Xiuqun Li

Sichuan University, Chengdu, Sichuan Sheng, China

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Publications (36)16.62 Total impact

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    ABSTRACT: To prepare human acellular adipose tissue matrix and to evaluate the cellular compatibility so as to explore a suitable bio-derived scaffold for adipose tissue engineering.
    03/2014; 28(3):377-83.
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    ABSTRACT: We have certified that the injectable thermosensitive ABM/PECE composite presented promising potential in bone regeneration benefited from the incorporation of the intrinsic osteoinductive acellular bone matrix (ABM) granules into the poly(ethylene glycol)-poly(ε-capro-lactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) hydrogel. In this study, the 12 mm × 8 mm × 2 mm cranial defects of the New Zealand white rabbits were fabricated to evaluate the bone regeneration effect. The ABM/PECE composite was injected into the defect while the pure PECE as control, and the bone regeneration was evaluated at 4, 12 and 20 weeks post-surgery by X-radiological examination, micro-computed tomography examination and histological analysis. In ABM/PECE composite treated group, the new bone formed originally from both the margin and the center of the defect, and the defect region had healed up to 20 weeks. Furthermore, the shadow density of the newly formed bone eventually approximated to host cortical bone. In comparison, the control group was filled with sparing new bone with low-density only from the periphery of the defect. Meanwhile, the quantitative determination of new bone by histomorphometry confirmed the excellent bone regeneration of ABM/PECE composite. All the results suggested that the ABM/PECE composite presented enhanced bone regeneration guidance in rabbit cranial defects.
    Biomaterials 10/2013; · 8.31 Impact Factor
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    ABSTRACT: Extracellular matrix is one of the focus researches of the adipose tissue engineering. To investigate the appropriate method to prepare the porcine skeletal muscle acellular matrix and to evaluate the biocompatibility of the matrix. The fresh skeletal muscle tissues were harvested from healthy adult porcine and were sliced into 2-3 mm thick sheets, which were treated by hypotonic-detergent method to remove the cells from the tissue. The matrix was then examined by histology, immunohistochemistry, and scanning electron microscopy. The toxic effects of the matrix were tested by MTT. Human adipoSe-derived stem cells (hADSCs) were isolated from adipose tissue donated by patients with breast cancer, and identified by morphology, flow cytometry, and differentiation ability. Then, hADSCs of passage 3 were seeded into the skeletal muscle acellular matrix, and cultured in the medium. The cellular behavior was assessed by calcein-AM (CA) and propidium iodide (PI) staining at 1st, 3rd, 5th, and 7th days after culturing. Histology, immunohistochemistry, and scanning electron microscopy showed that the muscle fibers were removed completely with the basement membrane structure; a large number of collagenous matrix presented as regular network, porous-like structure. The cytotoxicity score of the matrix was grade 1, which meant that the matrix had good cytocompatibility. The CA and PI staining showed the seeded hADSCs had the potential of spread and proliferation on the matrix. Porcine skeletal muscle acellular matrix has good biocompatibility and a potential to be used as an ideal biomaterial scaffold for adipose tissue engineering.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 06/2012; 26(6):749-54.
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    ABSTRACT: This paper is aimed to investigate the feasibility of applying the small intestine submucosa (SIS) as the scaffold in constructing tissue engineering cartilage in vitro. We obtained SIS from the small intestine of specific pathogen-free pigs. Then we isolated tunica submucosa layer from the mucosal, muscular, and serosal layers by gentle mechanic abrasion. The SIS was made acellular by combination of detergent and enzyme digestion. The chondrocytes were seeded onto the SIS and were cultured for 3 weeks. The cell growth, attachment and distribution were detected by histochemical stain, immunohistochemical stain and scan electron microscope. The chondrocytes could adhere and grow well on the matrix surface, and synthesize a large of the GAG and type U collagen. However, the chondrocytes grew only on the surface andsuperficial layer of the scaffold, they did not move into the inner part of the scaffold. It could be concluded that SIS has good cellular compatibility without cytotoxicity and provides temporary substrate to which these anchorage-dependent cells can adhere, and stimulate the chondrocytes anchored on the scaffold to proliferate and keep differentiated phenotype. Further study will be needed to promote the ability of chondrocyte chemotaxis in order to distribute the chondrocytes into the whole scaffold uniformly.
    Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 06/2011; 28(3):521-5.
