ObjectiveTo investigate the growth characteristics of pancreatic cancer cells in the twodimensional culture system (monolayer) and threedimensional culture system (type Ⅰ collagen and extracellular matrix gel). MethodsThree pancreatic cancer cell lines (SW1990, PCT, and ASPC1) were cultured in monolayer, type Ⅰ collagen, and extracellular matrix gel, respectively. The growth patterns were observed, growth curves were detected by CCK8 test, and the cell cycle distributions were analyzed by propidium iodide staining. Results In the twodimensional culture system, cells grew in monolayer. In the type Ⅰ collagen and the ECM gel threedimensional culture system, cells formed multicellular spheroids (MCS), of which the growth rates were slower than those of the cells in monolayer. The proportions of S phase of SW1990, PCT, and ASPC1 cells in twodimensional culture system were significantly more than those in the type Ⅰ collagen on 4 d and 8 d 〔(29.6±3.0)% vs. (18.2±5.1)%, (33.6±2.1)% vs. (14.5±3.2)%, (33.1±1.8)% vs. (24.7±2.6)%; Plt;0.05〕, while the difference of proportion of three cell lines in G2/M phase was not different between twodimensional culture system and type Ⅰ collagen (Pgt;0.05). The proportions of G0/G1 phase of SW1990 and PCT cells cultured in the type Ⅰ collagen on 4 d and 8 d and ASPC1 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in twodimensional culture system (Plt;0.05). The proportions of S phase of ASPC1 cells and SW1990 cells cultured in the type Ⅰ collagen on 4 d were significant more than those cultured in the type Ⅰ collagen on 8 d (Plt;0.05). ConclusionsThe characteristics of pancreatic cancer cells in twodimensional and threedimensional culture systems are different. MCS culture system can better mimic the in vivo growth environment of cells in tumors.
Abstract： Objective To investigate the extracellular matrix （ECM） gene expression profile of saphenous vein （SV） in end-stage renal disease （ESRD） patients undergoing coronary artery bypass grafting （CABG）. Methods Sixty-eight patients who were diagnosed as coronary artery disease by coronary angiography and admitted to Department of Cardiovascular Surgery，Zhongshan Hospital of Fudan University from July 2004 to December 2010 were enrolled in this study. According to whether or not they had preoperative ESRD history，all the 68 patients were divided into 2 groups，the ESRD group with 30 ESRD patients who needed maintenance hemodialysis，and the control group with 38 patients without preoperative renal disease. Preoperative clinical data of all the patients were collected in detail. SV samples were obtained at the time of CABG. Microarray，immunohistochemistry and Western blotting were used to investigate the expression profile of ECM genes of SV in ESRD patients undergoing CABG. Results There was no statistical difference in preoperative clinical variables between the 2 groups except the variables which were directly related to their kidney disease （P＞0.05）. There were 16 genes that were up-regulated at least 3-fold and 3 genes that were down-regulated at least 3-fold in the ECM gene expression profile of SV in the ESRD group patients before CABG. The expressions of matrix metalloproteinases-2 （MMP-2） and matrix metalloproteinases-9 （MMP-9） of the ESRD group were significantly higher than those of the control group （2.60±0.50 vs. 0.70±0.16，1.80±0.40 vs. 0.60±0.15，P＜0.01）. The expressions of tissue inhibitor of metalloproteinase-2 （TIMP-2） and tissue inhibitor of metalloproteinase-3 （TIMP-3） of the ESRD group were significantly lower than those of the control group （0.60±0.19 vs. 2.20±0.30，0.90±0.28 vs. 2.40±0.70，P＜ 0.05）. Conclusion A variety of ESRD-related risk factors of cardiovascular diseases may severely influence on the balance of ECM gene expression of SV before CABG，and the resulting imbalance is a risk factor to aggravate SV graft disease after CABG.
Patients with pathological tracheal loss more than a certain length may need tracheal transplantation.Traditional natural tissue and autologous tissue have failed to produce satisfactory clinical outcomes to replace the trachea because of local infection，tracheal stenosis，tracheomalacia，immune rejection et al. In recent years，the emergence oftissue engineering trachea provides a new idea for tracheal transplantation. But scientists have not yet reached a consensus about how to choose ideal extracellular matrix to construct tissue engineering trachea. At present research and applicationof tissue engineering trachea，extracellular matrices mainly include allogenic trachea，allogenic aorta and biologicalcomposite materials. Each allogenic matrix or biological composite material has its own advantages and disadvantages. Therefore，this article mainly summarizes recent application and research progress of extracellular matrix in long segmental tracheal defect and its future perspective.
