Rat Bone Marrow Research Articles

1H-NMR-Based Chemometric Analysis of Echinacea Species to Predict Effectors of Myeloid Progenitor Stimulation

Echinacea, a herbaceous, perennial flowering plant from the Compositae (Asteraceae) family, exhibits stimulating effects on myeloid progenitors (CFU-GMs) in rat bone marrow, as demonstrated in our previous study using a 75% (v v−1) ethanol extract of aerial parts. Expanding on this work, we have investigated eleven different Echinacea samples that belong to three species for their myeloid progenitor-stimulating activity. Simultaneously, we employed 1H-NMR spectroscopy (400 MHz, 0.02–10.02 ppm) and chemometric analysis to predict constituents responsible for activity. Female Sprague–Dawley rats received oral doses of ethanol extracts (0–200 mg extract dry weight kg−1 body weight) of Echinacea for seven days. Bone marrow cells were then cultured with CFU-GM growth factors. Extracts showing a statistically significant (p < 0.05) increase in CFU-GM, compared to the control, were considered active. Significant CFU-GM increases were observed in rats treated with seven Echinacea samples, ranging from 39% to 91% higher than the control, while four samples were inactive. All five Echinacea purpurea samples showed myeloid progenitor-stimulating activity, while one sample each of Echinacea angustifolia and Echinacea pallida also exhibited the same activity. By applying orthogonal partial least squares discriminant analysis (OPLS-DA) to the 1H-NMR spectra, we identified specific spectral bins (0.70–1.98 ppm aliphatic and 6.38–7.76 ppm aromatic) correlating with myelopoiesis stimulation. These findings highlight the potential of chemometric analysis using 1H-NMR spectroscopy to infer the chemical classes that could be responsible for the bioactive properties of complex herbal mixtures, like Echinacea.

A comprehensive stem cell laboratory module with blended learning for medical students at Tongji University.

The laboratory practice "Primary culture and directional differentiation of rat bone marrow mesenchymal stem cells (BMSCs)" is part of a required course for sophomore medical students at Tongji university, which has been conducted since 2012. Blended learning has been widely applied in medical courses. Based on a student-centered teaching philosophy, we reconstructed a comprehensive stem cell laboratory module with blended learning in 2021, aiming to facilitate students in enhancing their understanding of the multi-lineage differentiation potential of stem cells and improve their experimental skills, self-directed learning ability, and innovative thinking. First, we constructed in-depth online study resources, including videos demonstrating laboratory procedures, a PowerPoint slide deck, and published literature on student self-learning before class. In class, students performed a primary culture of BMSCs, freely chose among adipogenic, osteogenic, or chondrogenic differentiation, and used cytochemical or immunofluorescence staining for identification. After class, the extracurricular part involved performing quantitative polymerase chain reaction to examine the expression of multi-lineage differentiation marker genes, which was designed as an elective. After 2 years of practice, positive feedback was obtained from both students and faculty members who achieved, the learning goal as expected. The reconstructed stem cell laboratory module provides comprehensive practice opportunities for students. Students have a better understanding of BMSC at the molecular, cellular, and functional levels and have improved their experimental skills, which forms a basis for scientific research for medical students. Introducing blended learning into other medical laboratory practices thus seems valuable.

Stiffness and BMP-2 Mimetic Peptide Jointly Regulate the Osteogenic Differentiation of Rat Bone Marrow Stromal Cells in a Gelatin Cryogel.

Both biochemical and mechanical cues could regulate the function of stem cells, but the interaction mechanism of their signaling pathway remains unclear, especially in the three-dimensional (3D) culture mode. Higher matrix stiffness promotes osteogenic differentiation of stem cells, and bone morphogenic protein-2 (BMP-2) has been clinically applied to promote bone regeneration. Here, the crosstalk of extracellular mechanical signals on BMP-2 signaling was investigated in rat bone marrow stromal cells (rMSCs) cultured inside cryogels with interconnective pores. Stiff cryogel independently promoted osteogenic differentiation and enhanced the autocrine secretion of BMP-2, thus stimulating increased phosphorylation levels of the Smad1/5/8 complex. BMP-2 mimetic peptide (BMMP) and high cryogel stiffness jointly guided the osteogenic differentiation of rMSCs. Inhibition of rho-associated kinase (ROCK) by Y-27632 or inhibition of nonmuscle myosin II (NM II) by blebbistatin showed that osteogenesis induction by BMP-2 signaling, as well as autocrine secretion of BMP-2 and phosphorylation of the Smad complex, requires the involvement of cytoskeletal tension and ROCK pathway signaling. An interconnective microporous cryogel scaffold promoted rMSC osteogenic differentiation by combining matrix stiffness and BMMP, and it accelerated critical cranial defect repair in the rat model.

