Objective To summarize the regulations of Hedgehog signal ing pathway on the prol iferation and multidifferentiation of mesenchymal stem cells (MSCs). Methods The related l iterature in recent years concerning the regulations of Hedgehog signal ing pathway on the biological characteristics of MSCs was reviewed and analyzed. Results Hedgehog signal ing pathway promoted the prol iferation of MSCs, and played a major role in the induction of osteogenic and chondrogenic differentiations, but it inhibited the adi pocytic differentiation. Conclusion The regulations of Hedgehog signal ing pathway in MSCs multidifferentiation and prol iferation could be used as the new therapeutic targets of tissue ischemia, osteoporosis, achondroplasia, obesity, and so on.
Objective To study the effects of hypoxic preconditioning on the glucose metabol ism of rat BMSCs and its underlying mechanism so as to provide the theoretical basis for the optimization of the stem-cell based therapy. Methods Density gradient centrifugation method was adopted to isolate rat BMSCs from neonatal SD rats (aged 1-3 days). BMSCs were cultured to 4th passage and divided into 4 groups based on different culture conditions: group A in normoxia condition for 24 hours, group B in 1% O2 for 24 hours, group C in 2-methoxyestradiol (20 μmol/L) for 24 hours before hypoxic preconditioning, and group D in hypoxia-inducible factor 1 (HIF-1) specific siRNA (50 μmol/L) for 12 hours before hypoxicpreconditioning. MTT method was appl ied to evaluate the prol iferation of BMSCs. Biochemical analyzer and Real-timefluorescent quantitative PCR were appl ied to detect the glucose uptake, lactate production, and HIF-1α mRNA and Glut-1mRNA levels of BMSCs. Results MTT showed that the absorbance (A) values were 387.67 ± 58.92, 322.50 ± 50.60, 297.00 ± 53.00, and 286.00 ± 41.00 in groups A, B, C, and D, respectively, showing no significant difference among 4 groups (P gt; 0.05). The levels of glucose uptake and lactate production were higher in group B than in groups A, C, and D, showing significant differences (P lt; 0.05); the levels of groups C and D were higher than those of group A, but showing no significant difference (P gt; 0.05). The mRNA expressions of HIF-1α and Glut-1 elevated significantly in group B when compared with those in group A (P lt; 0.05); groups C and D were significantly lower than group B (P lt; 0.05) and were significantly higher than group A (P lt; 0.05). Conclusion Hypoxic preconditioning can stimulate the glucose uptake and metabol ism of rat BMSCs, whose mechanism is probably related to up-regulating the mRNA expressions of HIF-1α and Glut-1.
Objective To establish an effective model of myocardial infarction in black goat so as to provide a safe, convenient and credible model of myocardial infarction for treatment and research. Methods Sixteen black goats were made chronic myocardial infarction by ligation of far end of left anterior descending coronary artery through incision below xiphoidprocess. Electrocardiogram(ECG) and serum myocardial enzymes were investigated before and after occlusion. Echocardiographic measurements were performed, and left coronary artery angiography was performed with digital subtraction angiography (DSA) before infarction and 6 weeks after infarction. The myocardial ultrastructure were observed. Results All goats survived more than 6 weeks. ECG showed ambulatory change, ST-segment elevated half an hour after occlusion and pathologic Q waves 6 weeks after infarction, CK-MB significantly increased. Echocardiographic indexes showed significant decrease of maximal peak A, percent wall thickening(WHT) and ejecting fraction (EF), increase ofend-systolic volume (ESV), end-diastolic volume (EDV), and dilation of left ventricle. DSA showed block or decrease of perfusion of far end of left anterior descending coronary artery. Conclusion It is safe, convenient and credible to establish model of myocardial infarction by ligation of far end of left anterior descending coronary artery through incision below xiphoidprocess in black goat.
Objective To review the information of platelet gel used in the basic and clinical research in reparative and reconstructive surgery.Methods Literature about platelet gel used on the basic and clinical research was obtained through searching medical data and Internet. The effect of platelet gel on repairing and reconstructing the function and structure of tissue and organ was analyzed. Results Platelet gel had many growth factors and had the ability to improve wound healing and regenesis of bone and other tissues. Conclusion Platelet gel is widely available and almost genuine and is able to improve regenesis of many kinds of tissues. Extensive and intensive research should be made on itsclinical application.
