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ABSTRACT: Porous titanium was modified by anodic oxidation and heat treatment method. Scanning electron microscopy and X-ray diffraction examinations revealed that the modified surface of porous titanium was covered by anatase nanotubes. In vitro, the bioactivity of specimens before and after modification was evaluated by immersing into the double-concentration simulated body fluid for 7 days. The porous titanium specimens were implanted into the femurs of dogs for 3 months. The osteointegration of the implants was investigated by push-out test and histological examination. The results showed that the porous titanium with anatase nanotubes has the superior ability of apatite formation and a higher push-out force when compared with the other implants. The histological analysis indicated that the implant with anatase nanotubes had excellent ability to facilitate the osteointegration in vivo. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3422-3427, 2012.
Journal of Biomedical Materials Research Part A 07/2012; 100(12):3422-7. · 2.63 Impact Factor
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ABSTRACT: Tissue-engineered bones (TEBs) constructed with bone-marrow-derived mesenchymal stem cells (BMSCs) seeded on biomaterial scaffolds have achieved good results for bone defect repair in both animal experiments and clinical trials. This has been limited, however, by the source and quantity of BMSCs. We here explored TEBs constructed by placenta-derived mesenchymal stem cells (PMSCs) and compared their effect for the repair of critical-sized segmental osteoperiosteal defects with TEBs constructed with BMSCs. PMSCs were isolated from rabbit placenta by gradient centrifugation and in vitro monolayer culturing, and BMSCs were isolated from the hindlimb bone marrow of newborn rabbit. Primary cultured PMSCs and BMSCs were uniformly in a spindle shape. Immunocytochemistry indicated that both types of cells are positive for CD44 and CD105, and negative for CD34 and CD40L, confirming that they are mesenchymal stem cells. BrdU-labeled PMSCs and BMSCs were respectively co-cultured with bio-derived bone materials to construct TEBs in vitro. Critical-sized segmental osteoperiosteal defects of radii were created in 24 rabbits by surgery. The defects were repaired with TEBs constructed with PMSCs and BMSCs. The results showed that TEBs constructed by both PMSCs and BMSCs could repair the osteoperiosteal defects in a 'multipoint' manner. Measurement of radiography, histology, immunohistochemistry, alkaline phosphatase activity, osteocalcin assaying and biomechanical properties have found no significant difference between the two groups at 2, 4, 8 and 12 weeks after the transplantation (P > 0.05). Taken together, our results indicate that PMSCs have similar biological characteristics and osteogenic capacity to BMSCs and can be used as a new source of seeding cells for TEBs.
FEBS Journal 05/2012; 279(13):2455-65. · 3.79 Impact Factor
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ABSTRACT: Porous hydroxyapatite (HA) ceramic cylinder tubes coated with polylactic acid on the exposed surfaces were implanted in four nonskeletal sites (omentum, peritoneum, vastus lateralis, and side of femur). Six months postoperatively, proper amount of Chinese ink was injected to dye the implanting areas. Decalcified and nondecalcified sections were observed under inverted microscope. The results showed that the soft tissues around the HA cylinder tubes in peritoneum, vastus lateralis, and side of femur groups appeared visible black. Some small blacked vascular architectures were also discernible. However in omentum group, only small number of blacked vessels existed. Histological observations indicated that vascularization and ossification occurred in peritoneum, vastus lateralis, and side of femur groups. In omentum group, there was no any sign of vascularization and ossification. A conclusion could be made in this study that excepting bones and muscles, parietal peritoneum could serve as a potential spot for culturing histoengineering hydroxyapatite (HA)-polylactic acid (PLA) ceramic bone substitutes.