The establishing of myocardial tissue engineering techniques not only solve a series of issues that generate in cell and tissue transplantation after myocardial infarction, but also create a platform for selecting better materials and transplantation techniques. However, both experimental animal studies and recent clinical trials indicate that current transplantation techniques still have many defects, mainly including lack of suitable seed cells, low survival rate and low differentiation rate after transplantation. In this context, extracellular matrix （ECM）, as myocardial tissue engineering scaffold materials, has gained increasing attention and become a frontier and focus of medical research in recent years. ECM is no longer merely regarded as a scaffold or a tissue, but plays an important role in providing essential signals to influence major intracellular pathways such as cell proliferation, differentiation and metabolism. The involved models of ECM can be classified into following types：natural biological scaffold materials, synthetic polymer scaffold materials and composite scaffold materials with more balanced physical and biological properties. This review mainly introduces research progress of ECM in myocardial tissue engineering and ECM materials.
Objective To investigate the way and process of degradation behavior of acellular porcine aortic valve in vitro. Methods Acellular porcine aortic valve（n=90）were randomly divided into 3 groups （collagenase group, elastase group, control group）, 30 piece in each group . Behavior of acellular porcine aortic valve was degradated with 0.05mg/ml collagenase Ⅰ, 0. 05mg/ml elastase, phosphate buffered solution in collagenase group, elastase group and control group. The histomorphology, weight loss, value of protein and hydroxyproline were observed at 3,6,9, 12, 15 and 30d after degradation. Results The behavior of acellular porcine aortic valve of collagenase group and elastase group became poorer, looser and broken gradually in degradation. The weight loss of valve, the value of protein and hydroxyproline in vehiculum became greater gradually in collagenase group and elastase group（P〈0. 01）. Furthermore the effect of collagenase Ⅰ was b than elastase in degradation. Conclusion The effect of collagenase Ⅰ and elastase can degradate the acellular porcine aortic valve in vitro. Collagenase Ⅰ is b than elastase in degradation.
Objective To review the research progress of promoting the bone formation at early stage by components of the extracellular matrix (ECM). Methods Recent literature concerning the influence of these components on new bone formation and bone/implant contact was extensively reviewed and summarized. Results Coating of titanium or hydroxyapatite implants with organic components of the ECM (such as collagen type I, chondroitin sulfate, and Arg-Gly-Asp peptide) offers great potential to improve new bone formation and enhance bone/implant contact, which in turn will shorten recovery time and improve implant stability. Conclusion The increasing knowledge about the role of the ECM for recruitment, proliferation, differentiation of cells, and regeneration of tissue will eventually deal to the creating of an artificial ECM on the implant that could allow a defined adjustment of the required properties to support the healing process.
Objective To observe the expressions of extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinase 9 (MMP-9) around the prosthesis, and to study the relationship between the expressions of EMMPRIN and MMP-9 and osteolysis around prosthesis. Methods Interface tissues were obtained at three Delee-Charnley acetabular sections and seven Gruen femur sections from 8 cases (8 hips) undergoing revision after total hip arthroplasty between February 2010 and January 2012, and were divided into osteolysis group and non-osteolysis group based on preoperative X-ray film and intraoperative observation; the tissues from another 8 patients with osteoarthritis undergoing total hip arthroplasty as the control group. The immunohistochemical staining and RT-PCR assays were used to determine the expressions of EMMPRIN and MMP- 9. The correlation between the positive cells and the severity of osteolysis were analyzed and compared. Results Histological examination showed that many macrophages, multinucleated giant cells assembled in the membrane of osteolysis zone, but many fibroblasts and synovial cells in non-osteolysis zones. EMMPRIN and MMP- 9 positive cells and gene expressions were observed in every group. The percentage of positive cells and gene expression of EMMPRIN and MMP-9 in osteolysis group were significantly higher than those in non-osteolysis and control groups (P lt; 0.05), but no significant difference was found between non-osteolysis group and control group (P gt; 0.05). The percentage of positive cells of EMMPRIN in zone III of acetabular was higher than that in zone I and zone II