Objective To observe the effect of You Gui Yin and MSCs interventional therapy on the early stage of avascular necrosis of the femoral head (ANFH) and its role of improving revascularization and reossification. Methods Twenty-four adult Beagle dogs weighing (10.0 ± 0.5) kg were divided into 4 groups (n=6): group A (model group), group B (You Gui Yin group), group C (MSCs intervention group), group D(You Gui Yin and MSCs intervention group). The model of ANFH at early stage was establ ished by l iquid-nitrogen cryopreservation method, and MSCs were isolated, cultured and labeled by BrdU. Three weeks after model ing, groups C and D received 1 mL MSCs with artery perfusion [(0.5-1.0) × 106/mL)], groups B and D received intragastric administration of 100 mL You Gui Yin per day, groups A and C received intragastric administration of 100 mL distilled water. Gross observation on femoral head was conducted 4 and 8 weeks after continuous treatment. Meanwhile, DSA and MRI were adopted to observe the quantity and the diameter of femoralhead blood vessel, histology and immunohistochemistry staining were performed to observe the expression of VEGF and BrdU, and the expression of VEGF mRNA was detected by real-time fluorescence quantitative RT-PCR. Results At 4 and 8 weeks after treatment, the figuration of the femoral head in group A was flat and mushroom-shaped, while it was relatively normal in groups B, C and D. DSA observation: the number and the diameter of blood vessel in groups C and D were increased, and the obstructed blood vessel was open. At 4 and 8 weeks after treatment, significant differences between group C and group D were evident in the number and the diameter of blood vessel (P lt; 0.05); compared with before treatment, the diameter of blood vessel in two groups were significantly improved (P lt; 0.05) and the number of blood vessel in group D was significantly increased (P lt; 0.05). MRI observation: compared with group A, groups B, C and D were obviously improved, especially group D, T1W showed lower signal, T2W and STIR showed no abnormal changes of signal. Histopathology and immunohistochemistry staining: compared with group A, the structure of groups B, C and D were obviously improved, the positive expression of VEGF in group D was significantly higher than that of other groups (P lt; 0.05), the positive rate of BrdU, the number of positive osteoblast and the number of positive blood vessel in group D was obviously higher than that of group C (P lt; 0.05). Real-time fluorescence quantitative RT-PCR detection: the expression of VEGF mRNA in group D was significantly higher than that of other groups (P lt; 0.05), the expression of VEGF in groups B, C and D was higher than that of group A (P lt; 0.05). Conclusion The combination of You Gui Yin and MSCs interventional treatment has significant therapeutic effects on the early-stage ANFH, can improve the blood supply of the necrotic femoral head, promote repair and prevent collapse.
【Abstract】 Objective To review the recent progress of BMSCs acting as seeding cell for tissue engineeredcartilage. Methods The recent ten years l iterature about BMSCs acting as seeding cell for tissue engineered cartilage was extensively reviewed. Results Scaffold provided an optimal environment for the growth of BMSCs. Cytokine and gene del ivery could promote BMSCs to differentiate toward chondrocytes. All of them played important roles in the field of cartilage tissue engineering. Conclusion The improvement of three-dimensional scaffolds, the rational use of cytokine, and the enhancement of gene del ivery will promote the development of cl inical cartilage reconstruction.