Injectable cartilage microtissues based on 3D culture using porous gelatin microcarriers for cartilage defect treatment.

Cartilage tissues possess an extremely limited capacity for self-repair, and current clinical surgical approaches for treating articular cartilage defects can only provide short-term relief. Despite significant advances in the field of cartilage tissue engineering, avoiding secondary damage caused by invasive surgical procedures remains a challenge. In this study, injectable cartilage microtissues were developed through 3D culture of rat bone marrow mesenchymal stem cells (BMSCs) within porous gelatin microcarriers (GMs) and induced differentiation. These microtissues were then injected for the purpose of treating cartilage defects in vivo, via a minimally invasive approach. GMs were found to be noncytotoxic and favorable for cell attachment, proliferation and migration evaluated with BMSCs. Moreover, cartilage microtissues with a considerable number of cells and abundant extracellular matrix components were obtained from BMSC-laden GMs after induction differentiation culture for 28 days. Notably, ATDC5 cells were complementally tested to verify that the GMs were conducive to cell attachment, proliferation, migration and chondrogenic differentiation. The microtissues obtained from BMSC-laden GMs were then injected into articular cartilage defect areas in rats and achieved superior performance in alleviating inflammation and repairing cartilage. These findings suggest that the use of injectable cartilage microtissues in this study may hold promise for enhancing the long-term outcomes of cartilage defect treatments while minimizing the risk of secondary damage associated with traditional surgical techniques.

Physicochemical and Biological Characterization of Gelatin/Alginate Scaffolds Reinforced with β-TCP, FDBA, and SrHA: Insights into Stem Cell Behavior and Osteogenic Differentiation.

Bone tissue engineering necessitates the development of scaffolds with optimal properties to provide a suitable microenvironment for cell adhesion, proliferation, and osteogenic differentiation. The selection of appropriate scaffold materials remains a critical challenge in this field. In this study, we aimed to address this challenge by evaluating and comparing the performance of hydrogel scaffolds reinforced with β-tricalcium phosphate (β-TCP), allograft, and a combination of allograft and strontium hydroxyapatite (SrHA). In this study, scaffolds containing the following compounds with a weight ratio of 75 : 25 : 50 were made using a 3D printer: group (1) alginate + gelatin + β-TCP (TCP), group (2) alginate + gelatin + allograft (Allo), and group (3) alginate + gelatin + allograft + strontium hydroxyapatite (Str). Stem cells extracted from rat bone marrow (rBMSCs) were cultured on scaffolds, and cell proliferation and differentiation tests were performed. Also, the physical and chemical properties of the scaffolds were investigated. The two/one-way analysis of variance (ANOVA) by Tukey's post hoc test was performed. There was no significant difference between scaffolds with pore size and porosity. TCP scaffolds' mechanical strength and degradation rate were significantly lower than the other two groups (P < 0.05). Also, the swelling ratio of Allo scaffolds was higher than in other samples. The amount of cell proliferation in the samples of the TCP group was lower than the other two, and the Allo samples had the best results in this concern (P < 0.01). However, the scaffolds containing strontium hydroxyapatite had significantly higher bone differentiation compared to the other two groups, and the lowest results were related to the scaffolds containing β-TCP. Hydrogel scaffolds reinforced with allograft or its combination with strontium showed better physicochemical and biological behavior compared to those reinforced with β-TCP. Besides, adding strontium had a limited impact on the physicochemical features of allograft-containing scaffolds while improving their potential to induce osteogenic differentiation.

Photobiomodulation Effect of Diode Laser on Differentiation of Osteoprogenitor Cells in Rat Bone Marrow.

