Rat Bone Marrow-derived Mesenchymal Stem Research Articles

Optimal regenerative repair of large segmental bone defect in a goat model with osteoinductive calcium phosphate bioceramic implants

So far, how to achieve the optimal regenerative repair of large load-bearing bone defects using artificial bone grafts is a huge challenge in clinic. In this study, a strategy of combining osteoinductive biphasic calcium phosphate (BCP) bioceramic scaffolds with intramedullary nail fixation for creating stable osteogenic microenvironment was applied to repair large segmental bone defects (3.0 cm in length) in goat femur model. The material characterization results showed that the BCP scaffold had the initial compressive strength of over 2.0 MPa, and total porosity of 84%. The cell culture experiments demonstrated that the scaffold had the excellent ability to promote the proliferation and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs). The in vivo results showed that the intramedullary nail fixation maintained the initial stability and structural integrity of the implants at early stage, promoting the osteogenic process both guided and induced by the BCP scaffolds. At 9 months postoperatively, good integration between the implants and host bone was observed, and a large amount of newborn bones formed, accompanying with the degradation of the material. At 18 months postoperatively, almost the complete new bone substitution in the defect area was achieved. The maximum bending strength of the repaired bone defects reached to the 100% of normal femur at 18 months post-surgery. Our results demonstrated the good potential of osteoinductive BCP bioceramics in the regenerative repair of large load-bearing bone defects. The current study could provide an effective method to treat the clinical large segmental bone defects.

Estrogen receptor alpha regulates the expression of adipogenic genes genetically and epigenetically in rat bone marrow-derived mesenchymal stem cells.

Regulation of the efficacy of epigenetic modifiers is regarded as an important control mechanism in the determination and differentiation of stem cell fate. Studies are showing that the effect of estrogen is important in the differentiation of mesenchymal stem cells (MSCs) into adipocytes, osteocytes, and chondrocytes. Activation of certain transcription factors and epigenetic modifications in related genes play an active role in the initiation and completion of adipogenic differentiation. Understanding the role of estrogen in diseases such as obesity, which increases with the onset of menopause, will pave the way for more effective use of estrogen as a therapeutic option. Demonstration of the differentiation tendencies of MSCs change in the presence/absence of estrogen, especially the evaluation of reversible epigenetic changes, will provide valuable information for clinical applications. In this study, the effect of estrogen on the expression of genes involved in adipogenic differentiation of MSCs and accompanying epigenetic modifications was investigated. Our results showed that estrogen affects the expression of adipogenesis-related transcription factors such as PPARy, C/EBPα and Adipsin. In addition, after estrogen treatment, increased accumulation of estrogen receptor alpha (ERα) and repressive epigenetic markers such as H3K27me2 and H3K27me3 were observed on the promoter of given transcription factors. By using co-immunoprecipitation experiments we were also able to show that ERα physically interacts with the zeste homolog 2 (EZH2) H3K27 methyltransferase in MSCs. We propose that the increase of H3K27me2 and H3K27me3 markers on adipogenic genes upon estrogen treatment may be mediated by the direct interaction of ERα and EZH2. Taken together, these findings suggest that estrogen has a role as an epigenetic switcher in the regulation of H3K27 methylation leading to suppression of adipogenic differentiation of MSC.

Evaluation of the osteogenic potential of crocin-incorporated collagen scaffold on the bone marrow mesenchymal stem cells