Objective To confirm the stimulating effect of simvastatin on BMSCs of SD rats osteogenic differentiation, and to further study the role of Wnt signal ing pathway in this process. Methods BMSCs derived from the tibia and femur of 6-week-old female SD rats were cultured in vitro．Two groups were establ ished: control group and experimental group. After the 2nd passage, the cells of experimental group were treated with simvastatin (1 × 10-7mol/L) and the cells of control group with absolute ethyl alcohol and PBS. ALP staining was used at 7 days and von Kossa staining was appl ied at 28 days to assess osteoblastic differentiation and mineral ization. Real-time quantitative PCR was performed to evaluate theexpressions of Axin2, β-catenin, osteocalcin (OC), frizzled-2, Lef-1, and Wnt5a mRNA at 7 days and 14 days after simvastatin treatment. Results The observation of inverted phase contrast microscope showed that the majority of cells were polygonal and triangular in the experimental group, and were spindle-shaped in the control group at 7 days. The ALP staining showed blue cytoplasm, the positive cells for ALP staining in the experimental group were more than those in the control group at 7 days. The von Kossa staining showed that mineral ization of extracelluar matrix at 28 days in two groups, but the mineral ization in the experimental group was more obvious than that in the control group. The expression of Axin2 mRNA was significantly lower, and frizzled-2, Lef-1 mRNA were significantly higher in the experimental group than in the control group (P lt; 0.05) at 7 days, while the mRNA expressions of Axin2, OC, frizzled-2, Lef-1, and Wnt5a were significantly higher in the experimental group than in the control group at 14 days (P lt; 0.05). Conclusion Simvastatin can promote the osteogenic differentiation of BMSCs and change the expression of mRNA of some components of Wnt signal ing pathway.
【Abstract】 Objective To approach the possibil ity of combination of simvastatin and BMSCs transplantation forsteroid-associated osteonecrosis of femoral head. Methods The BMSCs harvested from 24 rabbits were prepared for cell suspension at a concentration of 1 × 107/mL, and combined with gelatin sponge. Seventy New Zealand white rabbits received one intravenous injection of l ipopolysaccharide (10 μg/ kg). After 24 hours, three injections of 20 mg/kg of methylprednisolone were given intramuscularly at a time interval of 24 hours. Forty-eight rabbits diagnosed as having femoral head necrosis by MRI were divided into 4 groups randomly, group A: no treatment; group B: only decompression; group C: decompression and BMSCs transplantation; and group D: simvastatin drench (10 mg/kg.d) decompression and BMSCs transplantation. The general information of animals were recorded; after 4 and 8 weeks of operation, 6 rabbits of each group were chosen randomly to do MRI scan, and femoral heads were harvested to do histopathology and scanning electron microscope examination. Results After 8 weeks, rabbits became more active than before treatment, and walking way became normal gradually in groups C and D. Fourweeks after operation, the MRI low signal region of all groups had no obvious changes, but 8 weeks later, the necrosis signal region of group A magnified while it reduced obviously in group D. Histopathological observation: 4 weeks after operation, diffuse presence of empty lacunae and pyknotic nuclei of osteocytes were found in the trabeculae, and few newborn micrangium could been seen in group A; lots of empty lacunae and a small quantity of newborn micrangium could been found in group B; and large amounts of osteoblats and newborn micrangium were found around the necrosis regions in groups C and D. The positive ratio of empty lacunae and microvessel density in group D were 19.30 ± 1.52 and 7.08 ± 1.09, showing significant difference compared with other groups (P lt; 0.05). After 8 weeks of treatment, the bone trabecula collapsed in many regions in group A; there was fibra callus formation along the decompression channel in group B; few empty lacunae was in the bone trabecular, but the shape of marrow cavity was not normal in group C; and it showed almost normal appearance in group D. The positive ratio of empty lacunae and microvessel density in group D were 11.31 ± 1.28 and 12.37 ± 1.32, showing significant differences compared with other groups (P lt; 0.05), meanwhile, showing significant difference compared with that of 4 weeks after operation(P lt; 0.05). Scanning electron microscope: 8 weeks after operation, the bone trabecula collapsed in many regions, and few osteoblasts could be found on the surface, a great quantity of fat cells cumulated in the bone marrow in group A; cracked bone trabecula could be found occasionally in group B; the density of bone trabecula was lower than the normal in group C; and the shape of the marrow avity and thedensity of bone trabecula were similar to the normal in group D. Conclusion Simvastatin can promote the differentiation of osteocyte and vascular endothel ial cell from MSCs, the combination of simvastatin and marrow stem cells transplantation for the treatment of steroid-associated osteonecrosis of femoral head have good appl ication prospects.