Objective To explore the method of preparing spongy and porous scaffold materials with swine articular cartilage acellular matrix and to investigate its appl icabil ity for tissue engineered articular cartilage scaffold. Methods Fresh swine articular cartilage was freeze-dried and freeze-ground into microparticles. The microparticles with diameter of less than 90 μm were sieved and treated sequentially with TNE, pepsin and hypotonic solution for decellularization at cryogenic temperatures. Colloidal suspension with a mass/volume ratio of 2% was prepared by dissolving the microparticles into 1.5% HAc, and then was lyophil ized for molding and cross-l inked by UV radiation to prepare the decellularized cartilage matrix sponge. Physicochemical property detection was performed to identify aperture, porosity and water absorption rate. Histology and scanning electron microscope observations were conducted. The prepared acellular cartilage matrix sponge was implanted into the bilateral area of spine in 24 SD rats subcutaneously (experimental group), and the implantation of Col I sponge served as control group. The rats were killed 1, 2, 4, and 8 weeks after operation to receive histology observation, and the absorption and degeneration conditions of the sponge in vivo were analyzed. BMSCsobtained from femoral marrow of 1-week-old New Zealand white rabbits were cultured. The cells at passage 3 were cultured with acellular cartilage matrix sponge l ixivium at 50% (group A), acellular cartilage matrix sponge l ixivium at 100% (group B), and DMEM culture medium (group C), respectively. Cell prol iferation was detected by MTT method 2, 4, and 6 days after culture. Results The prepared acellular cartilage matrix sponge was white and porous. Histology observation suggested that the sponge scaffold consisted primarily of collagen without chondrocyte fragments. Scanning electron microscope demonstrated that the scaffold had porous and honeycomb-shaped structure, the pores were interconnected and even in size. The water absorption rate was 20.29% ± 25.30%, the aperture was (90.66 ± 21.26) μm, and the porosity of the scaffold was 90.10% ± 2.42%. The tissue grew into the scaffold after the subcutaneous implantation of scaffold into the SD rats, angiogenesis was observed, inflammatory reaction was mild compared with the control group, and the scaffold was degraded and absorbed at a certain rate. MTT detection suggested that there were no significant differences among three groups in terms of absorbance (A) value 2 and 4 days after culturing with the l ixivium (P gt; 0.05), but significant differences were evident among three groups 6 days after culturing with the l ixivium (P lt; 0.05). Conclusion With modified treatment and processing, the cartilage acellular matrix sponge scaffold reserves the main components of cartilage extracellular matrix after thorough decellularization, has appropriate aperture and porosity, and provides even distribution of pores and good biocompatibil ity without cytotoxicity. It can be used as an ideal scaffold for cartilage tissue engineering.
Objective To study the biological characteristic of rabbit bone marrow mesenchymal stem cells (BMSCs) double-labeled by PKH26 and BrdU in vitro, and to construct tissue engineered cardiac patch in vitro. Methods The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensitywere observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. Thecharacteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkal ine phosphatase (ALP) staining, Al izarin red staining, Oil red O staining, immunocytochemical technique of collagen type I, and osteocalcin expression. The labeled BMSCs were seeded on the small intestinal submucosa (SIS) and co-cultured for 5-7 days to construct tissue engineered cardiac patch. The patches were tested by inverted phase contrast microscope, fluorescent microscope, scanning electron microscope, and HE staining to observe the cell prol iferation. Results The double-labeled cells grew well and showed red fluorescence. There was no significant difference in the growth characteristic between the labeled and unlabeled cells. There was no significant difference in the expression of stem cell specific surface antigen between before lebel ing and after lebel ing. After osteogenic induction of labeled BMSCs, ALP staining and Al izarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, l ipid droplets could be observed in cytoplasm by Oil red O staining. After the co-culture in vitro for 5-7 days, the double-labeled cells grew well, showing a multi-layer cellular structure on the surface of SIS. Conclusion Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal abil ity and multipotent differentiation abil ity; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