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    ABSTRACT: To study the possibility of bone marrow mesenchymal stem cells (BMSCs) differentiation into tenocytes (TCs) under strain stimulation by co-culture of BMSCs-small intestinal submucosa (SIS) composites in vitro. BMSCs were isolated by adherent culture from the bone marrow of 1-week-old SD rats. Inducing method of multiple differentiation and flow cytometry were applied to identify the cells. The stress-strain curve of SIS was measured with Instron machine. Purified BMSCs (2nd passage, 2.5 x 10(5) cells/cm2) were seeded on SIS (3 cm x 1 cm at size) and cultured for 2 days and then continued for another 5 days under strain stimulation (stretching frequency was 0.02 Hz, action time was 15 minutes/hour and 12 hours/day, strain amplitude was 5%) as experimental group, while the BMSCs-SIS composites were sustained static culture as control group. TCs were isolated from tail of 1-week-old SD rats. TCs-SIS composites were cultured under non-strained as positive control group. Scanning electron microscope (SEM) was used to examine the morphological changes of BMSCs after strain stimulation. The contents of Scleraxis and Tenomodulin in supernatant were tested by ELISA kit. Results The BMSCs could be induced to differentiate into osteoblasts and lipocytes, and showed the results of CD34-, CD45-, and CD90+, which were accorded with the biological characteristics of BMSCs. The failure test of SIS showed that the average elastic strain was 39.5%. SEM observation showed that the strain-stimulated BMSCs had the TCs-like morphological characteristics. The contents of Scleraxis and Tenomodulin in supernatant of experimental group, control group, and positive control group were (3.56 +/- 0.91) micromol/L and (4.27 +/- 1.10) micromol/L, (0.23 +/- 0.14) micromol/L and (0.16 +/- 0.10) micromol/L, and (14.73 +/- 2.30) micromol/L and (10.65 +/- 1.51) micromol/L, respectively. There were significant differences among 3 groups (P < 0.05). Appropriate strain stimulation could induce BMSCs differentiate into TCs, and the best conditions of strain stimulation need more experiments.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 07/2010; 24(7):817-21.
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    ABSTRACT: 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. The BMSCs were harvested from 6-month-old New Zealand rabbits and labeled with PKH26 and BrdU. The growth and fluorescent intensity were observed by inverted phase contrast microscope, fluorescent microscope, flow cytometry, and MTT detection. The characteristics of double-labeled BMSCs differentiating into osteoblasts and adipocytes, respectively, in vitro were identified by alkaline phosphatase (ALP) staining, Alizarin 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 proliferation. 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 labeling and after labeling. After osteogenic induction of labeled BMSCs, ALP staining and Alizarin red staining were positive, and the cells expressed collagen type I and osteocalcin. After adipocytes induction, lipid 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. Rabbit BMSCs can be double-labeled with PKH26 and BrdU stably. The labeled cells still have the potential of self-renewal ability and multi potent differentiation ability; tissue engineered cardiac patch can be constructed by co-culturing labeled BMSCs and SIS in vitro.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 07/2010; 24(7):828-33.
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    ABSTRACT: 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. SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articular cartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3 weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multiplication method were diluted by sodium alginate to (5-7) x 10(7) 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 established 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. 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 interface 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 the implant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly and active cell metabolism and proliferation were evident 9 months after operation. While in the control group, no repair of the defect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9 months 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 < 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P < 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P < 0.05). 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 line with physiological repair process of articular cartilage.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 08/2009; 23(8):974-9.
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    ABSTRACT: To investigate the feasibility of inducing canine BMSCs to differentiate into epithelial cells in vitro with epithelial cell conditioned medium (ECCM). Five mL BMSCs were obtained from iliac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured. The oral mucosa was harvested and cut into 4 mm x 4 mm after the submucosa tissue was eliminated; ECCM was prepared. BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group. The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting. The cells were identified by immunohistochemical staining through detecting cytokeratin 19 (CK-19) and anti-cytokeratin AE1/AE3 on the 21st day of induction. The ultra-structure characteristics were observed under transmission electron microscope. The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope. The cell growth curves of two groups presented S type. The cell growth curve of the experimental group was right shifted, showing cell proliferation inhibition in ECCM. The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, confirming the epithelial phenotype of the cells; while the result was negative in the control group. The cells were characterized by tight junction under transmission electron microscope. The canine ECCM can induce allogenic BMSCs to differentiate into epithelial cells in vitro.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 06/2009; 23(5):612-6.
