ObjectiveTo investigate the changes of fibrinogen and classical markers of collagen metabolism [carboxy-terminal propeptide of type Ⅰ procollagen (PICP) and carboxy-terminal cross-linked peptide of type Ⅰ collagen (ICTP)] in peripheral blood and pericardial drainage after coronary artery bypass grafting (CABG) and/or heart valve replacement (VR), and to evaluate their relationship with postoperative atrial fibrillation (POAF) after cardiac surgery. MethodsPatients who underwent CABG and/or VR in the Heart Center of Beijing Chao-Yang Hospital from March to June 2021 were included. Peripheral blood and pericardial drainage fluid samples were collected before surgery and at 0 h, 6 h, 24 h and 48 h after surgery to detect PICP, ICTP and fibrinogen levels, and preoperative, intraoperative and postoperative confounding factors were also collected. PICP, ICTP and fibrinogen levels were measured by enzyme-linked immunosorbent assay (ELISA). ResultsA total of 26 patients with 125 blood samples and 78 drainage samples were collected. There were 18 males and 8 females with an average age of 64.04±7.27 years. The incidence rate of POAF was 34.6%. Among the factors, the fibrinogen level in pericardial drainage showed two peaks within 48 h after operation (0 hand 24 h after operation) in the POAF group, while it showed a continuous downward trend in the sinus rhythm (SR) group, and the change trend of fibrinogen in pericardial drainage was significantly different over time between the two groups (P=0.022). Fibrinogen in blood, PICP and ICTP in blood and drainage showed an overall decreasing trend, and their trends over time were not significantly different between the two groups of patients (P>0.05). Univariate analysis showed that fibrinogen at 24 h and 48 h after pericardial drainage, fibrinogen in preoperative blood, PICP immediately after surgery and right atrial long axis diameter were significantly higher or longer in the POAF group than those in the SR group. Multiple regression showed that fibrinogen≥11.47 ng/mL in pericardial drainage 24 h after surgery (OR=14.911, 95%CI 1.371-162.122, P=0.026), right atrial long axis diameter≥46 mm (OR=10.801, 95%CI 1.011-115.391, P=0.049) were independent predictors of POAF. ConclusionThis study finds the regularity of changes in fibrinogen and collagen metabolic markers after CABG and/or VR surgery, and to find that fibrinogen in pericardial drainage 24 h after surgery is a potential novel and predictive factor for POAF. The results provide a new idea for exploring the mechanism of POAF, and provide a research basis for the accurate prediction and prevention of clinical POAF.
Objective To investigate the curative effects of homograft of the mesenchymal stem cells(MSCs) compbined with the medical collagen membrane of the guided tissue regeneration(MCMG) on the full thickness defects of the articular cartilage. Methods MSCs derived from New Zealand rabbits aged 3-4 months weighing 2.1-3.4 kg were cultured in vitro with a density of 5.5×108/ml and seeded onto MCMG. The MSC/MCMG complex was cultured for 48 h and transplanted into the fullthickness defects on the inboardcondyle and trochlea. Twenty-seven healthy New Zealand rabbits were randomly divided into 3 groups of 9rabbits in each. The cartilage defects in the inboard condyle and trochlea werefilled with the auto bone marrow MSCs and MCMG complex (MSCs/ MCMG) in Group A (Management A), with only MCMG in Group B (Management B)and with nothing in Group C (Management C). Three rabbits were killed at 4, 8 and 12 weeks after operation in each group, and the reparative tissue samples evaluated grossly,histologically and immunohistochemically were graded according tothe gross and histological scale. Results Four weeks after transplantation, the cartilage and subchondralbone were regenerated in Group A;for 12 weeks, the regenerated cartilage gradually thicked; 12 week after transplantation, the defect was repaired and the structures of the carticular surface and subchondral bone was in integrity.The defects in Group A were repaired by the hylinelike tissue and the defects in Groups B and C were repaired by the fibrous tissues. Glycosaminoglycan and type Ⅱcollagen in Groups A,B and C were reduced gradually.The statistical analysis on the gross at 12 weeks and the histologicalgradings at 4 weeks,8 weeks and 12 weeks showed that the inboardcondylar repairhad no significant difference compared with the rochlearepair(Pgt;0.05).Management A was significantly better than Managements B and C (Plt;0.05), and Management B was better than Management C(Plt;0.05). Conclusion Transplantation of the MSCs combined with MCMG on the full thickness defects of the articular cartilage is a promising approach to the the treatment of cartilage defects. MCMG can satisfy the demands of the scaffold for the tissue-engineered cartilage.