Bone marrow cells contain nonhematopoietic cells with the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Mechanical stress influences osteoblast differentiation of bone marrow cells into osteogenic, chondrogenic, and adipogenic lineages, measurable as the abundance of alkaline phosphatase-positive (ALP+) colony-forming unit-fibroblasts (CFU-F); however, the effect of diode laser irradiation on osteoblast differentiation is unknown. The aim of this study was to analyze the effects of photobiomodulation on the osteogenic differentiation of mesenchymal stem cells in the bone marrow, using the CFU-F assay. Bone marrow cells isolated from rat tibiae were cultured and irradiated with a diode laser (wavelength 808 nm) at a total energy of 0 J (control), 50 J, and 150 J. On day 7 after irradiation, ALP+ CFU-F were most abundant in the 50 J group and the least abundant in the 150 J group. Mineralized nodule formation was observed after long-term culture (21 days). Compared with the control group, there were significantly more nodules in the 50 J group and significantly fewer nodules in the 150 J group. Osteocalcin mRNA expression was highest in the 50 J group, and there was no difference between the control and 150 J groups. Irradiation with 50 J was effective in stimulating osteogenesis in bone marrow stem cells. These findings suggest that diode laser irradiation can induce osteogenesis in rat bone marrow cells in an energy-dependent manner, and appears suitable for application in bone regeneration therapy.

Thermo-photobiomodulation of stem cells

The most important task of cell transplantology is to activate the proliferative potential of mesenchymal stem cells (MSCs) before receiving bone marrow cells from a donor. This is necessary to increase a sufficient number of MSCs in early passages, when the probability of chromosomal mutations is still low. The proliferative activity of cells can be activated using photobiomodulation (PBM) by exposure to lowintensity laser radiation in the visible and near-infrared ranges. Recently, it was shown in vitro that the combination of PBM and moderate laserinduced heating can lead to a significant increase in the efficiency of MSC colony formation. The main objectives of the study are to find the optimal parameters for such a combined effect and answer the question about the possibility of a synergistic effect of thermal heating and laser radiation. MSCs isolated from rat bone marrow were used for the experiments. MSCs were exposed to short-term laser radiation of moderate power with a wavelength of 980 nm and an energy density of 68-340 J/cm2, accompanied by moderate heating of the cell suspension. Vials with grown colonies were photographed, then their number, size and number of cells in individual colonies were determined using special digital image processing methods. It was found that under optimal parameters, exposure to laser radiation of moderate power leads to an increase in the number of colonies by 4.1±0.5 times, and the total number of cells by 3.3±0.4 times compared to the control. It has been shown that this increase in cell number occurs as a result of the synergistic effect of photobiomodulation and moderate heating. Activation of colony formation after laser stimulation of MSCs occurs due to the migration of cells from the initially formed colonies with the subsequent formation of additional colonies by separated cells.

Isomangiferin promotes the migration and osteogenic differentiation of rat bone marrow mesenchymal stem cells.

Delayed or failed bone healing is a significant clinical challenge worldwide. Bone marrow mesenchymal stem cells (BMSCs) offer a promising approach for improving fracture healing. Isomangiferin, a xanthone C-glucoside, is known for its pharmacological activities, but its role in fracture healing remains unclear. In this study, we investigated the effects of isomangiferin on BMSCs under oxidative stress conditions induced by hydrogen peroxide (H2O2). Our results showed that isomangiferin promotes osteogenic differentiation and migration of H2O2-treated BMSCs, reduces apoptosis and reactive oxygen species production, and activates the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC) pathway. These findings suggest that isomangiferin may be a potential therapeutic agent for enhancing bone healing by modulating BMSC function.

Fibromodulin facilitates the osteogenic effect of Masquelet's induced membrane by inhibiting the TGF-β/SMAD signaling pathway.

Masquelet's induced membrane (IM) technique is a promising treatment strategy for the repair of substantial bone defects. The formation of an IM around polymethylmethacrylate bone cement plays a crucial role in this technique. Several studies have indicated that IMs have bioactivity because they contain abundant blood vessels, a variety of cells, and biological factors. The bioactivity of an IM increases during the initial stages of formation, thereby facilitating bone regeneration and remodeling. Nevertheless, the precise mechanisms underlying the enhancement of IM bioactivity and the promotion of bone regeneration necessitate further investigation. In this study, we successfully developed a Masquelet IM model of critical femur defects in rats. By employing proteomics analysis and biological detection techniques, we identified fibromodulin (FMOD) as a pivotal factor contributing to angiogenesis and the enhanced bioactivity of the IM. A significant increase in angiogenesis and the expression of bioactive factors in the IM was also observed with the upregulation of FMOD expression. Furthermore, this effect is mediated through the inhibition of the transforming growth factor beta (TGF-β)/SMAD signaling pathway. We also demonstrated that administering recombinant human FMOD enhanced osteogenesis in rat bone marrow mesenchymal stem cells and angiogenesis in human umbilical vein endothelial cells in vitro. Furthermore, the negative regulatory effect of the TGF-β signaling pathway was verified. In conclusion, this study provides a novel theoretical basis for the application of IMs in bone-defect reconstruction and explores possible new mechanisms that may play an important role in promoting the bioactivity and osteogenic potential of IMs.