Objective: Present study aimed to evaluate the effect of crocin-loaded collagen scaffold on the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BM-MSCs). Significance: Different studies have been conducted to develop an efficient strategy to accelerate and improve the recovery process of bone defects. It was shown that crocin (CRO), extracted from saffron, could induce osteogenic differentiation of rat BM-MSCs. Scaffolds can also provide a 3-dimensional environment for migration, adhesion, growth, and proliferation of MSCs. Methods: Collagen (COL) scaffolds were fabricated through freeze-drying followed by cross-linking by dehydrothermal method. Then, crocin was incorporated into the scaffolds. Physicochemical characterization of the scaffolds was evaluated. Rat BM- MSCs were seeded on crocin-loaded collagen scaffolds and cultured for 14 days. Osteogenic differentiation was evaluated using alizarin red (ALZ) staining and alkaline phosphatase (ALP) activity assay and compared to the positive control group. Results: The average pore size of the collagen scaffolds was about 97 ± 6.7 µm. Formation of amide cross-links was confirmed by FTIR. The scaffolds were capable of uptaking water up to 50 times more than their initial dry weight and releasing above 90% of their uploaded crocin during 24 h. Collagen scaffolds containing crocin (25 and 50 μM) increased ALZ intensity (3.16 ± 0.3 and 7.32 ± 0.3 folds, respectively) and ALP activity (13.7 ± 1.1 and 12.2 ± 9.4 folds, respectively) in comparison with the positive control group. Conclusion: Crocin-loaded collagen scaffold could effectively enhance calcium deposition and ALP activity in BM-MSCs and therefore proposed as a good candidate to accelerate the healing process of vital bone defects.

Electrospun PLGA scaffold loaded with osteogenic growth peptide accelerates cranial bone repair in rats

To study the feasibility of electrospun poly(D, L-lactide-co-glycolide) (PLGA) scaffold loaded with osteogenic growth peptide (OGP) for bone tissue engineering. PLGA scaffolds were prepared by electrospinning PLGA solution without OGP(control group)or with 0.1%, 0.2%and 0.4%OGP(0.1%OGP@PLGA, 0.2%OGP@PLGA, and 0.4%OGP@PLGA scaffolds, respectively).The microstructure of the scaffolds was observed by scanning electron microscopy(SEM).The scaffolds were soaked in PBS to confirm the release pattern of OGP.The biocompatibility of the scaffolds was evaluated using CCK-8 assay and live/dead staining after a 7-day coculture with rat bone marrow-derived mesenchymal stem cells(BMSCs).ALP assay and ARS staining were used to evaluate osteoinduction capacity of the scaffolds co-cultured with rat BMSCs for 14 days.In a male SD rat model of skull defect(5 mm in diameter), bone defect repair was evaluated 8 weeks after implantation of the scaffolds using Micro-CT, HE and Masson staining. The electrospun scaffolds had a fibrous structure similar to extracellular matrix(ECM)and were capable of sustained release of OGP for at least one month.Co-culture with 0.2%OGP@PLGA and 0.4%OGP@PLGA scaffolds, as compared with pure PLGA scaffold, significantly promoted the growth of rat BMSCs ((P < 0.01).The cells co-cultured with 0.4%OGP@PLGA scaffold showed the highest ALP activity and the greatest number of calcium nodules, indicating its strong osteoinduction ability (P < 0.01).Micro-CT and HE and Masson staining results showed that 0.4%OGP@PLGA scaffold had significantly better ability for promoting bone repair than the other two OGP-loaded scaffolds(P < 0.01). The electrospun PLGA scaffold loaded with OGP effectively mimics the structure of ECM and has a good biocompatibility and osteoinduction ability, suggesting its potential as a new bone tissue engineering scaffold for bone defect repair.

PKD1 alleviates oxidative stress-inhibited osteogenesis of rat bone marrow-derived mesenchymal stem cells through TAZ activation.

