ObjectiveTo review the research progress of different cell seeding densities and cell ratios in cartilage tissue engineering. MethodsThe literature about tissue engineered cartilage constructed with three-dimensional scaffold was extensively reviewed, and the seeding densities and ratios of most commonly used seed cells were summarized. ResultsArticular chondrocytes (ACHs) and bone marrow mesenchymal stem cells (BMSCs) are the most commonly used seed cells, and they can induce hyaline cartilage formation in vitro and in vivo. Cell seeding density and cell ratio both play important roles in cartilage formation. Tissue engineered cartilage with good quality can be produced when the cell seeding density of ACHs or BMSCs reaches or exceeds that in normal articular cartilage. Under the same culture conditions, the ability of pure BMSCs to build hyaline cartilage is weeker than that of pure ACHs or co-culture of both. ConclusionDue to the effect of scaffold materials, growth factors, and cell passages, optimal cell seeding density and cell ratio need further study.
Objective To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. Methods BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). Results CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic (P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group (P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group (P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group (P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant (P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP (P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group (P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group (P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group (P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased (P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group (P<0.05) except Tb.Th. Conclusion Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.
To investigate the mechanism of cAMP/Ca2+ signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into neuronal cells, we cultured the bone marrow mesenchymal stem cells D1 cells in the present study. D1 cells were divided into two groups: control group and salidroside inducing groups. Control group was cultured with complete culture solution D/F12, while salidroside inducing groups were induced with 100 mg·L–1 salidroside for different time periods (24, 48 and 72 hours). PCR-array assay was used to detect expression of 84 calcium related mRNA, and significantly different genes were chosen to analyse. As a result, there were 4 significantly upregulated mRNAs inclu-ding DNA damage-inducible transcript 3 (Ddit3), heat shock protein 5 (Hspa5), protein phosphatase 1 regulatory subunit (Ppp1r15a) and prostaglandin-endoperoxide synthase 2 (Ptgs-2), and 4 significantly downregulated mRNAs including glucagon (Gcg), interleukin 2 (Il2), tumor necrosis factor (Tnf) and somatostatin (Sst) in the cAMP/Ca2+ signaling pathway. They probably had an effect on the process of salidroside induced D1 cells differentiating into neuronal cells.
Objective To analyze the changes of gene expression profiles during the process that human bonemarrow mesenchymal stem cells (hBMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine (5-aza). Methods hBMSCs were isolated from marrow of obsolete ribs and induced with 5-aza. Then immunocytochemicalstaining was used to detect the expressions of α-actin, cardiac troponin T (cTnT), and connexin 43, and the percentage ofcTnT positive cells was tested with flow cytometry. In the process of differentiation, variation of gene expression was screenedwith Genechi ps Operating System of human gene expression profiles. And the differentially expressed genes were functionallyanalyzed and hierarchical clustered. Results When BMSCs were induced in vitro with 5-aza, part of the cells turnedinto myogenic cells morphologically. Before induction, immunocytochemical staining for α-actin and cTnT showed sl ightpositive and for connexin 43 showed negative. While after 3 weeks of induction, immunocytochemical staining for α-actin,cTnT, and connexin 43 showed all positive. With flow cytometry, the percentage of cTnT positive cells was 7.43% ± 0.02%before induction, but it was 49.64% ± 0.05% after induction. During differentiation, 1 814 differentially expressed geneswere reported by gene chi ps. Of them, 647 genes were divided into 5 groups with hierarchical clustering. They had variousbiological functions, involving signal transduction, cell metabol ism, prol iferation, differentiation, development, andtopogenesis. Conclusion hBMSCs can differentiate into cardiomyogenic cells with the induction of 5-aza in vitro. Multi plegenes related with signal transduction, transcri ption, and growth factors are involved during this process.
ObjectiveTo investigate the effect of graphene oxide (GO)-carboxymethyl chitosan (CMC) hydrogel loaded with interleukin 4 (IL-4) and bone morphogenetic protein 2 (BMP-2) on macrophages M2 type differentiation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).MethodsGO solution was mixed with CMC, then the phosphate buffered saline (PBS), IL-4, BMP-2, or IL-4+BMP-2 were added to prepare different GO-CMC hydrogel scaffolds with or without different cytokines under crosslinking agents. The characteristics of pure GO-CMC hydrogel were characterized by gross observation, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR), and the CMC hydrogel was used as control. The sustained release of GO-CMC hydrogels with different cytokines was also tested. Macrophages were isolated and cultured from female Sprague Dawley rats aged 4-5 weeks, and then cultured with GO-CMC hydrogels with and without different cytokines, respectively. CD206 immunofluorescence staining was used to detect the differentiation of macrophages after 24 hours. The 3rd generation of rats BMSCs were cultured with GO-CMC hydrogels with and without different cytokines respectively for osteogenic induction. The early osteogenesis was observed by alkaline phosphatase (ALP) staining after 10 days, and the late osteogenesis was observed by alizarin red staining after 21 days.ResultsGenerally, GO-CMC hydrogel was brown and translucent. SEM showed that the pore diameter and wall thickness of GO-CMC hydrogel were similar to that of CMC hydrogel, but the inner wall roughness increased. FTIR test showed that CMC polymerized to form hydrogel. In vitro, the sustained release experiments showed that the properties of GO-CMC hydrogels loaded with different cytokines were similar. CD206 immunofluorescence detection showed that GO-CMC hydrogels could induce macrophages differentiation into M2-type. ALP and alizarin red staining showed that GO-CMC hydrogels could induce BMSCs osteogenic differentiation, in which GO-CMC hydrogel loaded with IL-4+BMP-2 showed the most significant effect (P<0.05).ConclusionThe GO-CMC hydrogel loaded with IL-4 and BMP-2 can induce macrophages differentiation into M2-type and enhance the ability of BMSCs with osteogenic differentiation in vitro, which provide a new strategy for bone defect repair and immune regulation.