Static magnetic field enhances the bone remodelling capacity of human demineralized bone matrix in a rat animal model of cranial bone defects.

The regeneration of bone defects that exceed 2 cm is a challenge for the human body, necessitating interventional therapies. Demineralized bone matrices (DBM) derived from biological tissues have been employed for bone regeneration and possess notable osteoinductive and osteoconductive characteristics. Nevertheless, their efficiency in regenerating critically sized injuries is limited, and therefore additional signaling cues are required. Thanks to the piezoelectric properties of the bone, external physical stimulation is shown to accelerate tissue healing. We have implanted human DBM in critically sized cranial bone defects in rat animal models and exposed them to an external magnetic field (1 T) to enhance endogenous bone formation. Our in vitro experiments showed the superior cytocompatibility of DBM compared to cell culture plates. Furthermore, alkaline phosphatase activity after 14 days and Alizarin red staining at 28 days demonstrated differentiation of rat bone marrow mesenchymal stem cells into bone lineage on DBM. Computer tomography images together with histological analyses showed that implanting DBM in the injured rats significantly enhanced bone regeneration. Notably, combining DBM transplantation with a 2 h daily exposure to a 1 T magnetic field for 2 weeks (day 7 to 21 post-surgery) significantly improved bone regeneration compared to DBM transplantation alone. This research indicates that utilizing external magnetic stimulation significantly enhances the potential of bone allografts to regenerate critically sized bone defects.

Combined Light and Thermal Stimulation of Bone Marrow Stem Cells.

Introduction: The purpose of this study is to achieve a significant increase in the proliferative activity of mesenchymal stem cells (MSCs) of the bone marrow (BM) at early passages after laser exposure to a suspension of these cells and to estimate the effect of light and heat components of laser radiation on the proliferation of BM MSCs. Methods: The studies were performed on rats BM MSCs. MSC suspension was placed into the wells and heated by using laser radiation (980 nm wavelength) or a water bath at 70 °C providing similar temperature dynamics. The studies were carried out in 3 comparison groups: (1) control suspension of MSCs, which was not subjected to heating in a water bath or laser exposure; (2) MSC suspension, which was heated for in a water bath; and (3) suspension of MSCs, which was subjected to laser exposure. The exposure times for the 2nd and 3rd experimental groups were 10- 50 seconds. Results: Under optimal parameters of laser action on the suspension of BM MSCs, a six-fold increase in the number of BM MSCs colonies was registered compared to the control. The role of the light and heat components of laser exposure to MSCs was determined by comparable heating of a suspension of BM MSCs in a water bath, at which only a twofold increase in the number of colonies was maximally obtained. Conclusion: The increase in the MSC proliferation activity occurs due to their Thermo-Photobiomodulation. The result obtained is important for practical use in cell transplantation in the treatment of traumatic injuries of bone, cartilage, and tendon tissues when a rapid and multiple increase in the initial number of autologous BM MSCs is required.

In Vivo Genotoxicity Evaluation of a No-Pain Pharmacopuncture Extract Using the Micronucleus Test.

We aimed to evaluate the genotoxicity of a recently developed no-pain pharmacopuncture (NPP) targeting muscle relaxation and analgesia using the micronucleus test. To evaluate the potential of NPP extracts to induce micronuclei in rat bone marrow cells, a micronucleus test was performed using male Sprague-Dawley rats. The test substance NPP was administered intramuscularly at concentrations of 0.25, 0.5, and 1 mL/animal. Saline was used as the negative control and cyclophosphamide as the positive control. No NPP treatment-related deaths or abnormal changes in general appearance were observed at any dose level during the experimental period. No statistically significant differences in body weight were observed in any of the NPP dose groups compared to the saline negative control group. NPP did not cause a significant increase in the incidence of micronucleated polychromatic erythrocytes (PCEs) and PCEs or in the ratio of PCE-to-total erythrocytes. The NPP extract did not exhibit genotoxic in Sprague-Dawley rat bone marrow cells under the conditions of this study. Further toxicity studies of the NPP extract are required.