Oxidative stress is known to inhibit osteogenesis and PKD1 is implicated in bone remodeling and skeletogenesis. In the present study, we explored the role of PKD1 in osteogenesis under oxidative stress. H2O2 was used to induce oxidative stress in rat bone marrow (BM)‐mesenchymal stem cells (MSCs) during osteoblast differentiation. Alkaline phosphatase (ALP) activity, calcium deposits, and the RUNX2 marker were assayed to determine osteogenic differentiation. The correlation of PKD1, Sirt1, c‐MYC, and TAZ was further confirmed by chromatin immunoprecipitation (ChIP) and dual‐luciferase reporter assay. We found that H2O2 induced the downregulation of PKD1 expression and the upregulation of c‐MYC, and Sirt1 was accompanied by decreasing cell viability in BM‐MSCs. During osteogenic differentiation, the expression of PKD1 was upregulated significantly whereas Sirt1 tended to be upregulated mildly under normal conditions. Both PKD1 and Sirt1 were upregulated upon oxidative stress. The positive correlation of PKD1 expression with osteogenic differentiation under normal conditions might be hindered by oxidative stress and PKD1 could interact with TAZ under oxidative stress to regulate osteogenic differentiation. Our results suggest that PKD1 may alleviate oxidative stress‐inhibited osteogenesis of rat BM‐MSCs through TAZ activation.

Effect of the Internal Septum Extract of the Walnut Kernel on the Mesenchymal Stem Cells Cycle and MSCs-derived Insulin-Producing Β-cells Differentiation and Glucose Uptake

Background: For years, scientists have been striving to identify compounds to improve the characteristics and functions of cells for therapeutic applications. Objectives: This study aimed to evaluate the effect of the aqueous decoction of the internal septum of the walnut kernel (DISWK) on the survival, total antioxidant capacity (TAC), and cell cycle of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs). We also evaluated the expression of genes involved in pancreatic insulin-producing β-cell (IPCs) commitment and glucose uptake in MSCs-derived IPCs. Methods: The rBM-MSCs were isolated, and then the cytotoxic and antioxidant effects of DISWK on the cells were examined by the MTT and TAC assays, respectively. Moreover, the influence of DISWK on the rBM-MSCs cell cycle was evaluated by PI-staining and flow-cytometry analysis. Next, the rBM-MSCs were transdifferentiated into IPCs treated with DISWK. Finally, Ins1/2, Insr, Glut1, Glut4, and Pdx1 genes expression was investigated in the DISWK-treated IPCs by qRT-PCR. Results: The results indicated that DISWK significantly reduced the viability of rBM-MSCs in a dose- and time-dependent manner and decreased their TAC in long-term cultures. Moreover, it suppressed the rBM-MSCs cell cycle at S and G2 phases. The gene expression analysis showed that DISWK significantly upregulated Ins1/2, Insr, and Glut1 genes and downregulated Pdx1 gene in the rBM-MSCs-derived IPCs. Conclusions: Decoction of the internal septum of walnut kernel suppresses the MSCs cell cycle and may prevent IPCs development via inhibiting Pdx1. It may also help glucose uptake by upregulating Ins1/2, Insr, and Glut1 genes expression in MSCs-derived IPCs.

Fabrication and Characterization of Collagen/PVA Dual-Layer Membranes for Periodontal Bone Regeneration.

Guided tissue regeneration (GTR) is a promising treatment for periodontal tissue defects, which generally uses a membrane to build a mechanical barrier from the gingival epithelium and hold space for the periodontal regeneration especially the tooth-supporting bone. However, existing membranes possess insufficient mechanical properties and limited bioactivity for periodontal bone regenerate. Herein, fish collagen and polyvinyl alcohol (Col/PVA) dual-layer membrane were developed via a combined freezing/thawing and layer coating method. This dual-layer membrane had a clear but contact boundary line between collagen and PVA layers, which were both hydrophilic. The dual membrane had an elongation at break of 193 ± 27% and would undergo an in vitro degradation duration of more than 17 days. Further cell experiments showed that compared with the PVA layer, the collagen layer not only presented good cytocompatibility with rat bone marrow-derived mesenchymal stem cells (BMSCs), but also promoted the osteogenic genes (RUNX2, ALP, OCN, and COL1) and protein (ALP) expression of BMSCs. Hence, the currently developed dual-layer membranes could be used as a stable barrier with a stable degradation rate and selectively favor the bone tissue to repopulate the periodontal defect. The membranes could meet the challenges encountered by GTR for superior defect repair, demonstrating great potential in clinical applications.