Objective To investigate the effects of intermittent negative pressure on the mRNA expression of osteoprotegerin (OPG) and osteoprotegerin l igand (OPGL) in human BMSCs cultured in vitro. Methods BMSCs were isolated from adult marrow donated by 2 hip osteoarthritis patients with prosthetic replacement in January 2008 and cultured in vitro. The third passage cells were divided into experimental group and control group. The experimental group was induced by negative pressure intermittently for 2 weeks (pressure: 50 kPa, 30 minutes each time, twice per day) and the control groupwas routinely cultured. After 2 weeks of culture, cell morphology was observed by inverted phase contrast microscope, and the mRNA expressions of OPG and OPGL in BMSCs were analyzed by real-time PCR. Results The cell prol iferation speed of the experimental group was slower than that of the control group. The cell morph changed from shuttle to megagon with some prominences in experimental group and the cell morph kept shuttle in the control. The mRNA expression of OPG in experimental group increased significantly (P lt; 0.01) and the mRNA expression of OPGL in experimental group decreased significantly compared with control group (P lt; 0.01) 2 weeks later. Conclusion Intermittent negative pressure is capable of promoting the expression of OPG, while inhibiting the expression of OPGL in human BMSCs.
Objective To explore the label ing efficiency and cellular viabil ity of rabbit BMSCs labeled with different concentrations of superparamagnetic iron oxide (SPIO) particles, and to determine the feasibil ity of magnetically labeled stem cells with MR imaging. Methods The BMSCs were collected from il iac marrow of 10 adult rabbits (weighing 2.5-3.0 kg) and cultured. The SPIO-poly-L-lysine compound by different ratios mixed with medium, therefore, the final concentration of Fe2+ was 150 (group A), 100 (group B), 50 (group C) and 25 μg (group D) per mL, respectively, the 3rd generation BMSCs culture edium was added to lable; non-labeled cells served as a control (group E). MR imaging of cell suspensions was performed by using T1WI and T2WI sequences at a cl inical 1.5 T MRI system. Results BMSCs were efficiently labeled with SPIO, labeled SPIO particles were stained in all cytoplasms of groups A, B, C and D. With the increasing of Fe2+ concentration, blue dye particles increased. The staining result was negative in group E. The cell viabil ity in groups A, B, C, D and E was 69.20% ± 6.11%, 80.41% ± 2.42%, 94.32% ± 0.67%, 96.24% ± 0.34% and 97.43% ± 0.33%, respectively. There were statistically significant differences between groups A, B and groups C, D and E (P lt; 0.05), and between group A and group B (P lt; 0.05). T1WI images had no specific difference among 5 groups, T2WI images decreased significantly in groups A, B, C, decreased sl ightly in group D, and had l ittle change in group E. The T2WI signal intensities of groups A, B, C, D and E were 23.37 ± 6.21, 26.73 ± 3.60, 29.63 ± 2.82, 45.03 ± 6.76 and 783.15 ± 7.38, respectively, showing significant difference between groups A, B, C, D and group E (P lt; 0.05), and between groups A, B, C and group D (Plt; 0.05). Conclusion BMSCs can be easily and efficiently labeled by SPIO without interference on the cell viabil ity in labled concentration of 20-50 μg Fe2+ per mL. MRI visual ization of SPIO labeled BMSCs is feasible, which may be critical for future experimental studies.
