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.
ObjectiveTo explore the effects of concentrated growth factor (CGF) combined with mineralized collagen (MC) materials on the adhesion, proliferation, and differentiation of bone marrow mesenchymal stem cells (BMSCs) and their osteogenic effects in vivo, and to provide a theoretical basis for the combined application of CGF and MC materials in bone defect regeneration and repair.MethodsCGF was prepared from venous blood of healthy volunteers, and then CGF extracts (CGFe) were prepared. In vitro experiment: human BMSCs (hBMSCs) were divided into 4 groups. Groups A, B, and C were cultured with α-MEM medium [containing 10% fetal bovine serum (FBS) and 1% double antibody] containing 2%, 5%, and 10%CGFe, respectively; group D was cultured with α-MEM medium (containing 10%FBS and 1% double antibody) without CGFe. Scanning electron microscopy was used to observe the effect of CGFe on cell adhesion. Cell counting kit 8 (CCK-8) was used to detect the effect of CGFe on cell proliferation. After osteogenic induction, alkaline phosphatase (ALP) activity was detected and Western blot was performed to detect osteopontin (OPN) expression. In vivo experiment: Eighteen New Zealand big-eared rabbits were used to prepare circular bone defect models on the left and right mandibles, and implant CGF gel (prepared from autologous venous blood)+MC material (volume ratio 1∶1, experimental group) and simple MC material (control group), respectively. At 4, 8, and 12 weeks after operation, 6 rabbits were sacrificed respectively to obtain materials, and Micro-CT scanning was performed to observe the formation of new bone and material degradation in vivo.ResultsIn vitro experiments: Scanning electron microscopy showed that the cells of groups A, B, and C spread better on MC materials than group D, with more pseudopodia. CCK-8 method showed that different concentrations of CGFe could promote cell proliferation, and the absorbance (A) value of cells cultured for 2, 3, 5, and 7 days was in the order of group C>group B>group A>group D, the differences were significant (P<0.05). ALP activity test showed that its activity was proportional to the osteogenic induction time and CGFe concentration (P<0.05). Western blot analysis of osteogenic induction culture for 14 days showed that the relative expression of OPN protein in groups A, B, and C was significantly higher than that in group D, and the higher the CGFe concentration, the higher the relative expression of OPN protein (P<0.05). In vivo experiment: Micro-CT observation showed that the new bone formation and material degradation of the experimental group were better than those of the control group at 4, 8, and 12 weeks after operation. Quantitative detection showed that the volume of new bone volume, new bone volume fraction, trabeculae number, and trabecular thickness of the experimental group were significantly higher than those of the control group at each time point, the residual material volume, residual material volume fraction, and trabecular separation were significantly lower than those of the control group, all showing significant differences (P<0.05).ConclusionCGF can effectively promote the adhesion, proliferation, and osteogenic differentiation of BMSCs on MC materials, and 10%CGFe has the most significant effect. The combined application of CGF and MC material can significantly promote bone formation in vivo.
Objective To compare the cl inical outcomes of the core decompression combined with autologous bone marrow mesenchymal stem cells (BMSCs) transplantation with the isolated core decompression for the treatment of earlyavascular necrosis of the femoral head (ANFH). Methods From May 2006 to October 2008, 8 patients (16 hips) with earlyANFH were treated. There were 7 males and 1 female with an average age of 35.7 years (range, 19-43 years). According to the system of the Association Research Circulation Osseous (ARCO): 4 hips were classified as stage II a, 2 as stage II b, 1 as stage II c, and 1 as stage III a in group A; 2 hips were classified as stage II a, 2 as stage II b, 3 as stage II c, and 1 as stage III a in group B. The average disease course was 1.1 years (range, 4 months to 2 years). The patients were randomly divided into 2 groups according to left or right side: group A, only the core decompression was used; group B, both the core decompression and autologous BMSCs transplantation were used. The Harris score and visual analogue scale (VAS) score were determined, imaging evaluation was carried out by X-rays and MRI pre- and post-operatively. The erythrocyte sedimentation rate, C-reactive protein, l iver function, renal function, and immunoglobul in were detected for safety evaluation. Results All incisions healed by first intention. Eight patients were followed up 12-42 months (23.5 months on average). The cl inical symptoms of pain and claudication were gradually improved. The Harris scores and VAS scores of all patients were increased significantly at 3, 6, and 12 months after operation (P lt; 0.05). There was no significant difference between groups A and B 3 and 6 months after operation (P gt; 0.05), but there was significant difference between groups A and B 12 months after operation (P lt; 0.05). The necrosis area of femoral head in groups A and B were 18.13% ± 2.59% and 13.25% ± 2.12%, respectively, showing significant difference (P lt; 0.05). In group A, femoral head collapsed 12 months after operation in 1 case of stage III. No compl ication of fever, local infectionoccurred. Conclusion The core decompression and the core decompression combined with BMSCs transplantation are both effective for the treatment of early ANFH. The core decompression combined with BMSCs transplantation is better than core decompression in the rel ief of pain and postponing head collapse.