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    ABSTRACT: To detect the influence of raloxifene (RLX) on fracture healing in rabbit. Eight healthy New Zealand white rabbits (44 females and 36 males) weighing 1.9-2.1 kg were used. A 0.5-cm bone defect model in the mid-diaphysis of the left forelimb radius was established in 72 rabbits, which thereafter were divided into 4 groups (n = 18 per group, 10 females and 8 males): groups A, B and C received 7.5, 15.0 and 30.0 mg/(kg x d) RLX, respectively, from the 2nd to the 50th postoperative day; group D received no further treatment. The rest untreated 8 rabbits (4 females and 4 males) served as normal control for serum osteocalcin detection. At different postoperative time points, bone mineral density detection, X-ray scanning, biomechanics measurement, histology and immunohistochemistry observations were conducted; serum estradiol, plasma cholesterol, serum osteocalcin and the ratio of uterine weight to body weight were detected. The bone mineral density of each group reached a peak 20 days after operation, showing a significant difference between groups A, B and C and group D (P < 0.05), and no significant differences among groups A, B and C (P > 0.05). On the 30th and 50th postoperative day, the maximum failure load and the maximum displacement of groups A, B and C were greater than those of group D (P < 0.05), but no significant differences among groups A, B and C were evident (P > 0.05). On the 7th, 20th and 30th postoperative day, the X-ray score of fracture healing of groups A, B and C was greater than group D (P < 0.05); on the 50th postoperative day, there was significant difference between groups B and C and group D, and between group A and group C (P < 0.05), and no significant difference was evident between group B and group C (P > 0.05). The percentage of new bone formation in the fractured area of groups A, B and C was greater than that of group D on the 30th and 50th postoperative day (P < 0.05). For the type II collagen protein secretion in the fractured area, groups B and C were superior to group D on the 30th postoperative day (P < 0.05), and there was no significant difference between group A and group D (P > 0.05); no significant differences among four groups were evident on the 50th postoperative day (P > 0.05). On the 10th, 30th and 50th postoperative day, the serum osteocalcin of groups A, B, C and D was higher than that of normal control (P < 0.05), groups B and C were higher than group D (P < 0.05), and there was no significant difference between groups A, B and C, and between group A and group D (P > 0.05). For the plasma cholesterol, on the 30th postoperative day, no significant change was detected in each group (P > 0.05); on the 50th postoperative day, obvious decrease was observed in groups A, B and C, showing a significant difference compared with group D (P < 0.05). On the 30th and 50th postoperative day, there was significant difference between groups B and C and group D in serum estradiol (P < 0.05), and no significant differences were evident among other groups (P > 0.05). On the 30th and 50th postoperative day, the ratio of uterine weight to body weight in groups B and C was less than that of group D (P < 0.05), and no significant difference was evident between group A and group C (P > 0.05). Oral administration of 7.5 mg/(kg x d) RLX can promote the fracture healing of rabbit radius defect models safely and effectively.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 06/2009; 23(6):683-9.
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    ABSTRACT: To investigate the feasibility of polypropylene mesh (PPM) coated with SIS to reconstruct tracheal defect and the efficiency of SIS in improving epithelialization of the reconstructed trachea and reducing the postoperative complications. Twelve New Zealand white rabbits were chosen and divided randomly into 2 groups: PPM reconstruction group (n=6) and SIS-PPM reconstruction group (n=6). A tracheal full defect with a size of 1.2 cm x 0.6 cm was created. A PPM coated with SIS of 1.4 cm x 0.8 cm was inserted to the defect in SIS-PPM reconstruction group, pure PPM of 1.4 cm x 0.8 cm in PPM reconstruction group. Complications such as death, local inflammation, tracheal stenosis and subcutaneous emphysema were documented. After 4, 8 and 12 weeks, tracheal histological examination and SEM examination were performed. In SIS-PPM reconstruction group, all animals survived at the end of the experiment, no infection, subcutaneous emphysema and breathing difficulties occurred. In PPM reconstruction group, there was 2 deaths because of infection of lumina and suffocation after 6 and 18 days of implantation; all rabbits had local subcutaneous emphysema. The histological examination showed that there was no obvious granulation tissue and scar in two groups and that the mucous membrane and cilia grew more normally in SIS-PPM reconstruction group than in PPM reconstruction group. SEM observation: At 8 weeks after implantation, most of the reconstructed area of the trachea in SIS-PPM reconstruction group was covered by relatively mature cilia; the corresponding area in PPM reconstruction group was covered by the naive cilia. At 12 weeks after implantation, the cilia in SIS-PPM reconstruction group grew orderly without obvious secretion adherence; while the cilia in PPM reconstruction group grew in a disorderly manner and were attached by an abundance of secretion. Good epithelial regeneration can be achieved after repair of tracheal defect using PPM coated with SIS. SIS can promote regeneration of ciliated epithelium, decrease postoperative complications, such as subcutaneous emphysema. It may be a prospective biomaterial for circumferential tracheal defect reconstruction clinically.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 04/2009; 23(3):328-31.