The inhibitory effects of vancomycin on rat bone marrow-derived mesenchymal stem cell differentiation.

The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%-60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

Cyclic Adenosine Monophosphate-Enhanced Calvarial Regeneration by Bone Marrow-Derived Mesenchymal Stem Cells on a Hydroxyapatite/Gelatin Scaffold.

Cyclic adenosine monophosphate (cAMP) plays a significant role in inducing new bone formation by mediating various signal pathways. However, cAMP, combined with biomaterials, is rarely investigated to reconstruct calvarial defects. In this study, cAMP was loaded into a hydroxyapatite (HA)/gelatin (Gel) construct and implanted into critical skull defects in rats to evaluate the potential for enhancing skull regeneration. The physiochemical characteristics, the biocompatibility of Gel and HA/Gel scaffolds, and the regenerated bone tissue were assessed. The resulting HA/Gel scaffolds possessed a 3D interconnected porous structure with extensively distributed HA crystals and favorable physiochemical properties. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) within the HA/Gel scaffold showed greater biocompatibility. Compared with the Gel and HA/Gel groups, the cAMP-HA/Gel group revealed the highest bone density, more mature mineralized tissue, and more favorable integration between the new bone and inherent bone as analyzed by cone beam computed tomography and hematoxylin & eosin and Masson staining, respectively. Collectively, our study verified HA/Gel scaffolds as a prospective biomimetic treatment with biocompatibility and the therapeutic potential of cAMP in promoting new bone growth of a skull, which indicates its promise as a growth factor for bone tissue engineering.

Cyclic Strain and Electrical Co-stimulation Improve Neural Differentiation of Marrow-Derived Mesenchymal Stem Cells.

The current study investigated the combinatorial effect of cyclic strain and electrical stimulation on neural differentiation potential of rat bone marrow-derived mesenchymal stem cells (BMSCs) under epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) inductions in vitro. We developed a prototype device which can provide cyclic strain and electrical signal synchronously. Using this system, we demonstrated that cyclic strain and electrical co-stimulation promote the differentiation of BMCSs into neural cells with more branches and longer neurites than strain or electrical stimulation alone. Strain and electrical co-stimulation can also induce a higher expression of neural markers in terms of transcription and protein level. Neurotrophic factors and the intracellular cyclic AMP (cAMP) are also upregulated with co-stimulation. Importantly, the co-stimulation further enhances the calcium influx of neural differentiated BMSCs when responding to acetylcholine and potassium chloride (KCl). Finally, the phosphorylation of extracellular-signal-regulated kinase (ERK) 1 and 2 and protein kinase B (AKT) was elevated under co-stimulation treatment. The present work suggests a synergistic effect of the combination of cyclic strain and electrical stimulation on BMSC neuronal differentiation and provides an alternative approach to physically manipulate stem cell differentiation into mature and functional neural cells in vitro.

Therapeutic Role of Bone Marrow-Derived Mesenchymal Stem Cells in Controlling Prognosis of Hepatocellular Carcinoma in a Murine Model.

Hepatocellular carcinoma is the fourth leading cause of cancer deaths in the world. Conven - tional methods of cancer therapy are either invasive or have undesirable side effects. Therefore, exploring new therapeutic strategies to control the progression of hepatocellular carcinoma, such as cell-based therapies, is a key issue for prolonging patient survival. In this study, we aimed to evaluate tumor suppressive effects of mesenchymal stem cells on the in vivo pro - gression of hepatocellular carcinoma in murine model. Hepatocellular carcinoma was induced in 40 rats with diethylnitrosamine. Rats were divided into 4 groups: 1 group injected with diethylnitrosamine only, 1 group injected with diethylnitrosamine and 1 dose of rat bone marrowderived mesenchymal stem cells, 1 group injected with diethylnitrosamine and 2 doses of rat bone marrowderived mesenchymal stem cells, and 1 group was injected with diethylnitrosamine and 3 doses of rat bone marrow-derived mesenchymal stem cells. Rats were killed after 1 month of dose 3. Liver specimens were histopathologically examined, and serum samples were examined for liver function and cytokines. Histopathological examination revealed that mesenchymal stem cell transplant induced liver regeneration. It also improved liver function as revealed by decreased levels of alanine and aspartate aminotransferase. Mesenchymal stem cells also repaired the immunopathology of the liver environment, as it decreased levels of interleukin 2 and 10, tumor necrosis factor α, and interferon γ. Mesenchymal stem cell infusion significantly enhanced hepatic structure and function of livers in a rat hepatocellular carcinoma model.