ObjectiveTo explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology.MethodsRabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression.ResultsHDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a " honeycomb” network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant (P>0.05).ConclusionThree-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.
This study aimed to characterize and magnetic resonance imaging (MRI) track the mesenchymal stem cells labeled with polylysine-coated superparamagnetic iron oxide (PLL-SPIO). Rat bone marrow derived mesenchymal stem cells (rMSCs) were labeled with 25, 50 and 100 μg/mL PLL-SPIO for 24 hours. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity and differentiation potential. Electron microscopic observations and Prussian blue staining revealed that 75%-100% of cells were labeled with iron particles. PLL-SPIO did not show any cytotoxicity up to 100 μg/mL concentration. Both 25 μg/mL and 50 μg/mL PLL-SPIO labeled stem cells did not exhibit any significant alterations in the adipo/osteo/chondrogenic differentiation potential compared to unlabeled control cells. The lower concentration of 25 μg/mL iron labeled cells emitted an obvious dark signal in T1W, T2WI and T2*WI MR image. The novel PLL-SPIO enables to label and track rMSCs for in vitro MRI without cellular alteration. Therefore PLL-SPIO may potentially become a better MR contrast agent especially in tracking the transplanted stem cells and other cells without compromising cell functional quality.
ObjectiveTo evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. Methods EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group (n=6) and porous β-TCP scaffold as the control group (n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. ResultsThe isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel (P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group (P<0.05), except for area fraction at 4 weeks after implantation (P>0.05). ConclusionThe prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.
ObjectiveTo prepare dopamine modified and cartilage derived morphogenetic protein 1 (CDMP1) laden polycaprolactone-hydroxyapatite (PCL-HA) composite scaffolds by three-dimensional (3D) printing and evaluate the effect of 3D scaffolds on in vitro chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsA dimensional porous PCL-HA scaffold was fabricated by 3D printing. Dopamine was used to modify the surface of PCL-HA and then CDMP-1 was loaded into scaffolds. The surface microstructure was observed by scanning electron microscope (SEM) and porosity and water static contact angle were also detected. The cytological experiment in vitro were randomly divided into 3 groups: group A (PCL-HA scaffolds), group B (dopamine modified PCL-HA scaffolds), and group C (dopamine modified and CDMP-1 laden PCL-HA scaffolds). The hBMSCs were seeded into three scaffolds, in chondrogenic culture conditions, the cell adhesive rate, the cell proliferation (MTT assay), and cell activity (Live-Dead staining) were analyzed; and the gene expressions of collagen type Ⅱ and Aggrecan were detected by real-time fluorescent quantitative PCR.ResultsThe scaffolds in 3 groups were all showed a cross-linked and pore interconnected with pore size of 400–500 μm, porosity of 56%, and fiber orientation of 0°/90°. For dopamine modification, the scaffolds in groups B and C were dark brown while in group A was white. Similarly, water static contact angle was from 76° of group A to 0° of groups B and C. After cultured for 24 hours, the cell adhesion rate of groups A, B, and C was 34.3%±3.5%, 48.3%±1.5%, and 57.4%±2.5% respectively, showing significant differences between groups (P<0.05). Live/Dead staining showed good cell activity of cells in 3 groups. MTT test showed that hBMSCs proliferated well in 3 groups and the absorbance (A) value was increased with time. The A value in group C was significantly higher than that in groups B and A, and in group B than in group A after cultured for 4, 7, 14, and 21 days, all showing significant differences (P<0.05). The mRNA relative expression of collagen type Ⅱ and Aggrecan increased gradually with time in 3 groups. The mRNA relative expression of collagen type Ⅱafter cultured for 7, 14, and 21 days, and the mRNA relative expression of Aggrecan after cultured for 14 and 21 days in group C were significantly higher than those in groups A and B, and in group B than in group A, all showing significant differences (P<0.05).ConclusionCo-culture of dopamine modified and CDMP1 laden PCL-HA scaffolds and hBMSCs in vitro can promote hBMSCs’ adhesion, proliferation, and chondrogenic differentiation.
ObjectiveTo investigate the effect of micro RNA (miR)-335-5p regulating bone morphogenetic protein 2 (BMP-2) on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs were cultured in vitro and randomly divided into control group (group A), miR-335-5p mimics group (group B), miR-335-5p mimics negative control group (group C), miR-335-5p inhibitor group (group D), and miR-335-5p inhibitor negative control group (group E). After grouping treatment and induction of osteogenic differentiation, the osteogenic differentiation of cells in each group was detected by alkaline phosphatase (ALP) and alizarin red staining; the expressions of miR-335-5p and BMP-2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) mRNAs were detected by real-time fluorescence quantitative PCR analysis; the expressions of Runx2, OPN, OCN, and BMP-2 proteins were detected by Western blot.ResultsCompared with group A, the relative proportion of ALP positive cells and the relative content of mineralized nodules, the relative expressions of BMP-2, miR-335-5p, OPN, OCN, Runx2 mRNAs, the relative expressions of Runx2, OPN, OCN, and BMP-2 proteins in group B were significantly increased (P<0.05); the above indexes in group D were significantly decreased (P<0.05); the above indexes between groups C, E and group A were not significantly different (P>0.05).ConclusionmiR-335-5p can up-regulate BMP-2 expression and promote osteogenic differentiation of hBMSCs.