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    ABSTRACT: 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. 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 co-cultured BMSCs were tested by immunohistochemistry staining, and the expressions of Col I and OCN mRNA were detected by RT-PCR. 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-like 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 < 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. Rat osteoblast conditioned culture medium can significantly induce the differentiation of allogeneic rats' BMSCs towards osteoblasts.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 02/2009; 23(2):145-50.
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    ABSTRACT: To study the effect of hypoxia on the proliferation of hBMSCs and human placental decidua basalis-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs, flow cytometry (FCM) was applied to detect cell surface marker. After establishing the experimental model of CoCl2 chemical hypoxia, MTT method was applied to evaluate the proliferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoCl2 concentration (0, 50, 75, 100, 125, 150, 175, 200 micromol/L). 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 proliferations 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 proliferated 24 hours after hypoxia with CoCl2 concentration of 150 micromol/L (P < 0.05), while hPDB-MSCs were significantly proliferated 12 hours after hypoxia with CoCl2 concentration of 75 micromol/L (P < 0.05). Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the proliferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 02/2009; 23(2):136-9.
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    ABSTRACT: To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-beta3 and BMP-7 in vitro. The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors' iliac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures of hBMSCs were divided into four groups: 10 ng/mL TGF-beta3 group (group A), 200 ng/mL BMP-7 group (group B), combination group of TGF-beta3 and BMP-7 (group C) and blank group as the control (group D). Histological observation, RT-PCR and RQ-PCR were applied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviously changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day (P < 0.05). The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day (P < 0.05). The expressions of collagen type X only was positive in group A. Combination of TGF-beta3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 01/2009; 22(12):1470-5.
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    ABSTRACT: To provide an ideal seed cell for tissue engineered urinary bladder and urethra by serially culturing canine smooth muscle cells from urinary bladder in vitro and compare biological characteristics of different passages of cells. Bladder smooth muscle cells of 12-month-old male dogs weighing 10-12 kg were isolated from adult dogs' urinary bladders by collagenase and trypsin digestion and serially cultured in DMEM medium supplemented with 10% serum of newborn bovines. Morphology and proliferation of the cells were observed and the serially-cultured cells were identified with the transmission electron microscope and immunohistochemistry. The cells appeared spindle in parallel rows when they grew to the degree of subconfluency, and showed the "peak-valley" structure under the inverted phase contrast microscope. The cells could be proliferated serially to the 12th passage in vitro. The growth curve showed the cells before the 7th passage had the similar proliferation characteristics and the growth cycle was about 40 hours. The TEM showed myofilament and the dense body in cytoplasm of smooth muscle cells. Smooth muscle actin was positive by immunohistochemical staining. After the 7th passage, the cells' growth became slow, and myofilament and the dense body in cytoplasm vanished. The canine smooth muscle cells from urinary bladder can be serially cultured in vitro and highly purified and largely proliferated by the appropriate method. The cells before the 7th passage can be used as optimal seed cells for tissue engineered urinary bladder and urethra.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 01/2009; 22(12):1476-80.