Berberine ameliorates the LPS-induced imbalance of osteogenic and adipogenic differentiation in rat bone marrow-derived mesenchymal stem cells.

Lipopolysaccharide (LPS) from oral pathogenic bacteria is an important factor leading to alveolar bone absorption and the implant failure. The present study aimed to evaluate the modulation of berberine hydrochloride (BBR) on the LPS-mediated osteogenesis and adipogenesis imbalance in rat bone marrow-derived mesenchymal stem cells (BMSCs). Cell viability, osteoblastic and adipogenic differentiation levels were measured using the Cell Counting Kit-8 assay, alkaline phosphatase (ALP) staining and content assay, and oil red O staining, respectively. Reverse transcription-quantitative PCR and immunoblotting were used to detect the related gene and protein expression levels. In undifferentiated cells, BBR increased the mRNA expression levels of the osteoblastic genes (Alp, RUNX family transcription factor 2, osteocalcin and secreted phosphoprotein 1) but not the adipogenic genes (fatty acid binding protein 4, Adipsin and peroxisome proliferator-activated receptorγ). LPS-induced osteoblastic gene downregulation, adipogenic gene enhancement and NF-κB activation were reversed by BBR treatment. In osteoblastic differentiated cells, decreased ALP production by LPS treatment was recovered with BBR co-incubation. In adipogenic differentiated cells, LPS-mediated lipid accumulation was decreased by BBR administration. The mRNA expression levels of the pro-inflammatory factors (MCP-1, TNF-α, IL-6 and IL-1β) were increased by LPS under both adipogenic and osteoblastic conditions, which were effectively ameliorated by BBR. The actions of BBR were attenuated by compound C, suggesting that the role of BBR may be partly due to AMP-activated protein kinase activation. The results demonstrated notable pro-osteogenic and anti-adipogenic actions of BBR in a LPS-stimulated inflammatory environment. This indicated a potential role of BBR for bacterial infected-related peri-implantitis medication.

Dual-crosslinked 3D printed gelatin scaffolds with potential for temporomandibular joint cartilage regeneration

A promising alternative to current treatment options for degenerative conditions of the temporomandibular joint (TMJ) is cartilage tissue engineering, using 3D printed scaffolds and mesenchymal stem cells. Gelatin, with its inherent biocompatibility and printability has been proposed as a scaffold biomaterial, but because of its thermoreversible properties, rapid degradation and inadequate strength it must be crosslinked to be stable in physiological conditions. The aim of this study was to identify non-toxic and effective crosslinking methods intended to improve the physical properties of 3D printed gelatin scaffolds for cartilage regeneration. Dehydrothermal (DHT), ribose glycation and dual crosslinking with both DHT and ribose treatments were tested. The crosslinked scaffolds were characterized by chemical, mechanical, and physical analysis. The dual-crosslinked scaffolds had the highest degree of crosslinking and the greatest resistance to hydrolytic and enzymatic degradation. Compared to the dual-crosslinked group, the ribose-crosslinked scaffolds had thinner printed strands, larger pore surface area and higher fluid uptake. The compressive modulus values were 2 kPa for ribose, 37.6 kPa for DHT and 30.9 kPa for dual-crosslinked scaffolds. None of the crosslinking methods had cytotoxic effects on the seeded rat bone marrow-derived mesenchymal stem cells (rBMSC). After 4 and 7 d, the dual-crosslinked scaffolds exhibited better cell proliferation than the other groups. Although all scaffolds supported chondrogenic differentiation of rBMSC, dual-crosslinked scaffolds demonstrated the lowest expression of the hypertrophy-related collagen 10 gene after 21 d. The results show that 3D printed gelatin scaffolds, when dually crosslinked with ribose and DHT methods, are not toxic, promote chondrogenic differentiation of rBMSC and have potential application in tissue engineering of TMJ condylar cartilage.

