To observe the collagen-hydroxylaptite composite in the repair of bone defect, ten minipigs were chosen to make a mandibular dafect measuring 2 cm in diameter and the composite was implanted, while the use of autogenous bone graft and the blank wese served as control. On the 4, 8, 12, 24 and 48 weeks after the operation, the animals were sacrificed and the samples were examined under light microscope. The result showed that: no infection or necrosis occurred. The composite coalesced with host bone and the outcome was similar to that of the autogenous bone graft. No foreign body giant cells or vacuum left from osteonecrosis was observed. It was suggested that the composite had the advantage of abundant supply, easy to handle and no harm. The biocompatibility was good and might be hopeful as a bone substitute.
Objective To evaluate the feasibility and the value of the layered cylindric collagenhydroxyapatite composite as a scaffold for the cartilage tissue engineering after an observation of how it absorbs the chondrocytes and affe cts the cell behaviors. Methods The chondrocytes were isolated and multiplied in vitro, and then the chondrocytes were seeded onto the porous collagen／h ydro xyapatite composite scaffold and were cultured in a three-dimensional environme n t for 3 weeks. The effects of the composite scaffold on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by the phase-contrast microscopy, histology, scanning electron micros copy, and immunohistochemistry. Results The pore diameter of the upper layer of the collagen-hydroxyapatite composite scaffold was about 147 μm. and the porosity was 89%; the pore diameter of the bottom layer was about 85 μm and the porosity was 85%. The layered cylindric collagenhydroxyapatite composite scaffold had good hydrophilia. The chondrocytes that adhered to the surface of the scaffold, proliferated and migrated into the scaffold after 24 hours. The chondrocytesattached to the wall of the microholes of the scaffold maintained a rounded morphology and could secrete the extracellular matrix on the porous scaffold. Conclusion The layered cylindric collagenhydroxyapatite composite scaffold has a good cellular compatibility, and it is ber in the mechanical property than the pure collagen. It will be an ideal scaffold for the cartilage tissue enginee ring.
OBJECTIVE: To build the trestle of tissue engineering for skin with the collagen. METHODS: The collagen was obtained from the baby cattle hide pretreated by Na2S and elastinase and Protease M, then the collagen was dissolved in 0.5 mol/L acetic acid solution. The collagen was treated with Protease N to minimize its immunogenicity. The resulting collagen could be used to build the trestle of tissue engineering for skin because of good biocompatibility. The collagen molecular weight and structure were analyzed by SDS-PAGE. The bioactivity of trestle was tested in the experiment of the mice wound healing and the cell implantation. RESULTS: The SDS-PAGE result of the collagen treated by Protease M showed the typical spectrum of type I collagen. The built trestle was a collagen sponge matrix in which micropore size was 50-200 microns. It could accelerate wound healing and the implanted fibroblasts could proliferate well. CONCLUSION: The collagen treated by Protease N can get good biocompatibilily and is suitable for building the trestles of tissue engineering for skin with good bioactivity.
OBJECTIVE: To validate the hemostatic properties of collagen sponge made in China. METHODS: The experimental model of superficial cut of liver was established in 20 Sprague-Dawley adult rats, which were divided into two groups randomly. Collagen sponge or gelatin sponge was used to cover the cut respectively. Hemostatic result was observed. Afterwards, standard liver trauma model by resection left front liver lobe was made, wound was treated with collagen sponge or gelatin sponge respectively. Hemostatic result was observed. Concurrent hemostatic time and bleeding amount were noted. At 7, 14 and 20 days after operation, intra-abdominal adhension, infection and healing state of liver were observed by exploratory laparotomy. The histological changes of regenerate liver tissue were observed by microscopy. RESULTS: Collagen sponge adhered to wound well. Concurrent hemostatic time and bleeding amount in collagen sponge group were superior to those of gelatin sponge (P lt; 0.05). The histological examination showed that collagen sponge was absorbed and degraded rapidly, regenerative hepatocytes could be induced. CONCLUSION: Collagen sponge has fine hemostatic properties and can induce regeneration of hepatocytes effectively. It is worth popularizing for its convenience in clinical application and its properties of rapid degradation and absorption.