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    ABSTRACT: To evaluate the biocompatibility of manufactured heterogeneous demineralized bone matrix (DBM) particles and to provide basis for further experimental study and clinical application. Heterogeneous DBM particles A (decreased and demineralized) and B (decreased, demineralized and acellular), particle size from 250 to 810 microm, and leaching liquor were made with a series of physical and chemical methods from pig limbs cortical bone. The residual calcium and phosphorus contents of bone particles were measured after decreased and demineralized. The acute toxicity test, skin stimulating test, pyrogeneous test, hemolysis test, cellular toxicity test and muscular embedded test were carried out according standard toxicological method. The contents of calcium and phosphorus in cortical bone were (189.09 +/- 3.12) mg/g and (124.73 +/- 2.87) mg/g, and in demineralized bone matrix particles were (3.48 +/- 0.09) mg/g and (3.46 +/- 0.07) mg/g. The residual calcium content was 1.87%, of phosphorus was 2.69%. The activity of mice was normal in the acute toxicity test. No animal died and no toxicity symptom or adverse effects were shown within 7 days. The mean weight daily increased showed no statistically significant difference (P > 0.05) between two groups after 7 days. Skin stimulating reactions were not found in the two experimental groups and negative control group by intradermal stimulation test. The maximal increase of body temperature in two experimental groups were 0.4 degree C, which meet the national standard (< 0.6 degree C). The rate of haemolysis to the leaching liquor was 1.14% (A) and 0.93% (B), which was lower than the national standard (< 5%). The cell proliferation rates of two experimental groups when compared with control group showed no statistically significant difference (P > 0.05). The toxicity of DBM particles leaching liquor was graded from 0 to 1, which means the material has no cytotoxicity. All the animals survived well. There was no tissue necrosis, effusion or inflammation at all implantation sites. For the index of HE and Masson staining, there were no effusion around the material and inflammatory cell infiltrate obviously in two experimental groups. Inflammatory cell infiltrate is slight in control group 2 weeks postoperatively. The inflammatory cell infiltration was mitigate gradually over time in two experimental groups after 4, 8 and 12 weeks. New bone and collagen fibers formation were observed when the material was degraded and absorbed. Score evaluation of local cellular immune response at different time after operation of two experimental groups showed no statistically significant difference (P > 0.05). Heterogeneous DBM has no obvious toxicity, skin irritation, pyrogenicity, and no cytotoxicity with a rate of haemolysis < 5%, so it has good biocompatibility and partial osteoinductive.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 01/2009; 23(1):76-81.
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    ABSTRACT: To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to evaluate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell proliferation and phenotype. The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) and co-cultured with notochord cells (group B) (1:1), and cell proliferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 microm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 microm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P > 0.05). The co-cultured cells (group B) increased in cell proliferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell proliferation in group B was more than that in group A after 4-day culture (P < 0.05). The co-cultured cells retained their phenotype for 5 passages, while parallel-cultured chondroid cells lost the expression of proteoglycan and collagen II after the third passage. The notochord cells are conducive for the proliferation and phenotype-keeping of the chondroid cells and may play a key role in preventing degeneration of the disc.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 08/2008; 22(8):939-43.
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    ABSTRACT: To explore an effective method to cultivate esophageal mucosa epithelial cells (EMECs) of canine in vitro, and to observe the biological characteristics of EMECs growing on SIS in order to provide an experimental basis for esophagus tissue engineering. Esophageal tissues were obtained from five healthy dogs aged 2 to 5 weeks under sterile conditions. The primary EMECs were cultivated with defined keratinocyte serum free medium (DKSFM) containing 6% FBS. The morphological characteristics and the growth curve of EMECs of the 2nd generation were observed for 1 to 5 days. The expressions of the EMECs marker (cytokeratin 19, CK-19) were examined by immunocytochemistry. The 2nd generation of EMECs was seeded on SIS and observed by HE staining, immunohistochemical staining, and SEM for 4 and 8 days. The primary culture of canine EMECs arranged like slabstone. Immunohistochemical staining of CK-19 of the 2nd generation EMECs showed positive broadly. The cells growth reached the peak level at 2 days by MTT method. EMECs were polygon in shape and arranged like slabstone, and formed a single layer on the surface of SIS. The cells were contacted closely with each other for 4 days. Eight days later, 2 to 3 layers stratified structure was formed. Lots of EMECs were grown on SIS, and showed laminate arrangement. With mixed enzymatic digestion, the culture of EMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS shows good biocompatibility and can be used as a good scaffold material in the tissue engineered esophagus.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 06/2008; 22(6):742-6.
