating the recovery of ROS-induced transcriptional repression.

Preparation, characterization and biological activities of egg white peptides-calcium chelate

The optimal conditions for preparation of egg white peptides-calcium chelate (EWPs-Ca) were determined by response surface methodology (RSM). Under the optimal condition (mass ratio of peptide: calcium of 4:1 at 53 °C and pH 8.2 for 30 min), the calcium content of EWPs-Ca was 44.1 mg/kg. The results of Ultraviolet–Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that the carboxyl oxygen and amino nitrogen atoms of the egg white peptides (EWPs) may chelate with calcium during the chelation. Amino acid analysis suggested that glutamate, aspartic acid, cysteine, threonine, glycine and lysine played important roles during the chelation. The scanning electron microscopy showed that due to EWPs combined with calcium, the structure of EWPs-Ca became loose porous sponge. Moreover, the amount of transferred calcium of EWPs-Ca was 46.18 ± 2.12, 63.86 ± 0.36, 106.91 ± 0.44, 123.61 ± 1.59 μg/mL at 30, 60, 120, 180 min in Caco-2 cell monolayers, respectively. Compared with the blank control, the proliferation rate of Human fetal osteoblast (HFOB) cells reached the maximum value of 119.55 ± 1.36%, 110.20 ± 1.56% and 112.58 ± 1.62% at 24, 48 and 72 h, respectively and EWPs-Ca also improved the activity of alkaline phosphatase (ALP).

Nobiletin promotes osteogenic differentiation of human osteoblastic cell line (MG-63) through activating the BMP-2/RUNX-2 signaling pathway.

Nobiletin (NOB) is polymethoxy flavonoids, which plentifully there in Citrus depressa and they demonstrate numerous pharmacological effects. NOB has an anti-proliferative effect, attenuates ovalbumin-treated eosinophilic airway inflammation and Type II collagen treated arthritis. NOB noticeably inhibits bone resorption and renovates bone loss in mice model, but role of NOB in bone metabolism is unclear. Human bone is a important organ that sustains its homeostasis among bone resorpting osteoclasts and bone developing osteoblasts. The balances of among these two kind of cell outcomes are implicated in bone remodeling. The current study designed to explore possessions of NOB on differentiation and proliferation of MG-63 cells and contribution of morphogenetic protein signaling. Cell proliferation was analyzed by MTT, mineralization analysis by alizarin red staining and morphogenetic signaling protein by RT-PCR. No stimulus outcome of NOB on cell proliferation was found at days of 1, 3 and 7. Accumulation of calcium was augmented after that treatment of NOB. The mRNA expression of BMP-2, COL-I, ALP, OCN, RUNX2 and COL1A1 augmented markedly with NOB supplement. Hence, NOB can stimulate osteogenic differentiation of MG-63, almost certainly by promoting RUNX2 and BMP-2 signaling and this result might provide to its action on stimulation of osteoblast development, differentiation and augments of bone mass.

Preliminary evaluation of the in vitro biocompatibility of magnetic bone cement composites

Polymethyl methacrylate (PMMA) bone cement with the addition of magnetic glass-ceramic (MGC) has attracted much attention for the treatment of deep-seated tumour tissues. Since PMMA bone cement has been a gold standard in orthopaedic surgery and magnetic glass-ceramic can generate heat under a magnetic field, the combination of these two materials could show promising results for magnetic induction hyperthermia treatment.Thus, magnetic glass-ceramic powders were mixed with the commercial Palacos MV® cement in amounts up to 40 ​wt%. The resulted magnetic bone cement composites were assessed in vitro for bioactivity and cytocompatibility. In vitro bioactivity was evaluated using a simulated body fluid (SBF). The effect of magnetic cement composition on the viability of cancerous and normal (non-cancerous) cells was assessed using U2OS osteosarcoma and OBS human osteoblast cells, respectively. Cell attachment to the surface of the magnetic composite samples was observed using fluorescence and scanning electron microscopy.The magnetic composites showed bioactive properties. After two weeks of immersion in SBF, a non-uniform layer of hydroxyapatite was observed on their surface. Alamar blue cytotoxicity tests after 3 days in cell culture indicated that magnetic composites are cytotoxic for U2OS cancer cells, but they are cytocompatible to OBS normal cells.