Myeloprotective and Hematinic Influence of Harungana madagascariensis in Benzene-initiated Onco-hematologic Process in Wistar Rats

Background: Harungana madagascariensis is a medicinal plant that is traditionally utilized for the treatment of anaemia. Benzene constitutes an occupational toxicant due to its adverse effect on hematology of exposed industrial workers leading to onco-hematology. Therefore, the efficacy of Harungana madagascariensis against benzene-induced pre-leukaemic condition was investigated in rat model.&#x0D; Methodology: Hematological disturbance leading to pre-leukemic conditions was induced in Wistar rats by intraperitoneal administration of 400mg/kg of benzene in propan-2-ol:water (1:1 v/v) every other day for 28 days. Following benzene intoxication, 200 mg/ kg HM was administered orally for 14 days. Hematological parameters and blood cell morphology were compared between baseline control and benzene-intoxicated rats with or without HM extract treatment.&#x0D; Results: BZ-induced hematologically disturbed rats exhibited anaemic symptoms marked by reduction in hemoglobin level, red blood cells, and packed cell volume with morphologic blast cell appearance, polychromasia, hypersegmented neutrophil, anisocytosis and poikilocytosis. Moreover, there was an altered redox status depicted by a significant reduction in plasma level of total sulfhydryl content with concomitant increase in advanced oxidation protein products (AOPPs) that were accompanied by increased frequency of micronucleated polychromatic erythrocytes and hypercellularity in the bone marrow of benzene intoxicated rats. However, treatment with Harungana madagascariensis restored blood hematology and alleviated the blood cell morphological alteration induced by benzene. It also improved plasma redox status, reduced the frequency of micronucleus and improve the architecture of bone marrow cellularity in treated intoxicated rats.&#x0D; Conclusion: Harungana madagascariensis protected against benzene-induced hematological alterations leading to pre-leukemic conditions in Wistar rats.

Effect of Er:YAG Pulsed Laser-Deposited Hydroxyapatite Film on Titanium Implants on M2 Macrophage Polarization In Vitro and Osteogenesis In Vivo.

In a previous study, we successfully coated hydroxyapatite (HAp) onto titanium (Ti) plates using the erbium-doped yttrium aluminum garnet pulsed-laser deposition (Er:YAG-PLD) method. In this study, we performed further experiments to validate the in vitro osteogenic properties, macrophage polarization, and in vivo osseointegration activity of HAp-coated Ti (HAp-Ti) plates and screws. Briefly, we coated a HAp film onto the surfaces of Ti plates and screws via Er:YAG-PLD. The surface morphological, elemental, and crystallographic analyses confirmed the successful surface coating. The macrophage polarization and osteogenic induction were evaluated in macrophages and rat bone marrow mesenchymal stem cells, and the in vivo osteogenic properties were studied. The results showed that needle-shaped nano-HAp promoted the early expression of osteogenic and immunogenic genes in the macrophages and induced excellent M2 polarization properties. The calcium deposition and osteocalcin production were significantly higher in the HAp-Ti than in the uncoated Ti. The implantation into rat femurs revealed that the HAp-coated materials had superior osteoinductive and osseointegration activities compared with the Ti, as assessed by microcomputed tomography and histology. Thus, HAp film on sandblasted Ti plates and screws via Er:YAG-PLD enhances hard-tissue differentiation, macrophage polarization, and new bone formation in tissues surrounding implants both in vitro and in vivo.