Semaphorin 3A promotes the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in inflammatory environments by suppressing the Wnt/β-catenin signaling pathway.

After periodontal treatment, the local inflammatory environment surrounding periodontal tissues cannot be entirely eliminated. The means by which alveolar bone repair and regeneration are promoted in inflammatory environments have important clinical significance. As a powerful protein that promotes the differentiation of osteocytes, semaphorin 3A (Sema3A) shows potential for bone regeneration therapy. However, the effect of Sema3A on osteogenic differentiation in an inflammatory environment, as well as the underlying mechanism, have not yet been explored. We used lentivirus to transduce rat bone marrow-derived mesenchymal stem cells (rBMSCs) to stably overexpress Sema3A. Lipopolysaccharide from Escherichia coli (E. coli LPS) was used to stimulate rBMSCs to establish an inflammatory environment. ALP staining, Alizarin red staining, ALP activity tests, quantitative RT-PCR (qRT-PCR), and Western blotting were used to elucidate the effect of Sema3A on the osteogenesis of rBMSCs in inflammatory environments. XAV939 and LiCl were used to determine whether the Wnt/β-catenin signaling pathway was involved in attenuating the inhibition of Sema3A-induced osteogenic differentiation by LPS. The qRT-PCR and Western blot results demonstrated that the lentiviral vector (LV-NC) and lentiviral-Sema3A (LV-Sema3A) were successfully transduced into rBMSCs. An inflammatory environment could be established by stimulating rBMSCs with 1 μg/ml E. coli LPS. After Sema3A overexpression, mineral deposition was exacerbated, and the BSP and Runx2 gene and protein expression levels were increased. Furthermore, E. coli LPS activated the Wnt/β-catenin signaling pathway and decreased rBMSC osteogenesis, but these effects were attenuated by Sema3A. In conclusion, Sema3A could protect BMSCs from LPS-mediated inhibition of osteogenic differentiation in inflammatory environments by suppressing the Wnt/β-catenin pathway.

E7-Modified Substrates to Promote Adhesion and Maintain Stemness of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have drawn great attention in clinical applications due to the self-renewal ability, multi-differentiation potential, and low immunogenicity. However, there are challenges in the ex vivo expansion of MSCs, including low efficiency, stemness loss, and safety. Therefore, it is crucial to construct a substrate that can show an alterable affinity to MSCs, and induce efficient cell expansion with minimal stemness loss. In this study, EPLQLKM (E7)-modified substrates with tunable E7 densities are fabricated on PEGylated substrates. The PEG layer with an average thickness of 1.7 nm shows good antifouling ability. E7-modified substrates have an improving effect on adhesion and spreading of the rat bone marrow-derived mesenchymal stem cells (rBMSCs), along with the increase of E7 densities. rBMSCs on E7-modified substrates maintain the stem cell phenotypes, and shows robust proliferation and multilineage differentiation, especially on the substrates with high E7 densities. In summary, this study provides a novel strategy of E7 functionalization to promote adhesion and maintain stemness of MSCs, which holds great potentials in the functionalization of microcarriers for the expansion of MSCs.