Objective To introduce the development of the collagen materials in drug release and tissue engineering. Methods Literature review and complex analysis were adopted. Results In recent years, some good progress hasbeen made in the studies of collagen, and study on collagen-based materials has become an investigative hotspot especially in tissue engineering. Some new collagen-based drug delivery andengineered materials have come into clinically-demonstrated moment, which willpromote their clinical applications in tissue repairs.ConclusionCollagen has been considered a good potential material in drug release, especially in the tissue-engineering field. To give collagen new characters we should pay more attention to grafting with different function branches through chemistry technique in the future work, except- moderate cross-linking treatment or commingling withother nature or synthesized macromolecules.
Objective To explore the possibility of constructing tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid (HA) by electrospinning. Methods The three-dimensional porous nano-scaffolds were prepared by electrospinning techniques with collagen type II and HA (8 ∶ 1, W ∶ W), which was dissolved in mixed solvent of 3-trifluoroethanol and water (1 ∶ 1, V ∶ V). The morphology were observed by light microscope and scanning electron microscope (SEM). And the porosity, water absorption rate, contact angle, and degradation rate were detected. Chondrocytes were harvested from 1-week-old Japanese white rabbit, which was disgested by 0.25% trypsin 30 minutes and 1% collagenase overlight. The passage 2 chondrocytes were seeded on the nano-scaffold. The cell adhesion and proliferation were evaluated by cell counting kit 8 (CCK-8). The cell-scaffold composites were cultured for 2 weeks in vitro, and the biological morphology and extracelluar matrix (ECM) secretion were observed by histological analysis. Results The optimal electrospinning condition of nano-scaffold was 10% electrospinning solution concentration, 10 cm receiver distance, 5 mL/ h spinning injection speed. The scaffold had uniform diameter and good porosity through the light microscope and SEM. The diameter was 300-600 nm, and the porosity was 89.5% ± 25.0%. The contact angle was (35.6 ± 3.4)°, and the water absorption was 1 120% ± 34% at 24 hours, which indicated excellent hydrophilicity. The degradation rate was 42.24% ± 1.51% at 48 days. CCK-8 results showed that the adhesive rate of cells with scaffold was 169.14% ± 11.26% at 12 hours, and the cell survival rate was 126.03% ± 4.54% at 7 days. The histological and immunohistochemical staining results showed that the chondrocytes could grow well on the scaffold and secreted ECM. And the similar cartilage lacuma structure could be found at 2 weeks after co-culture, which suggested that hyaline cartilage formed. Conclusion The collage type II and HA complex three-dimensional nano-scaffold has good physicochemical properties and excellent biocompatibility, so it can be used as a tissue engineered cartilage scaffold.
ObjectiveTo explore the expression of collagen Ⅳ in breast cancer and its clinical significance. We analyzed the correlation of the results with other prognostic parameters which included tumor size, status of estrogen receptor, axillary nodal status, TNM grade, and 5 years survival. The expression of collagen Ⅳ in 93 cases of human primary breast cancer as well as 5 cases of benign breast masses were examined.MethodsUsing monoclonal antibodies of collagen Ⅳ, the expression of collagen Ⅳ in breast masses were detected with immunohistochemical technique (LSAB).ResultsThe absent expression of collagen Ⅳ in the tumor masses was correlated with axillary lymph node involvement, tumor size and poor prognosis (5 years survival). The patients who had no expression of collagen Ⅳ in tumor masses had a shorter survival. ConclusionThe expression of collagen Ⅳ in tumor samples are correlated with axillary node involvement and prognosis. Collagen Ⅳ would be helpful for evaluation of invasion and treatment in breast carcinoma.
Objective To develop a scaffold material containing collagen Ⅰ and sodium hyaluronate for the cartilage tissue engineering and to evaluate its biocompatibility by using the rabbit chondrocytes derived from amandibular condylar process. Methods The porous matrices containing collagen Ⅰ and sodium hyaluronate were fabricated by the freezedrying technique and were crosslinked by using 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC). The microstructure of the scaffold was observed under thescanning electron microscope (SEM), and the enzymatic degradation test was performed to compare the ability of the scaffold resistance to collagenase before and after the crosslinking. The chondrocytes from the rabbits’ condylar process were isolated and cultured before they were seeded into the scaffold, and cell attachment and proliferation were measured by the cell count 1, 3, 5, 7 and 10 daysafter the cell being seeded; then, the biocompatibility of the scaffold was evaluated by the light microscopic examination, histological examination, and the SEM exmination. Results The porous structure of the scaffold facilitated the penetration and attachment of the seeded cells. The porosity was 83.7% and the pore size was 100-120 μm. The cell number increased from 3.7×104 per scaffold 1 day after the cell being seeded to 8.2×104 per scaffold 10 days after the cell being seeded. The crosslinking treatment could significantly enhance the scaffold resistance to the collagenase activity. The examinations under the light microscope and SEM indicated that the chondrocyte adhered and spread well on the scaffold, and the extracellular matrices were also observed around the chondrocytes. Conclusion The porous scaffold composed of collagen Ⅰ and hyaluronan has anappropriate structure and a good biocompatibility for the attachment and proliferation of the chondrocytes, which can facilitate it to become a useful scaffold in the cartilage tissue engineering.