Glucocorticoids promote steroid-induced osteonecrosis of the femoral head by down-regulating serum alpha-2-macroglobulin to induce oxidative stress and facilitate SIRT2-mediated BMP2 deacetylation

Our study investigated the possible molecular mechanism of glucocorticoid in steroid-induced osteonecrosis of the femoral head (SINFH) through regulating serum alpha-2-macroglobulin and SIRT2-mediated BMP2 deacetylation. Essential genes involved in glucocorticoid-induced SINFH were screened by transcriptome sequencing and analyzed by bioinformatics, followed by identifying downstream regulatory targets. Rat bone marrow mesenchymal stem cells were isolated and treated with methylprednisolone (MP) for in vitro cell experiments. Besides, a glucocorticoid-induced rat ONFH was established using the treatment of MP and LPS. ChIP-PCR detected the enrichment of SIRT2 in the promoter region of BMP2, and the deacetylation modification of SIRT2 on BMP2 was determined. Bioinformatics analysis revealed that glucocorticoids may induce ONFH through the SIRT2/BMP2 axis. In vitro cell experiments showed that glucocorticoids up-regulated SIRT2 expression in BMSCs by inducing oxidative stress, thereby promoting cell apoptosis. The up-regulation of SIRT2 expression may be due to the decreased ability of α2 macroglobulin to inhibit oxidative stress, and the addition of NOX protein inhibitor DPI could significantly inhibit SIRT2 expression. SIRT2 could promote histone deacetylation of the BMP2 promoter and inhibit its expression. In vitro cell experiments further indicated that knocking down SIRT2 could protect BMSC from oxidative stress and cell apoptosis induced by glucocorticoids by promoting BMP2 expression. In addition, animal experiments conducted also demonstrated that the knockdown of SIRT2 could improve glucocorticoid-induced ONFH through up-regulating BMP2 expression. Glucocorticoids could induce oxidative stress by down-regulating serum α2M to promote SIRT2-mediated BMP2 deacetylation, leading to ONFH.

Effects of Argon Gas Plasma Treatment on Biocompatibility of Nanostructured Titanium.

In this study, we applied argon plasma treatment to titanium surfaces with nanostructures deposited by concentrated alkali treatment and investigated the effects on the surface of the material and the tissue surrounding an implant site. The results showed that the treatment with argon plasma removed carbon contaminants and increased the surface energy of the material while the nanoscale network structure deposited on the titanium surface remained in place. Reactive oxygen species reduced the oxidative stress of bone marrow cells on the treated titanium surface, creating a favorable environment for cell proliferation. Good results were observed in vitro evaluations using rat bone marrow cells. The group treated with argon plasma exhibited the highest apatite formation in experiments using simulated body fluids. The results of in vivo evaluation using rat femurs revealed that the treatment improved the amount of new bone formation around an implant. Thus, the results demonstrate that argon plasma treatment enhances the ability of nanostructured titanium surfaces to induce hard tissue differentiation and supports new bone formation around an implant site.

Effects of using collagen and aloe vera grafted fibroin scaffolds on osteogenic differentiation of rat bone marrow mesenchymal stem cells in SBF-enriched cell culture medium

In the study, collagen and aloe vera were grafted onto silk fibroin with two different methods, and 3D-microporous scaffolds (1F5C4A1 and 2F5C4A1) were formed by lyophilization. Three osteogenic cultures were started by seeding rat bone marrow mesenchymal stem cells (MSCs) and pre-induced MSC (osteoblast (OB)) on biopolymeric scaffolds. The osteogenic medium was enriched with 10% (v/v) simulated body fluid (SBF) to promote mineralization and osteogenic differentiation in one of the MSC cultures and the OB culture. X-ray diffraction (XRD), scanning electron microscopy (SEM), scanning electron microscopy- energy dispersive spectrum (SEM-EDS) analyses on cellular samples and histochemical (alizarin red, safranin-O, alcian blue) and immunohistochemical (anti-collagen-1, anti-osteocalcin, anti-osteopontin) staining showed that bone-like mineralization was occurred by both chemically and cellular activity. In addition, pre-osteogenic induction of MSCs in 2D-cultured was found to promote osteogenesis more rapidly when started 3D-cultured. These results indicated that enrichment of the cell culture medium with SBF is sufficient for in vitro mineralization rather than using high concentrations of SBF. The findings showed that OB cells on the 2F5C4A1 scaffold obtained the best osteogenic activity. Still, other culture media with 10% SBF content could be used for bone tissue engineering under osteogenic induction.