Journal of Biomedical Materials Research Part A 02/2012; 100(5):1203-8. · 2.63 Impact Factor
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ABSTRACT: Diabetic skin ulcer is difficult to heal due to the lack of cellular and molecular signals required for normal wound repair. Emulsion electrospinning was adopted to imbed basic fibroblast growth factor (bFGF) into ultrafine fibers with a core-sheath structure to promote the wound healing process. An initially burst release as low as 14.0 ± 2.2% was achieved, followed by gradual release for around 4 wk. In vitro investigations on mouse embryo fibroblasts indicated that bFGF-loaded fibrous mats enhanced cell adhesion, proliferation, and secretion of extracellular matrix (ECM). Skin wounds were created in the dorsal area of diabetic rats for in vivo evaluation of skin regeneration after covered with bFGF-loaded fibrous mats. Compared with fibrous mats infiltrated with free bFGF, bFGF-loaded scaffolds revealed significantly higher wound recovery rate with complete re-epithelialization and regeneration of skin appendages. Higher density and mature capillary vessels were generated during 2 wk after treatment with bFGF-loaded fibers, and there was no fiber fragment observed in the histological sections at week 4 after operation. The gradual release of bFGF from fibrous mats enhanced collagen deposition and ECM remodeling, and the arrangement and component of collagen fibers were similar to normal tissues. The above results demonstrate the potential use of bFGF-loaded electrospun fibrous mats to rapidly restore the structural and functional properties of wounded skin for patients with diabetic mellitus.
Biomaterials 03/2011; 32(18):4243-54. · 7.40 Impact Factor
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ABSTRACT: In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a "biomimetic niche." The CD34(+) cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2-5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34(+) cells from umbilical cord blood in the 3D system increased 3.3-4.8 folds when compared with the initial CD34(+) cells. CD34(+)/CD38(-) cells accounted for 82-90% of CD34(+) cells. After 5 weeks, CD34(+)/CD38(-) cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10(3) vs. 1.0 ± 0.5 × 10(4), p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10(3) vs. 2.5 ± 0.7 × 10(2), p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6-9.3 folds vs. 1.0-1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2-7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.
Cytotechnology 10/2010; 62(5):439-48. · 1.21 Impact Factor
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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 an effective method to culture oral mucosa epithelial cells (OMECs) of canine in vitro, and to observe the biological characteristics of OMECs growing on small intestinal submucosa (SIS) in order to provide the experimental basis for epithelium tissue engineering.
The primary OMECs were cultivated with DKSFM (defined keratinocyte serum free medium) containing 6% fetal bovine serum (FBS). The morphological characteristics and the growth curve of OMECs were observed. The expressions of OMECs marker (CK19) were examined by immunocytochemistry. The 2nd passage of OMECs were seeded on SIS, OMECs co-cultured with SIS were observed by hematoxylin-eosin staining, immunohistochemical staining, and scanning electron microscope (SEM).
OMECs were grown well in DKSFM. Immunohistochemical staining of the 2nd passage cultured canine OMECs with broadly reacting anti-cytokeratin anyibodies (CK19) was positive. OMECs formed a single layer on the surface of SIS, and eight days later the cells were polygong and arranged like slabstone.
Culture of canine OMECs in DKSFM containing 6% FBS is a simple and feasible method. SIS has good biocompatibility, it is a kind of good bioscafold in the tissue-engineered epithelium.
Hua xi kou qiang yi xue za zhi = Huaxi kouqiang yixue zazhi = West China journal of stomatology 02/2010; 28(1):76-80.
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ABSTRACT: Biodegradable polymer particles present a promising approach for intracellular delivery of drugs, proteins, and nucleic acids. Poly-d,l-lactide-poly(ethylene glycol) (PELA) copolymers with different weight ratios of polyethylene glycol (PEG) were used as drug carriers in the present study. PELA particles entrapped with fluorescein isothiocyanate (FITC) as a fluorescent marker were formulated using a double emulsion-solvent evaporation technique. The size and morphology of the particles were observed with scanning electron microscope (SEM), atomic force microscope (AFM), and laser diffraction particle size analyzer (LDPSA). The purpose in the present work was to investigate the cytotoxicity and the process of endocytosis of PELA particles with different contents of PEG and variable particle size using rat osteoblasts (OBs). The cytotoxicity of the particles was investigated using 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Results indicate that as the content of PEG in the polymer increased, so did cell survival. Endocytosis was observed through a light microscope and a fluorescence microscope; intracellular uptake and retention were determined quantitatively using fluorescence spectrophotometer (FSP). The results showed that as PEG content in PELA copolymer increased, there was a reduction in endocytosis of nanoparticles in osteoblasts.