The secondary anastomotic stenosis is often occured from the repair and reconstructive operation of the injured bile duct. It is difficult to treat and the outcome is serious. In order to prevent this complication, the fibrin glue instead of traditional suturing technique combined with inner support was used. Fifty-four hybrid dogs were divided into 3 groups. Group A received Roux-en-y choledochojejunostomy with fibrin glue; group B received Roux-en-y choledochojejunostomy, with a fibrin glue combined support left permanently in the bile duct and group C received Roux-en-y choledocholejejunostomy with fibrin glue combined a support left temporarily in the bile duct. The amount of collagen in the scar was measured at 3/4, 3, 6, 9, 12 months respectively after operation. The results showed: 1. the mature period of scar was shortened from 12 months to 9 months when fibrin glue instead of suture was used in choledochojejunostomy; 2. the mature period of scar was further shortened from 9 months to 6 months when fibrin glue combined with inner support instead of fibrin glue was used in choledochojejunostomy. The conclusions were as follows: 1. fibrin glue could facilitate the healing of wound by inhibiting the formation of scar and accelerrate the maturation of scar; 2. when the inner support was used with fibrin glue in the operation, the mature period of scar could be further shortened; 3. the mechanism of action of the fibrin glue included minimizing the injury, avoiding foreign-body reaction, modifying organization of hematoma, preventing formation of biliary fistular and enhancing intergration and cross-linkage of collagen.
To investigate the inhibitory effect of Col I A1 antisense ol igodeoxyneucleotide (ASODN) transfection mediated by cationic l iposome on Col I A1 expression in human hypertrophic scar fibroblasts. Methods Scar tissue was obtained from volunteer donor. Human hypertrophic scar fibroblasts were cultured by tissue block method. The cells at passage 4 were seeded in a 6 well cell culture plate at 32.25 × 104 cells/well, and then divided into 4 groups: group A, l iposomeand Col I A1 ASODN; group B, Col I A1 ASODN; group C, l iposome; group D, blank control. At 8 hours, 1, 2, 3 and 4 days after transfection, total RNA of the cells were extracted, the expression level of Col I A1 mRNA was detected by RT-PCR, the Col I A1 protein in ECM was extracted by pepsin-digestion method, its concentration was detected by ELISA method. Results Agarose gel electrophoresis detection of ampl ified products showed clear bands without occurrence of indistinct band, obvious primer dimmer and tailing phenomenon. Relative expression level of Col I A1 mRNA: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), and groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (Pgt; 0.05); at 1 day after transfection, groups A and B were less than groups C and D (P lt; 0.05), and there was no significant difference between group A and group B, and between group C and group D (P gt; 0.05 ); at 2 days after transfection, there were significant differences among four groups (P lt; 0.05); at 3 and 4 days after transfection, group A was less than groups B, C and D (P lt; 0.05), group B was less than groups C and D (P lt; 0.05), and no significant difference was evident between group C and group D (P gt; 0.05). Concentration of Col I protein: at 8 hours after transfection, group A was less than groups B, C and D (P lt; 0.05), groups B and C were less than group D (P lt; 0.05), and no significant difference was evident between group B and group C (P gt; 0.05); at 1 day after transfection, significant differences were evident among four groups (P lt; 0.05); at 2, 3 and 4 days after tranfection, groups A and B were less than groups C and D (P lt; 0.05), and no significant difference was evident between group A and group B (P gt; 0.05). Conclusion Col I A1 ASODN can inhibit mRNA and protein expression level of Col I A1. Cationic l iposome, as the carrier, can enhance the inhibition by facil itating the entry of ASODN into cells and introducing ASODN into cell nucleus.