of revision hip (P lt; 0.05), but no significant difference between zone I and zone II (P gt; 0.05). The percentage of positive cells of MMP-9 in zone I and zone III was significantly higher than that in zone II of revision hip (P lt; 0.05), but no significant difference between zone I and zone III (P gt; 0.05). The expression of EMMPRIN from high to low in order was zones 1, 7, 4, 2, 3, 5, and 6 at femur; the values of zones 1, 7, and 4 were significantly higher than those of zones 2, 3, 5, and 6 (P lt; 0.05), but no significant difference among zones 1, 7, and 4, and among zones 2, 3, 5, and 6 (P gt; 0.05). The expression of MMP-9 from high to low in order was zones 1, 7, 4, 2, 3, 6, and 5 at femur; the values of zones 1 and 7 were significantly higher than those of zones 4, 2, 3, 6, and 5 (P lt; 0.05), and the values of zones 4 and 2 were significantly higher than those of zones 3, 6, and 5 (P lt; 0.05), but no significant difference between zone 1 and zone 7, between zone 4 and zone 2, and among zones 3, 5, and 6 (P gt; 0.05). Conclusion The expressions of EMMPRIN and MMP-9 have certain coherence. The over-expressions of EMMPRIN and MMP-9 may be one of the key points of inhibiting bone reconstruction and bone resorption at bone-implant interface under the stimulation of wear debris．
Objective To investigate the feasibility of fabricating an oriented scaffold combined with chondrogenic-induced bone marrow mesenchymal stem cells (BMSCs) for enhancement of the biomechanical property of tissue engineered cartilage in vivo. Methods Temperature gradient-guided thermal-induced phase separation was used to fabricate an oriented cartilage extracellular matrix-derived scaffold composed of microtubules arranged in parallel in vertical section. No-oriented scaffold was fabricated by simple freeze-drying. Mechanical property of oriented and non-oriented scaffold was determined by measurement of compressive modulus. Oriented and non-oriented scaffolds were seeded with chondrogenic-induced BMSCs, which were obtained from the New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope. Then cell-scaffold composites were implanted subcutaneously in the dorsa of nude mice. At 2 and 4 weeks after implantation, the samples were harvested for evaluating biochemical, histological, and biomechanical properties. Results The compressive modulus of oriented scaffold was significantly higher than that of non-oriented scaffold (t=201.099, P=0.000). The cell proliferation on the oriented scaffold was significantly higher than that on the non-oriented scaffold from 3 to 9 days (P lt; 0.05). At 4 weeks, collagen type II immunohistochemical staining, safranin O staining, and toluidine blue staining showed positive results in all samples, but negative for collagen type I. There were numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells on the oriented-structure constructs. Total DNA, glycosaminoglycan (GAG), and collagen contents increased with time, and no significant difference was found between 2 groups (P gt; 0.05). The compressive modulus of the oriented tissue engineered cartilage was significantly higher than that of the non-oriented tissue engineered cartilage at 2 and 4 weeks after implantation (P lt; 0.05). Total DNA, GAG, collagen contents, and compressive modulus in the 2 tissue engineered cartilages were significantly lower than those in normal cartilage (P lt; 0.05). Conclusion Oriented extracellular matrix-derived scaffold can enhance the biomechanical property of tissue engineered cartilage and thus it represents a promising approach to cartilage tissue engineering.
Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
Objective To review the decellularized methods for obtaining extracellular matrix (ECM) and the applications of decellularized ECM scaffold in tissue engineering. Methods Recent and related literature was extensively and comprehensively reviewed. The decellularized methods were summarized and classified. The effects of different sterilization methods on decellularized scaffolds were analyzed; the evaluation criterion of extent of decellularization was put forward; and the application of decellularized ECM scaffold in different tissues and organs engineering field was summarized. Results The decellularized methods mainly include physical methods, chemical methods, and biological methods, and different decellularization methods have different effects on the extent of cell removal and ECM composition and structure. Therefore, the best decellularization method will be chosen according to the characteristics of the tissues and decellularization methods to achieve the ideal result. Conclusion It is very important to choose the appropriate decellularized method for preparing the biological materials desired by tissue engineering. The biological scaffolds prepared by decellularized methods will play an important role in tissue engineering and regenerative medicine.