International Journal of Nanomedicine 01/2010; 5:557-66. · 3.13 Impact Factor
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Mei Yun Tan, Wei Zhi,
Ren Qian Wei,
Yong Can Huang,
Kun Peng Zhou,
Bo Tan,
Li Deng,
Jing Cong Luo,
Xiu Qun Li,
Hui Qi Xie,
Zhi Ming Yang
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ABSTRACT: Myocardial infarction (MI) remains a common and deadly disease. Using tissue-engineered cardiac grafts to repair infarcted myocrdium is considered to be a therapeutic approach. This study tested the feasibility of using MSCs-seeded SIS to repair chronic myocardial infarction in a rabbit model. MI in rabbits was created by ligation of the left anterior descending artery. BrdU-labeled mesenchymal stem cells (MSCs) were seeded on the small intestinal submucosa and cultured for 5-7 days prior to implantation. Four weeks after myocardial infarction, cardiac grafts were implanted onto the epicardial surface of infarcted myocardium. Four weeks after implantation of the membranes, a serial of tests including echocardiography, hemodynamics, histology and immunohistochemistry were undertaken to evaluate the effect of the implanted grafts on recovery of the infarcted myocardium. It was shown that left ventricular contractile function and dimension, the capillary density of the infarcted region, and myocardial pathological changes were significantly improved in rabbits implanted either SIS or MSCs-seeded SIS. But the MSCs-seeded SIS was more effective. Immunofluorescence staining demonstrated the migration of Brdu-labeled MSCs from the membrane into the infarcted area and their differentiation to cardiomyocytes and smooth muscle cells. Taken together, these results suggest that MSCs-seeded SIS can be used to repair chronic myocardial infarction, which enhances myocardial regeneration.
Biomaterials 04/2009; 30(19):3234-40. · 7.40 Impact Factor
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Ren-Qian Wei,
Bo Tan,
Mei-Yun Tan,
Jing-Cong Luo,
Li Deng,
Xiao-He Chen,
Xiu-Qun Li,
Xiao Zuo, Wei Zhi,
Peng Yang,
Hui-Qi Xie,
Zhi-Ming Yang
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ABSTRACT: The acellular porcine small intestinal submucosa (SIS) has been successfully used for esophagoplasty. However, it does not lead to a complete epithelialization in a canine model. A cellular component may be required for better reconstruction. The present study was undertaken to investigate the feasibility and effectiveness of the combination of SIS and autologous oral mucosal epithelial cells (OMECs) for esophageal reconstruction. The OMECs harvested from beagle dogs were cultured and propagated, and the 3rd passage cells were seeded on a single-layer SIS. Male beagle dogs were subjected to surgical resection to produce cervical esophageal defects (5 cm in length, 180 degrees in range). SIS with or without OMECs was patched on the esophageal defects. Barium esophagram, immunohistochemistry, and histology were performed to evaluate the therapeutic effectiveness. Four weeks after surgery, the histological examination showed a complete re-epithelialization and almost no inflammation in the SIS with OMECs group. But in the SIS group, only a partial epithelialization was observed along with inflammation. Eight weeks after surgery, the squamous epithelium was found to cover the entire graft surface in both groups; however, the muscular regeneration was observed in the SIS with OMECs, but not in the SIS group. The graft of SIS combined with autologous OMECs promotes re-epithelialization and muscular regeneration. It is an effective alternative method for esophageal repair.