Bone Regeneration by Homeopathic Symphytum officinale

Cell therapy has been proposed as an effective treatment method for bone regeneration using stem cells and osteogenic inducting mediators. In the present study, the ability of homeopathic remedy Symphytum officinale to induce osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rat BM-MSCs) was studied MSCs were isolated from the rat’s bone morrow. Cytotoxic effect of Symphytum on the MSCs was evaluated by MTT assay. The osteogenic induction ability of Symphytum has been assessed by alizarin red staining (ALZ) and quantitative alkaline phosphatase (ALP) activity assays and compared with the osteogenic standard medium. MTT assay results illustrated that Symphytum 200 C had a high cytotoxicity (to about 75.2 ± 3.12), so that lower potencies were used for the next experiments. Symphytum 6 C had the most color intensity in ALZ and ALP staining assays. Also, quantitative analyses of ALP activity revealed that Symphytum 6 C caused the most increase in the ALP activity up to 4.04 ± 0.34 and 7.86 ± 1.38 on day 7 and 21, respectively. According to this finding, Symphytum could enhance osteogenic differentiation of MSCs and can be considered as a safe, low cost, and highly effective drug for bone regeneration. Cell therapy has been proposed as an effective treatment method for bone regeneration using stem cells and osteogenic inducting mediators. In the present study, the ability of homeopathic remedy Symphytum officinale to induce osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rat BM-MSCs) was studied. The results illustrated that Symphytum 6 C had the most osteogenic induction effect compare with osteogenic standard medium. According to this finding, Symphytum officinale as a natural compound can significantly induce osteogenic differentiation of rat BM-MSCs. So, it could be considered as a safe and more cost-effective therapeutic agent for clinical treatment of bone disorders.

Hepatic stem cell Numb gene is a potential target of Huang Qi Decoction against cholestatic liver fibrosis

Numb is a negative regulator of Notch signal pathway. Previous study has demonstrated that Notch signal pathway activation is required for hepatic progenitor cell (HPC) differentiating into cholangiocytes in cholestatic liver fibrosis (CLF), and Huang Qi Decoction (HQD) could prevent CLF through inhibition of the Notch signal pathway. However, the role of Numb in HQD against CLF is yet unclear. Thus, CLF rats transplanted into rat bone marrow-derived mesenchymal stem cells with knocked down Numb gene (BMSCNumb-KD) were treated with HQD. Simultaneously, Numb gene knockdown was also performed in WB-F344 cell line and then treated with refined HQD in vitro. In vivo study revealed that liver fibrosis was inhibited by HQD plus BMSCNumb-KD treatment, while Hyp content in liver tissue, the gene and protein expression of α-SMA, gene expression of Col I, TNF-α, and TGF-β1 were increased compared to that in HQD group. Furthermore, Notch signal pathway was inhibited by HQD plus BMSCNumb-KD, while the protein expression of Numb was decreased and RBP-Jκ and Hes1 was increased compared to that in HQD group. In vitro, HQD reduced the differentiation of WB-F344 cells into cholangiocyte phenotype, while this effect was attenuated after Numb-knockdown. This study highlights that the absence of hepatic stem cell Numb gene decreases effect of HQD against CLF, which give rise the conclusion that Numb might be a potential target for HQD against CLF.

PEGylated gold nanoparticles promote osteogenic differentiation in in vitro and in vivo systems

Gold nanoparticles (GNPs) have attracted considerable attention in promoting osteogenic differentiation. However, little is known about their osteogenic effects on cells from various origins, and further in vivo investigation as a promising osteogenic agent upon hydrogel-conjugation is required. Herein, the effects of PEGylated GNPs with the sizes of 4, 18 and 45 nm on osteogenic differentiation in the pre-osteoblastic MC3T3-E1 cell line, human bone marrow-derived mesenchymal stem cells (hBMSCs) and rat bone marrow-derived mesenchymal stem cells (rBMSCs) were comprehensively evaluated. The results showed that GNPs with all of three different sizes had excellent biocompatibility, and their effects on induction of osteogenic differentiation of various cell types were dependent on size. Specifically, 45-nm GNPs showed more pronounced alkaline phosphatase (ALP) activity, mineralized nodule formation and expression of osteogenic marker genes compared with the 18-nm group, while 4-nm GNPs reduced the above osteogenic effects. Meanwhile, β-catenin and p-GSK-3β were upregulated by GNPs-45 nm and GNPs-18 nm, but suppressed by GNPs-4 nm, indicating that Wnt/β-catenin signaling pathway mighy play an important role in GNPs-induce osteogenic differentiation. In addition, specific blocking of Wnt/β-catenin signals by KYA1797K inhibited 45 nm GNPs-induced osteogenic differentiation of MC3T3-E1 cells. Furthermore, 45-nm GNPs conjugated with PEG-hydrogel were chosen for in vivo bone regeneration testing in rabbits with 4-mm femur defects. The results demonstrated that the GNPs stimulated bone regeneration rapidly and effectively. Such comprehensive evaluation of GNPs' effects on in vitro osteogenic differentiation and in vivo bone regeneration could promote their applications in clinical translation.