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    ABSTRACT: To investigate the role of myosin light chain (Myl) in myogenesis in vitro. The extraocular muscle, diaphragm and gastrocnemius muscle myoblasts (eMb, dMb and gMb) were isolated and purified from 12 3-week-old C57BL/6 mice by using the enzyme digestion and Preplate technique, and then were subcultivated. The Myl expression in Mb was detected by RT-PCR and Western blot analysis; the Mb proliferation activity was tested by methylene blue assay, and the myotube formation was observed. After anti-Myl antibody (1, 2, 3, 8, 16 ng/mL) was induced in the Mb culture (experimental group), the ability of proliferation of myoblasts and the myotube formation were identified. Meanwhile, the Mb which was cultured without anti-Myl antibody was identified as the control group. The results of RT-PCR and Western blot analysis showed that Myl1 and Myl4 mRNA and Myl protein were expressed in eMb, dMb and gMb at 24 hours after seeding, and their expression level were lower in eMb than in dMb and gMb (P < 0.01), and the latter two did not show any significant difference (P > 0.05). Myl2 and Myl3 mRNA was not detected in these three myoblasts. The proliferation assay showed that the eMb proliferated faster as compared with dMb and gMb (P < 0.01). eMb began to yield myotubes at 40 hours after seeding and dMb and gMb at 16 hours after seeding. At 6 days, the number of myotubes derived from eMb was (137.2 +/- 24.5)/field, which was significantly larger than that of myotubes from dMb [(47.6 +/- 15.5)/field ] and gMb [(39.8 +/- 5.1)/ field ] (P < 0.01). There was not statistically significant difference between the latter two groups (P > 0.05). After the antibody treatment, the absorbency values of the eMb, dMb and gMb in the experimental groups at each antibody concentration point were significantly higher than those in the corresponding control groups (P < 0.05), and the dose-dependent way was performed. The numbers of myotubes from dMb at 16 hours were (48.2 +/- 7.1)/ well in the experimental group and (23.4 +/- 4.9)/ well in the control group, and at 6 days were (40.6 +/- 10.2)/ field in the experimental group and (63.1 +/- 6.1)/ field in the control group. There was statistically significant difference between the experimental and control groups (P < 0.01). Myl may play a role in myogenesis through the negative effect on the myoblast proliferation.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 06/2008; 22(6):753-8.
  • Ping Han, Zhiming Yang, Xiuqun Li
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    ABSTRACT: To explore an effective method to culture and purify canine bladder transitional epithelial cells. Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0. 125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry. Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed microfilament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anti-cytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells. Different generations of cells showed diploid cells. A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 12/2007; 21(11):1238-42.
  • Yao Lu, Li Deng, Xiuqun Li
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    ABSTRACT: To explore a method to isolate, culture and multiplicate the placenta-derived mesenchymal stem cells (PMSCs) and the bone marrow-derived mesenchymal stem cells (BMSCs) of rabbit, and to compare their biological characteristics. PMSCs were isolated from placenta of 1 fetation rabbit by Percoll density gradient centrifuge and cultured in vitro. BMSCs were isolated from hindlimb bone marrow blood of 1 new born rabbit by direct plates culture method. The 3rd passage PMSCs and BMSCs were observed by inverted phase contrast microscope. The stem cell marker (CD44, CD105, CD34 and CD40L) were examined by immunohistochemistry. The 2nd passage PMSCs and BMSCs were co-cultured with biomaterials, (1.0-1.5) x 10(6) cells in one biomaterial, and then observed by haematoxylin staining after 5 days, and by SEM after 3 days and 8 days. PMSCs and BMSCs were both uniformly spondle-shaped in appearance and showed active proliferative capacity. The proliferative ability of PMSCs were quite strong and declined with passages. After cultured 10 passages in vitro, its growth slowed. Both PMSCs and BMSCs expressed CD44 and CD105, but did not express CD34 and CD40L immunoreactivity. PMSCs and BMSCs poliferated and adhered to the surface of biomaterials, and cell formed clumps and network; the cells proliferation and the matrix were seen in the pore after 5 days of culture. The observation of SEM showed that many cells adhered to the biomaterials with spindle-shape and polygon after 3 days; and that PMSCs and BMSCs grew, arranged in layers and secreted many matrices; the reticular collagen formed arround cells after 8 days. PMSCs and BMSCs have similar biological characteristics and PMSCs can be served as excellent seeding-cells for tissue engineering.
    Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 10/2007; 21(9):989-93.

Publication Stats

37 Citations
16.62 Total Impact Points

Institutions

  • 2004–2011
    • Sichuan University
      • • Department of Orthopedic Surgery
      • • Laboratory of Stem Cell and Tissue Engineering
      Chengdu, Sichuan Sheng, China
  • 2009
    • State Key Laboratory of Medical Genetics of China
      Ch’ang-sha-shih, Hunan, China