Experimental Biology and Medicine 02/2009; 234(4):453-61. · 2.64 Impact Factor
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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 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: The extraocular muscle (EOM) suffers much less injury from Duchenne muscular dystrophy (DMD) than other skeletal muscles such as diaphragm and gastrocnemius. The present study was undertaken to test the hypothesis that differential expression of regulatory proteins between the EOM and other skeletal muscles is responsible for the observed difference in the sensitivity to DMD-associated damage. Protein expression in the tissue samples obtained from EOM, diaphragm or gastrocnemius of C57BL/6 mice was analyzed by two-dimensional gel electrophoresis and mass spectrometry. There were 35 proteins that were identified to be differentially expressed among different skeletal muscle tissues. Among the 35 proteins, a fast skeletal muscle isoform myosin light chain 1 (MLC1f) protein was further studied in relation to muscle cell proliferation. The EOM-derived myoblasts had much lower levels of MLC1f and higher rate of cell proliferation in contrast to the myoblasts derived from diaphragm or gastrocnemius, which displayed a higher expression of MLC1f along with a slow proliferation. Deletion of MLC1f using siRNA targeting MLC1f resulted in an increased rate of cell proliferation in the myoblasts. Cell cycle analysis revealed that MLC1f inhibited the transition of the cell cycle from the G1 to the S phase. Therefore, the present study demonstrates that MLC1f may negatively regulate proliferation of myoblasts through inhibition of the transition from the G1 to the S phase of the cell cycle. Low levels of MLC1f in myoblasts of EOM may ensure cell proliferation and enhance the repair process for EOM under the DMD disease condition, thus making EOM suffer less injury from DMD.
Experimental Biology and Medicine 12/2008; 233(11):1374-84. · 2.64 Impact Factor
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ABSTRACT: As mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation, the feasibility and efficacy of co-culturing human MSCs (hMSCs) with rabbit articular chondrocytes (rACs) to promote chondrogenic and osteogenic differentiation of hMSCs for clinical osteoarthritic therapy were investigated in the present study. The two distinct cell types were encapsulated in alginate hydrogels singly or in one of three ratios (2:1, 1:1, 1:2 of hMSCs to rACs) and cultured under chondrogenic conditions for 28 days. The results demonstrated that newly synthesized cartilaginous extracellular matrix (ECM) and type II collagen (col-2) gene signal were upregulated with greater hMSC ratios and longer culture periods. However, a specific col-2 gene probe for human was found only in single hMSC group but absent in all co-culture groups, which indicate that the enhanced cartilaginous phenotype originated from the co-cultured rACs. Osseous phenotype was histologically detected only in the 2:1 group on day 28; and xenogenic osteocalcin assay showed that it originated from hMSCs. This suggests that variations in the ratios of co-cultured hMSC and rAC regulated the cartilaginous and osseous phenotype as well as the differentiation of hMSCs in alginate constructs. The study provides new insights into the role of cell-cell interactions in regulating both cell differentiation and cell function and highlights the importance of developing appropriate differentiation protocols for tissue engineering therapies.
Bone 08/2008; 45(1):42-51. · 4.02 Impact Factor
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ABSTRACT: To search for the best approaches to transfect plasmid pEGFP-N1 into human fibroblasts.
Cationic polymer mediated transfection, cationic liposome transfection and electrotransfection were compared for transfecting plasmid pEGFP-N1, a plasmid that expresses G418 resistance protein and Enhanced green fuluorescent protein (EGFP), into Human fibroblasts. The expression of EGFP was observed 24 hours after the transfection by fluorescence microscope. The transient transfection efficiency was measured by flow cytometry. The stable transfected cells were screened by G418.
Electrotransfection had the highest transient transfection efficiency (56.8%), with the most masc clones of stably transfection and the least time taken to form big masc clones (20 days). Cationic polymer mediated transient transfection had the lowest efficiency(1.7%), with the least masc clones and the longest time taken to form the big masc clones (30 days). Cationic liposome transfection had a transient transfection efficiency of 39.9% and took 25 days to form the big masc clones.
Electrotransfection is the best way of stably transfecting plasmid pEGFP-N1 into human fibroblasts.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 08/2008; 39(4):654-7.
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ABSTRACT: To develop a method for reconstructing urothelial structures using tissue engineering techniques.