Objective To observe the expressions of CXC chemokine receptor 4 (CXCR4) in muscle satell ite cells in situ of normal and cardiotoxin-intoxicated muscle tissues so as to further investigate the molecular mechanism involving inmuscle regeneration such as progressing muscular dystrophy (PMD) for seeking the way to cure muscle retrogression. Methods The muscle injured model of 12 C57 male mice was made by injecting cardiotoxin (5 μg per mouse) in left quadriceps femoris, their right quadriceps femoris was used as control without any injection. The histological, immunohistochemical analysis and RT-PCR were done to investigate the expression of CXCR4 in the quadriceps femoris in situ after 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks. Results HE staining results demonstrated that the muscle tissues experienced the process from muscle injury, repair to regeneration. The result of immunohistochemistry showed that the expressions of CXCR4 in injured muscle tissue were 1 955.6 ± 150.3, 2 223.2 ± 264.3, 2 317.6 ± 178.7, 3 066.5 ± 269.6, 1 770.9 ± 98.7 and 1 505.7 ± 107.1 at 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks after injection of cardiotoxin, there was significant difference when compared with normal muscle (640.3 ± 124.0, P lt; 0.001). The RT-PCR showed that the expressions of CXCR4 mRNA in injured muscle tissue were0.822 ± 0.013, 0.882 ± 0.025, 1.025 ± 0.028, 1.065 ± 0.041, 0.837 ± 0.011 and 0.777 ± 0.015 at 1 day, 4 days, 1 week, 2 weeks, 4 weeks and 6 weeks after injection of cardiotoxin, there was significant difference when compared with normal muscle (0.349 ± 0.006, P lt; 0.001). Conclusion CXCR4 may be the critical protein in the process of muscle impairment and reparation.
Objective To study the effect of hypoxia on the prol iferation of hBMSCs and human placental decidua basal is-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs,flow cytometry (FCM) was appl ied to detect cell surface marker. After establ ishing the experimental model of CoC12 chemical hypoxia, MTT method was appl ied to evaluate the prol iferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoC12 concentration (0, 50, 75, 100, 125, 150, 175, 200 μmol/L). Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC (HLA-ABC), but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The prol iferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably prol iferated 24 hours after hypoxia with CoC12 concentration of 150 µmol/L (P lt; 0.05), while hPDB-MSCs were significantly prol iferated 12 hours after hypoxia with CoC12 concentration of 75 µmol/L (P lt; 0.05). Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the prol iferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
Objective To study the effect of rat osteoblast conditioned culture medium on the BMSCs differentiation of allogeneic rat and to find a new approach to provide seed cells for bone tissue engineering. Methods BMSCs and osteoblasts were harvested from 10 healthy one-week-old SD rats (male and female, weighing 20-30 g) by adherent method and enzyme digestion method respectively. Cell identification was conducted. Osteoblast conditioned culture medium was prepared by mixing supernatant of osteoblasts at passage 1-5 with complete medium (1:1). Then, BMSCs at passage 2 were co-cultured with osteoblast conditioned culture medium (inducement group) and complete medium (control group), respectively. The morphological changes of co-cultured BMSCs were observed by inverted phase contrast microscope, the growth condition of BMSCs was detected by MTT method, the expressions of ALP, Col I and osteocalcin (OCN) in the cocultured BMSCs were tested by immunohistochemistry staining, and the expressions of Col I and OCN mRNA were detected by RT-PCR. Results In the inducement group, BMSCs grew bigger, changing from long fusiform to flat and polygon with protuberance 7 days after co-culture; the presence of cell colony-l ike growth was observed 9 days after co-culture. Cell growth curve demonstrated that the counts of BMSCs was increased with time, there were more cells in the control group than that of the inducement group, and there was a significant difference in cell counts between the control and the inducement group 4-7 days after co-culture (P lt; 0.05). For the inducement group, ALP staining was positive 12 days after co-culture, the calcium nodules were appeared 18 days after co-culture, Col I and OCN were positive 21 days after co-culture, and the expressions of Col I and OCN mRNA were detected by RT-PCR 21 days after co-culture. Conclusion Rat osteoblast conditioned culture medium can significantly induce the differentiation of allogeneic rats’ BMSCs towards osteoblasts.
Objective To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. Methods SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articularcartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multipl ication method were diluted by sodium alginate to (5-7) × 107 cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n=20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was establ ished in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. Results Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the inferface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in theimplant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly andactive cell metabol ism and prol iferation were evident 9 months after operation. While in the control group, no repair of thedefect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P lt; 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P lt; 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P lt; 0.05). Conclusion Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in l ine with physiological repair process of articular cartilage.