ObjectiveTo develop an anti-inflammatory poly (lactic-co-glycolic acid) (PLGA) scaffold by loading xanthohumol, and investigate its anti-inflammatory and cartilage regeneration effects in goats. Methods The PLGA porous scaffolds were prepared by pore-causing agent leaching method, and then placed in xanthohumol solution for 24 hours to prepare xanthohumol-PLGA scaffolds (hereinafter referred to as drug-loaded scaffolds). The PLGA scaffolds and drug-loaded scaffolds were taken for general observation, the pore diameter of the scaffolds was measured by scanning electron microscope, the porosity was calculated by the drainage method, and the loading of xanthohumol on the scaffolds was verified by Fourier transform infrared (FTIR) spectrometer. Then the two scaffolds were co-cultured with RAW264.7 macrophages induced by lipopolysaccharide for 24 hours, and the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by RT-PCR and Western blot to evaluate the anti-inflammatory properties in vitro of two scaffolds. Bone marrow mesenchymal stem cells (BMSCs) was obtained from bone marrow of a 6-month-old female healthy goat, cultured by adherent method, and passaged in vitro. The second passage cells were seeded on two scaffolds to construct BMSCs-scaffolds, and the cytocompatibility of scaffolds was observed by live/dead cell staining and cell counting kit 8 (CCK-8) assay. The BMSCs-scaffolds were cultured in vitro for 6 weeks, aiming to verify its feasibility of generating cartilage in vitro by gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, and biochemical analysis. Finally, the two kinds of BMSCs-scaffolds cultured in vitro for 6 weeks were implanted into the goat subcutaneously, respectively. After 4 weeks, gross observation, histological staining, collagen type Ⅱ immunohistochemical staining, biochemical analysis, and RT-PCR were performed to comprehensively evaluate the anti-inflammatory effect in vivo and promotion of cartilage regeneration of the drug-loaded scaffolds. Results The prepared drug-loaded scaffold had a white porous structure with abundant, continuous, and uniform pore structures. Compared with the PLGA scaffold, there was no significant difference in pore size and porosity (P>0.05). FTIR spectrometer analysis showed that xanthohumol was successfully loaded to PLGA scaffolds. The in vitro results demonstrated that the gene and protein expressions of inflammatory cytokines (IL-1β and TNF-α) in drug-loaded scaffold significantly decreased than those in PLGA scaffold (P<0.05). With the prolongation of culture, the number of live cells increased significantly, and there was no significant difference between the two scaffolds (P>0.05). The in vitro cartilage regeneration test indicated that the BMSCs-drug-loaded scaffolds displayed smooth and translucent appearance with yellow color after 6 weeks in vitro culture, and could basically maintained its original shape. The histological and immunohistochemical stainings revealed that the scaffolds displayed typical lacunar structure and cartilage-specific extracellular matrix. In addition, quantitative data revealed that the contents of glycosaminoglycan (GAG) and collagen type Ⅱ were not significantly different from BMSCs-PLGA scaffolds (P>0.05). The evaluation of cartilage regeneration in vivo showed that the BMSCs-drug-loaded scaffolds basically maintained their pre-implantation shape and size at 4 weeks after implantation in goat, while the BMSCs-PLGA scaffolds were severely deformed. The BMSCs-drug-loaded scaffolds had typical cartilage lacuna structure and cartilage specific extracellular matrix, and no obvious inflammatory cells infiltration; while the BMSCs-PLGA scaffolds had a messy fibrous structure, showing obvious inflammatory response. The contents of cartilage-specific GAG and collagen type Ⅱ in BMSCs-drug-loaded scaffolds were significantly higher than those in BMSCs-PLGA scaffolds (P<0.05); the relative gene expressions of IL-1β and TNF-α were significantly lower than those in BMSCs-PLGA scaffolds (P<0.05). ConclusionThe drug-loaded scaffolds have suitable pore size, porosity, cytocompatibility, and good anti-inflammatory properties, and can promote cartilage regeneration after implantation with BMSCs in goats.