The bladder mucosa of a puppy dog was mechanically separated, cutted into pieces, and digested with trypsin. The obtained bladder transitional epithelia were cultured with DKSFM and then transplanted to the surface of the small intestinal submucosa (SIS) framework. The compound of the epithelia and small intestinal submucosa (SIS) materials were planted under the back skin of the nude rats. The rats were sacrificed at week 1, 2, 4 and 8 after the transplant. Histological and immunohistochemical examinations were performed to the implanted samples.
The bladder transitional epithelia adhered to, and grew and proliferated on the surface of the SIS framework. The bladder transitional epithelia covered the entire surface of the framework after nine days of culture in vitro, showing a single-layer cellular structure. The bladder transitional epithelia planted on the SIS framework formed a multi-layer structure after four weeks or eight weeks of transplant to the nude rats. The brown cellular plasma staining was observed, indicating a positive reaction to the immunohistochemical staining with cytokeratin.
Urothelial structures can be reconstructed in vitro and in vivo with tissue engineering technologies, which lays a technical foundation for further urinary tract reconstructing experiments.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 06/2008; 39(3):481-4, 510.
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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 explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa(SIS) and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques.
The enzyme-treatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth muscle cells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co-cultured for a further observation. At 5,7 and 9 days of the co-culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold.
The primary bladder smooth muscle cells that had been harvested by the enzyme-treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitro for 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindle-shaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the well-developed actin filaments in the cytoplasm and the dense patches in the cell membrane under the transmission electron microscope. The immunohistochemical staining showed the canine bladder smooth muscle cells with positive reacting alpha-actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a single-layer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were (16.85 +/- 0.79) x 10(5), (39.74 +/- 2.16) x 10(5) and (37.15 +/- 2.02) x 10(5), respectively. The cell counts in the control group were (19.43 +/- 0.54) x 10(5), (34.50 +/- 1.85) x 10(5) and (33.07 +/- 1.31) x 10(5), respectively. There was a significant difference between the two groups at 5 days (P < 0.05).
With the enzyme-treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio-scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 12/2007; 21(12):1366-70.
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ABSTRACT: To review the research progress of osteoblasts in the hematopoietic microenvironment of bone marrow and regulatory pathways and mechanisms.
The advances in the osteoblasts as crucial components for hematopoietic microenvironment in bone marrow, regulation to osteoblasts and hematopoietic stem cells (HSCs), and correlative singal pathways and mechanisms were introduced based on the recent related literature.
Evidence indicates that osteoblasts are crucial components of the hematopoietic microenvironments in adult bone marrow. The osteoblasts maintain the quiescence of primitive HSCs by the signaling receptors-ligands, secreted cell factors and cell-adhesion molecules and by regulating other cells in the niche. The quiescent primitive HSCs persist stem cell characteristic which has unlimited selfrenewal and multipotent differentiation potential.
The further understanding of the relationship between osteoblasts and hematopoietic microenvironment should lead to development of new strategies directed toward clinical therapeutics of HSCs transplantation.
Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery 06/2007; 21(5):517-22.
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ABSTRACT: In order to explore the methods for commercialized bone tissue engineering, engineered bones should be cultivated in bioreactors to realize three-dimensional culture under well-defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1-month-old Zelanian rabbits were inoculated on to the BDBS (bio-derived bone scaffolds) to investigate the three-dimensional fabrication of engineered bone in an RWVB (rotating-wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent protein, were respectively seeded at 2 x 10(6) and 1 x 10(6) cells x ml(-1) on to the BDBS and then cultured in a T-flask and an RWVB for 1 week. The morphologies and structure of the fabricated bone were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von-Kossa staining on mineralized nodules, type I collagen and bone morphogenetic protein-2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell-seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T-flask. Bone tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three-dimensional fabrication of engineered bone could be realized.
Biotechnology and Applied Biochemistry 10/2006; 45(Pt 2):65-74. · 1.53 Impact Factor