【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
Objective To study the growth characteristics of umbil ical cord MSCs (UCMSCs) in vitro and its effect on the nerve regeneration after spinal cord injury (SCI). Methods UCMSCs isolated from pregnant rats umbil ical cord were cultured and purified in vitro. Sixty female Wistar rats weighing (300 ± 10) g were randomized into three groups (n=20per group). UCMSCs group (group A) in which UCMSCs suspension injection was conducted; DMEM control group (groupB) in which 10% DMEM injection was conducted; sham group (group C) in which the animal received laminectomy only.Establ ish acute SCI model (T10) by Impactor model-II device in group A and group B. The recovery of the lower extremity was observed using BBB locomotor scoring system, neurofilament 200 (NF-200) immunofluorescence staining was performed to detect the neural regeneration, and then the corticospinal tract (CST) was observed using the biotinylated dextran amine (BDA) tracing. Results Cultured UCMSCs were spindle-shaped fibrocyte-l ike adherent growth, swirl ing or parallelly. The USMSCs expressed CD29, but not CD31, CD45, and HLA-DR. The BBB score was higher in group A than group B 4, 5, and 6 weeks after operation, and there was a significant difference between two groups (P lt; 0.05). The BBB scores at different time points were significantly lower in groups A and B than that in group C (P lt; 0.05). UCMSCs was proved to survive and assemble around the injured place by frozen section of the cords 6 weeks after injury. NF-200 positive response area in groups A, B, and C was (11 943 ± 856), (7 986 ± 627), and (13 117 ± 945) pixels, respectively, suggesting there was a significant difference between groups A, C and group B (P lt; 0.05), and no significant difference was evident between group A and group C (P gt; 0.05). BDA anterograde tracing 10 weeks after operation demonstrated that more regenerated nerve fibers went through injured area in group A, but just quite few nerve fibers in group B went through the injuried cavity. The ratios of regenerative axons amount to T5 axons in group A and group B were smaller than that of group C (P lt; 0.05). Conclusion UCMSCs can prol iferate rapidly in vitro, survive and differentiate to neurons after being grafted into injured spinal cord. The transplantation of UCMSCs is effective in promoting functional recovery and axonal regeneration after SCI.
Objective To investigate a new composite matrix (BMSCs seeded on the denuded human amniotic membrane, BMSCs-DHAM) bridging the both stumps of spinal cord injury in rats to promote axon regeneration and improve motor function of hind l imbs. Methods The human amniotic membrane (HAM) was voluntarily donated by the healthy pregnant women after a caesarean section. The cells on the HAM were completely removed with a tryptic and mechanical approach to prepare DHAM. The BMSCs were separated and cultured from 4-week-old female rats (n=4), then the forth passage of BMSCs were labeled by PKH26 and seeded on DHAM (BMSCs-DHAM). The growing state of BMSCs was observed under themicroscopy. Moreover, 40 female rats (8-week-old, weighting 200-220 g) were made spinal cord injury models by transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). The both stumps were respectively wrapped by BMSCs- DHAM or simple DHAM in groups A and C, and the same dose of BMSCs or physiological sal ine were also respectively injected the central lesion in groups B and D. At 12 weeks after surgery, the functional recovery of the hindl imbs was evaluated by the BBB locomotor rating score, and other indexes were tested including cortical motion evoked potential (MEP), anterograde biopinylated dextan amine (BDA) tracing, and immunofluorescence of neurofilament protein 200 (NF-200). Results HE staining proved that the DHAM was devoid of cellular components by this way, and BMSCs grew well on the substrate under the microscopy. At 12 weeks after operation, the BBB score (12.50 ± 1.26) in group A was significantly higher than those of other groups (P lt; 0.05), and the recovery in latency (3.52 ± 2.45) ms and ampl itude (480.68 ± 18.41) μV of MEP was also obviously improved in group A (P lt; 0.05) when compared with other groups. In addition, anterograde BDA tracing revealed that the rate of the positive BDA axons 54.12% ± 3.30% under the lesion level in group A was higher than those of other groups (P lt; 0.05), and lots of the regeneration axons (positive NF-200) were found to grow into the spinal cord under the composite matrix in group A. Conclusion The BMSCs-DHAM composite matrix can improve hindl imb motor function to some extent after spinal cord injury. It will be widely appl ied as the matrix material in the future.
Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.