Integrin α2β1 as a negative regulator of the laminin receptors α6β1 and α6β4

Integrin α2β1 is a widely expressed collagen I receptor which also mediates laminin-111 binding in some cell types, but the functional relevance of collagen versus laminin binding for different cell types is poorly understood. Here we use AFM-based singe-cell force spectroscopy (SCFS) to compare α2β1-mediated adhesion strength to collagen and laminin in different cell types. Chinese Hamster Ovary (CHO) cells stably expressing integrin α2β1 (CHO-A2) displayed enhanced adhesion to collagen, but weak adhesion to laminin, consistent with a role of α2β1 as a receptor only for collagen in these cells. Inversely, the α2β1-deficient CHO wildtype cells (CHO-WT) showed weak adhesion to collagen, but strong adhesion to laminin-111, in turn suggesting that integrin α2β1 expression suppresses laminin binding. Analogous results were obtained in a pair of SAOS-2 human osteosarcoma cell lines. Again, wildtype cells (SAOS-WT) adhered strongly to laminin and poorly to collagen, while expression of integrin α2β1 (SAOS-A2) induced strong adhesion to collagen, but reduced adhesion to laminin. Expression of α2β1 also shifted cell spreading preference from laminin to collagen and suppressed laminin-dependent transmigration. In agreement with reduced laminin adhesion, α2β1 expression downregulated transcription and expression of integrin subunits α6 and β4, components of the main laminin-111 binding receptors integrin α6β1 and α6β4 in these cells. Integrin α6 and β4 expression was also reduced when α2 expression was chemically induced using tetradecanoyl-phorbol-acetate (TPA). Our results thus show that integrin α2β1 expression negatively regulates integrin α6β1 and α6β4-mediated adhesion, spreading and invasion on laminin in different cancer cell types. In contrast to SAOS-WT, but similar to SAOS-A2 osteosarcoma cells, primary Human osteoblasts (HOB) cells express α2 but only low levels of β4 integrin, preferentially adhere to and spread on collagen over laminin and show suppressed laminin-dependent transmigration. By enhancing collagen binding directly and suppressing laminin binding indirectly through laminin receptor downregulation, α2β1 expression may thus re-direct migrating cancer cells from laminin-rich to collagenous tissues and partially revert osteosarcoma cells towards an untransformed phenotype.

Osteo-mucosal engineered construct: In situ adhesion of hard-soft tissues

ObjectivesThe aim of this work was to combine engineered hard and soft tissue, adopting a new method for interfacial adhesion of osteo-mucosal construct. We hypothesized that the chemical procedure involved in this method not only adheres the components, but also improves the cell growth inside them. Methods3D-printed functionally-graded porous hard-tissue scaffolds were characterized, functionalized by aminolysis and tyrosinase, and accommodated by human osteoblast cells. Introducing amino groups through aminolysis and inducing dopaquinones by tyrosinase can take part in the Michael additions to cause the adhesion. Subsequently, fully-differentiated engineered oral mucosa was formed directly on the surface of hard tissue. Constructs were assessed in term of morphology, structure, chemical composition, histology, and cytocompatibility. Interfacial adhesion was compared to a control group prepared by using a biological glue for the attachment of the soft and hard tissues. ResultsThe data confirmed higher proliferation of osteoblast cells via aminolysis and improved osteoblast cells distribution and differentiation by incorporation of tyrosinase in collagen. There was evidence of multilayered, stratified epithelium on the osteo-mucosal model with viable fibroblasts and osteoblasts within the lamina propria and bone tissue layers. Our method of adhesion resulted in cohesive debonding within the engineered soft tissue; while in the control group, adhesive debonding and complete separation of the oral mucosa from the hard tissue was observed. Although the shear strength of the osteo-mucosal model (157.6 kDa ± 25.1) was slightly higher than that of the control group (149.4 kDa ± 23.1), there was no statistically significant difference between them (p > 0.05). However, the advantage of our in situ adhesion approach is the absence of a barrier like glue which can disrupt direct cellular communications between tissues. SignificanceThis study provides a novel method of directly combining tissue-engineered human bone with oral mucosa, which has the potential to improve cell-ingrowth and tissue integration. This engineered tissue construct, after further optimization, can be used clinically as a graft material in various oral surgeries and can also be employed as an in vitro model to investigate many aspects of oral diseases and examine dental materials and oral health care products as a replacement of in vivo models.

Bio-Functionalized Chitosan for Bone Tissue Engineering.

Hybrid biomaterials allow for the improvement of the biological properties of materials and have been successfully used for implantology in medical applications. The covalent and selective functionalization of materials with bioactive peptides provides favorable results in tissue engineering by supporting cell attachment to the biomaterial through biochemical cues and interaction with membrane receptors. Since the functionalization with bioactive peptides may alter the chemical and physical properties of the biomaterials, in this study we characterized the biological responses of differently functionalized chitosan analogs. Chitosan analogs were produced through the reaction of GRGDSPK (RGD) or FRHRNRKGY (HVP) sequences, both carrying an aldehyde-terminal group, to chitosan. The bio-functionalized polysaccharides, pure or “diluted” with chitosan, were chemically characterized in depth and evaluated for their antimicrobial activities and biocompatibility toward human primary osteoblast cells. The results obtained indicate that the bio-functionalization of chitosan increases human-osteoblast adhesion (p < 0.005) and proliferation (p < 0.005) as compared with chitosan. Overall, the 1:1 mixture of HVP functionalized-chitosan:chitosan is the best compromise between preserving the antibacterial properties of the material and supporting osteoblast differentiation and calcium deposition (p < 0.005 vs. RGD). In conclusion, our results reported that a selected concentration of HVP supported the biomimetic potential of functionalized chitosan better than RGD and preserved the antibacterial properties of chitosan.