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 whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
ObjectiveTo observe the change of stromal cell-derived factor 1α/cysteine X cysteine receptor 4 (SDF-1α/CXCR4) signaling pathway during the process of axial stress stimulation promoting bone regeneration, and to further explore its mechanism.MethodsA total of 72 male New Zealand white rabbits were selected to prepare the single cortical bone defect in diameter of 8 mm at the proximal end of the right tibia that repaired with deproteinized cancellous bone. All models were randomly divided into 3 groups (n=24). Group A was treated with intraperitoneally injection of PBS; Group B was treated with stress stimulation and intraperitoneally injection of PBS; Group C was treated with stress stimulation and intraperitoneally injection of AMD3100 solution. The X-ray films were taken and Lane-Sandhu scores of bone healing were scored at 2, 4, 8, and 12 weeks after operation, while specimens were harvested for HE staining, immunohistochemical staining of vascular endothelial growth factor (VEGF) and CXCR4, and Western blot (SDF-1α and CXCR4). The bone healing area was scanned by Micro-CT at 12 weeks after operation, and the volume and density of new bone were calculated.ResultsX-ray film showed that the Lane-Sandhu scores of bone healing in group B were significantly higher than those in groups A and C at 4, 8, and 12 weeks after operation (P<0.05). Micro-CT scan showed that the bone defect was repaired in group B and the pulp cavity was re-passed at 12 weeks after operation. The volume and density of new bone were higher in group B than in groups A and C (P<0.05). HE staining showed that the new bone growth in bone defect area and the degradation of scaffolds were faster in group B than in groups A and C after 4 weeks. The immunohistochemical staining showed that the expressions of VEGF and CXCR4 in 3 groups reached the peak at 4 weeks, and group B was higher than groups A and C (P<0.05). Western blot analysis showed that the expressions of SDF-1α and CXCR4 in group B were significantly higher than those in groups A and C at 4 and 8 weeks after operation (P<0.05).ConclusionAxial stress stimulation can promote the expression of SDF-1α in bone defect tissue, activate and regulate the CXCR4 signal collected by marrow mesenchymal stem cells, and accelerate bone regeneration in bone defect area.
ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.
ObjectiveTo evaluate the effect of bone morphogenetic protein 7 (BMP-7)/poly (lactide-co-glycolide) (PLGA) microspheres on in vitro proliferation and chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs).MethodsBMP-7/PLGA microspheres were fabricated by double emulsion-drying in liquid method. After mixing BMP-7/PLGA microspheres with the chondrogenic differentiation medium, the supernatant was collected on the 1st, 3rd, 7th, 14th, and 21st day as the releasing solution. The BMSCs were isolated from the bilateral femurs and tibias of 3-5 days old New Zealand rabbits, and the 3rd generation BMSCs were divided into 2 groups: microspheres group and control group. The BMSCs in microspheres group were cultured by 200 μL BMP-7/PLGA microspheres releasing solution in the process of changing liquid every 2-3 days, while in control group were cultured by chondrogenic medium. The cell proliferation (by MTT assay) and the glycosaminoglycan (GAG) contents (by Alician blue staining) were detected after chondrogenic cultured for 1, 3, 7, 14, and 21 days. The chondrogenic differentiation of BMSCs was observed by safranine O staining, toluidine blue staining, and collagen type Ⅱ immunohistochemistry staining at 21 days.ResultsMTT test showed that BMSCs proliferated rapidly in 2 groups at 1, 3, and 7 days; after 7 days, the proliferation of BMSCs in the control group was slow and the BMSCs in microspheres group continued to proliferate rapidly. There was no significant difference of the absorbance (A) value at 1, 3, and 7 days between 2 groups (P>0.05), but theA value at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05). Compared with control group at 21 days, in microsphere group, almost all nuclei were dyed bright red by safranine O staining, almost all the nuclei appeared metachromatic purple red by toluidine blue staining, and the most nuclei were yellow or brown by immunohistochemical staining of collagen type Ⅱ. Alcian blue staining showed that the content of GAG in 2 groups increased continuously at different time points; after 7 days, the increasing trend of the control group was slow and the microspheres group continued hypersecretion. There was no significant difference of the GAG content at 1, 3, and 7 days between 2 groups (P>0.05), but the GAG content at 14 and 21 days in microspheres group was significantly higher than that in control group (P<0.05).ConclusionBMP-7/PLGA microspheres prepared by double emulsion-drying in liquid method in vitro can promote proliferation and chondrogenic differentiation of rabbit BMSCs.
Seeding cells play an important role in the peripheral nerve damage repair. Seeding cells studied consequently in peripheral nerve are Schwann cells, bone marrow mesenchymal stem cells and neural stem cells. Schwann cells, the first seeding cells, are various unique glial cells in the peripheral nervous system, which can form the myelin sheath for insulation and package of the neuron projecting axons in the peripheral nervous system so that the conduction velocity of the nerve signal was accelerated. It can be proved that Schwann cells played an important role in the maintenance of peripheral nerve function and in the regeneration process after peripheral nerve injury. The second, bone marrow mesenchymal stem cells are the various mesenchymal stem cells mainly exist in the systemic connective tissues and organs. These functional stem cells are often studied at present, and it has been found that they have exuberant proliferation and differentiation potentials. Neural stem cells, mentioned the third in sequence, are the kind of pluripotent cells with multi-directional differentiation, which could conduct the self-renewal function, and generate and differentiate neurons, astrocytes and oligodendrocytes through asymmetric cell division. These three types of seed cells are discussed in this paper.
