【Abstract】 Objective To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation ofBMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, maleor female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Al izarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL reached (300 ± 30), (140 ± 25), (80 ± 35), (70 ± 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-l ike cells at 7 days. The cells in group A showed slower shape changesbut higher prol iferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense gro wth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells ofgroup A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P lt; 0.05). At the 20 days, Al izarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 ± 1.10, 12.87 ± 0.81 and 15.59 ± 0.25, respectively, while the area of mineral deposits were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24), and (415 921.70 ± 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P lt;0.05). But there was no statistically significant difference between groups B and C (P gt; 0.05). Conclusion PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.
Objective To observe the effect of BMSCs on the cardiac function in diabetes mellitus (DM) rats through injecting BMSCs into the ventricular wall of the diabetic rats and investigate its mechanism. Methods BMSCs isolated from male SD rats (3-4 months old) were cultured in vitro, and the cells at passage 5 underwent DAPI label ing. Thirty clean grade SD inbred strain male rats weighing about 250 g were randomized into the normal control group (group A), the DM group (group B), and the cell transplantation group (group C). The rats in groups B and C received high fat forage for 4 weeks and the intraperitoneal injection of 30 mg/kg streptozotocin to made the experimental model of type II DM. PBS and DAPI-labeledpassage 5 BMSCs (1 × 105/μL, 160 μL) were injected into the ventricular wall of the rats in groups B and C, respectively. After feeding those rats with high fat forage for another 8 weeks, the apoptosis of myocardial cells was detected by TUNEL, the cardiac function was evaluated with multi-channel physiology recorder, the myocardium APPL1 protein expression was detected by Western blot and immunohistochemistry test, and the NO content was detected by nitrate reductase method. Group C underwent all those tests 16 weeks after taking basic forage. Results In group A, the apoptosis rate was 6.14% ± 0.02%, the AAPL1 level was 2.79 ± 0.32, left ventricular -dP/dt (LV-dP/dt) was (613.27 ± 125.36) mm Hg/s (1 mm Hg=0.133 kPa), the left ventricular end-diastol ic pressure (LVEDP) was (10.06 ± 3.24) mm Hg, and the NO content was (91.54 ± 6.15) nmol/mL. In group B, the apoptosis rate was 45.71% ± 0.04%, the AAPL1 level 1.08 ± 0.24 decreased significantly when compared with group A, the LVdP/ dt was (437.58 ± 117.58) mm Hg/s, the LVEDP was (17.89 ± 2.35) mm Hg, and the NO content was (38.91±8.67) nmol/mL. In group C, the apoptosis rate was 27.43% ± 0.03%, the APPL1 expression level was 2.03 ± 0.22, the LV -dP/dt was (559.38 ± 97.37) mm Hg/ s, the LVEDP was (12.55 ± 2.87) mm Hg, and the NO content was (138.79 ± 7.23) nmol/ mL. For the above mentioned parameters, there was significant difference between group A and group B (P lt; 0.05), and between group B and group C (P lt; 0.05). Conclusion BMSCs transplantation can improve the cardiac function of diabetic rats. Its possible mechanismmay be related to the activation of APPL1 signaling pathway and the increase of NO content.
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.
Objective To study repair of osteochondral defects by using composite of autologous BMSCs and chitosan/HAP (CS/HAP) bilayered scaffold in rabbits and its feasibil ity as osteochondral tissue engineering scaffolds. Methods CS/HAP bilayered scaffolds were produced with CS and HAP using a lyophil ization and sintering method. The pore size of the scaffold was observed by scanning electron microscopy (SEM). Anhydrous ethanol substitution method determined its porosity. BMSCs were isolated from bone marrow and cultured by general bone marrow methods. Both CD44 and CD45 on the BMSCs surface were detected with immunocytochemistry to identify BMSCs. Cell-scaffold complex was made with BMSCs as seed cells and CS/HAP bilayered scaffold as carrier by fibrin glue planting technique. The distribution ofBMSCs in CS/HAP scaffold was tested by SEM. The osteochondral defect (4 mm in diameter and 3 mm in height) model was made in the right knee joint of 36 Japanese white rabbits, which were randomly divided into 3 groups. Defects were repaired with CS/HAP and BMSCs composite ( group A, n=12) and with CS/HAP implants (group B, n=12); defects were not treated as a control (group C, n=12). Histological evaluation and gross observation were carried out at 6 weeks (n=6 in each group) and 12 weeks (n=6 in each group) postoperatively. Semi-quantitative histomorphological analysis was done to evaluate the repair cartilage tissue according to the modified Wakitani grading scale. Results CS/HAP bilayered scaffold possessed a porosity of 76.00% ± 5.01% and pore size of 200-400 μm (mean 300 μm ) in CS layer, and 72.00% ± 4.23% and 200-500 μm (mean 350 μm) in HAP layer, respectively. BMSCs formed colonies within 10-14 days. Immunocytochemistry results showed BMSCs had positive CD44 expression and negative CD45 expression. At 6 and 12 weeks after operation, gross and histological observation showed that the cartilage defects were fully filled with regenerated tissue, but bone defects were partially repaired in group A; the cartilage and bone defects were partially filled with regenerated tissue in group B and group C. The modified Wakitani grading scale were 5.17 ± 1.17 and 3.20 ± 0.75 in group A, 9.00 ± 0.63 and 6.00 ± 0.89 in group B, and 10.00 ± 0.89 and 9.60 ± 0.82 in group C at 6 weeks and 12 weeks postoperatively, respectively; showing significant differences between group A and groups B, C (P lt; 0.05). Conclusion The novel CS/HAP bilayered scaffold possesses porous structure and will possibly become a newbiomaterial of osteochondral tissue engineering.
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.
【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 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.
【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 investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.