Bioinspired polydopamine/graphene oxide/collagen nanofilms as a controlled release carrier of bioactive substances

A single functional coating is far from enough to meet the high requirements of complex bone microenvironments for titanium (Ti)-based implants. In this study, multilayered polydopamine (PDA)/graphene oxide (GO)/type I collagen (Col I) (PGC) nanofilms on the basis of bioinspired design make it possible to develop a versatile, personalized, and biocompatible Ti-based implant. The nanofilms were successfully prepared by layer by layer (LBL) self-assembly of PDA, GO, and Col I, which was confirmed by surface physical and chemical characterization. In vitro biological assessments demonstrated the outstanding biological properties of PGC nanofilms coated Ti substrates, including enhanced protein adsorption, excellent biocompatibility, and accelerated osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs). Importantly, the more layers of PGC nanofilms were, the more significant osteogenic differentiation of rBMSCs was. In addition, PGC nanofilms displayed a powerful capacity of controllably releasing bioactive substances. And the encapsulated amount and release rate of bioactive substances (e.g., silver (Ag) ions and bovine serum albumin (BSA)) could be precisely controlled by the layer number of PGC nanofilms. The work provides a fairly feasible approach for developing a multifunctional bioactive coating on the surfaces of Ti-based implants.

THE CHARACTERISTICS OF RAT EMBRYONIC FIBROBLASTS (REFS) AND RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS (RAT-BMMSCS) FROM THE WISTAR RAT: A COMPARATIVE STUDY

Objective: Rat embryonic fibroblasts (REFs) and rat bone marrow-derived mesenchymal stem cells (rat-BMMSCs) an be used as in vitro models for a variety of studies, including for degenerative diseases such as arterial ischemia, tissue engineering and development of induced pluripotent stem cells (iPSCs). Therefore, the further developments of the use of these two cells of great importance.&#x0D; Methods: The experiments were performed with Wistar rat, those with 15-17 d gestation (aged 32 w) as a REFs source and those aged 12 w as a BMMSCs source. Dulbecco's modified eagle medium (DMEM) was used for both cell cultures but with different media supplements. Proliferation ability was determined for both by calculating population doubling time (PDT). Characterization was performed by differentiation testing into osteocyte, chondrocyte and adipocyte cells by staining with Alizarin Red, Alcian Blue and Oil Red O and by an investigation of specific antigen characteristics using flow cytometry with positive CD90 and CD29 and negative CD34 markers.&#x0D; Results: Morphologically, the REFs and rat-BMMSCs had the same fibroblasts like shape. PDT was higher for the REFs than the BMMSCs (p&lt;0.05), and both could differentiate into osteocytes, chondrocytes and adipocyte. The characteristics of the positive markers (CD29 and CD90) were higher in rat-BMMSCs than in REFs.&#x0D; Conclusion: In this study demonstrated that the explant method for REFs isolation and flushing method for rat-BMMSC isolation are both effective. It also showed that rat-BMMSC grow faster than REFs, and that both cells have the same differentiation ability as rat-BMMSCs but with different specific surface antigen characteristics.