Extracellular and intracellular effects of bioactive glass nanoparticles on osteogenic differentiation of bone marrow mesenchymal stem cells and bone regeneration in zebrafish osteoporosis model

Bioactive glass nanoparticles (BGNs) are well-recognized multifunctional biomaterials for bone tissue regeneration due to their capability to stimulate various cellular processes through released biologically active ions. Understanding the correlation between BGN composition and cellular responses is key to developing clinically usable BGN-based medical devices. This study investigated the influence of CaO content of binary SiO2-CaO BGNs (CaO ranging from 0 to 10 mol%) on osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and in vivo bone regeneration in zebrafish osteoporosis model. The results showed that BGNs could promote osteogenic differentiation of rBMSCs by indirectly releasing active ions or directly interacting with rBMSCs by internalization. In both situations, BGNs of a higher CaO content could promote the osteogenic differentiation of rBMSCs to a greater extent. The internalized BGNs could activate the transcription factors RUNX2 and OSX, leading to the expression of osteogenesis-related genes. The results in the zebrafish osteoporosis model indicated that the presence of BGNs of higher CaO contents could enhance bone regeneration and rescue dexamethasone-induced osteoporosis to a greater extent. These findings demonstrate that BGNs can stimulate osteogenic differentiation of rBMSCs by releasing active ions or internalization. A higher CaO content facilitates osteogenesis and bone regeneration of zebrafish as well as relieving dexamethasone-induced osteoporosis. The zebrafish osteoporosis model can be a potent tool for evaluating the in vivo bone regeneration effects of bioactive materials. Statement of significanceBioactive glass nanoparticles (BGNs) are increasingly used as fillers of nanocomposites or as delivery platforms of active ions to regenerate bone tissue. Various studies have shown that BGNs can enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by releasing active ions. However, the correlation between BGN composition and cellular responses and in vivo bone regeneration effect has still not been well investigated. Establishment of a suitable in vivo animal model for investigating this correlation is also challenging. The present study reports the influence of CaO content in binary SiO2-CaO BGNs on osteogenic differentiation of BMSCs extracellularly and intracellularly. This study also demonstrates the suitability of zebrafish osteoporosis model to investigate in vivo bone regeneration effect of BGNs.

Preparation of cerium-containing nanoporous titanium coatings for osseointegration and their promotion of osteogenic differentiation and anti-inflammatory properties

Conventional implants face many problems, such as failure to form good osseointegration and peri-implantitis in the early stage of implantation. It has been shown that hydroxyapatite (HA) has good osteoconductive ability. Also considering the potential of cerium oxide in osteogenesis and anti-inflammation, we prepared cerium-containing nanoporous HA coatings on the surface of pure titanium using the micro-arc oxidation (MAO) technique. The surface morphology of the coating was observed by scanning electron microscopy (SEM). The mechanical strength of the coating was examined by the ultrasonic oscillator. The elemental composition of the coating was examined by energy spectrum analysis (EDS). The ion release capacity of the cerium-containing coatings was examined by inductively coupled plasma emission spectrometry (ICP-OES). The hydrophilic properties of the coatings were examined by the hydrophilic angle test. In vitro, experiments were performed using rat bone marrow mesenchymal stem cells (BMSCs) and mouse mononuclear macrophages (Raw264.7). The biosafety of the cerium-containing coating was verified by live-dead staining and CCK-8 proliferation assay. The osteogenic and anti-inflammatory ability of the cerium-containing coating was examined by immunofluorescence staining and quantitative polymerase chain reaction (q-PCR). In addition, ion experiments were used to further demonstrate the osteogenic and anti-inflammatory potential of cerium ions. Subsequent animal experiments further demonstrated the osteoinductive potential of the cerium-containing HA coating. In conclusion, our cerium (HA)-containing coatings prepared by micro-arc oxidation on titanium surfaces show excellent osteogenic and anti-inflammatory properties, which are expected to improve the functionality of clinical implants and also lay the foundation for the clinical application of cerium ions.

ADVANCING THE FABRICATION OF 3D MINI-TISSUE ENTHESIS CONSTRUCTS: CHARACTERIZATION OF BONE, TENDON, AND BMSC SPHEROIDS

AbstractObjectivesThe enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production.MethodsCell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×104, 5×104, 1×105, 2×105 in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&amp;E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope.3D mini-tissue constructs15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix.ResultsRTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×105 RTFs and 3×105 BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage.ConclusionsThis study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model.Declaration of Interest(a) fully declare any financial or other potential conflict of interest