Objective To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage.Methods Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type Ⅱcollagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct wascross-linked by use of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensilestrength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performedto evaluate the degradability of the scaffold in vitro. Results A bulk scaffold with desired configuration was obtained. The mechanical test showed that the crosslinking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100250 μm. In vitro lysozyme degradation indicated that crosslinked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. Conclusion Chitosan and typeⅡcollagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite socaffold meets requirements for tissue engineered scaffold and may serve as an alternative cellcarrier for tissue engineering cartilage.
Riboflavin-ultraviolet A (UVA) collagen cross-linking has not only achieved good clinical efficacy in the treatment of corneal diseases such as dilatation keratopathy, bullae keratopathy, infectious keratopathy, and in the combined treatment of corneal refractive surgeries, but also its efficacy and safety in scleral collagen cross-linking have been initially confirmed. To better promote the application of cross-linking in the clinical treatment of corneal and scleral diseases, exploring controllability and predictability of the surgical efficacy are both important for evaluating the surgical efficacy and personalized precision treatment. In this paper, the progress on the cross-linking depth of riboflavin-UVA collagen cross-linking, and its relationship with the cross-linking effect will be reviewed. It will provide the reference for further application of this procedure in ophthalmology clinics.
Objective To observe the effect of dynamic mechanical loading on the proliferation, differentiation, and specific gene expression of MC3T3-E1 cells that on three-dimensional (3D) biomimetic composite scaffolds prepared by low temperature 3D printing technology combined with freeze-drying. Methods The silk fibroin, collagen type Ⅰ, and nano-hydroxyapatite (HA) were mixed at a mass ratio of 3∶9∶2 and were used to prepare the 3D biomimetic composite scaffolds via low temperature 3D printing technology combined with freeze-drying. General morphology of 3D biomimetic composite scaffold was observed. Micro-CT was used to observe the pore size and porosity of the scaffolds, and the water swelling rate, stress, strain, and elastic modulus were measured. Then, the MC3T3-E1 cells were seeded on the 3D biomimetic composite scaffolds and the cell-scaffold composites were randomly divided into 2 groups. The experimental group was subjected to dynamic mechanical loading (3 500 με, 1 Hz, 15 minutes per day); the control group was not subjected to loading treatment. After 7 days and 14 days, the cell-scaffold composites of 2 groups were harvested to observe the growth of cells on the scaffolds by HE staining and scanning electron microscope. And the gene and protein expressions of collagen type Ⅰ, BMP-2, and osteocalcin (OCN) were measured by real-time fluorescent quantitative PCR and Western blot. Results The 3D biomimetic composite scaffold was a white cubic grid. Micro-CT detection showed the pore network structure in the scaffold material with good pore connectivity. The diameters of large pore and micro-aperture were (506.37±18.63) μm and (62.14±17.35) μm, respectively. The porosity was 97.70%±1.37%, and the water absorption swelling rate was 1 341.97%±64.41%. Mechanical tests showed that the compression displacement of the scaffold was (0.376±0.004) mm, the compressive stress was (0.016±0.002) MPa, and the elastic modulus was (162.418±18.754) kPa when the scaffold was compressed to 10%. At 7 days and 14 days, HE staining and scanning electron microscope observation showed that the cells grew inside the scaffold, mainly distributed around the scaffold pore wall. The cells in experimental group were more than control group, and the cells morphology changed from shuttle to flat. There was no significant difference in the cell counting between 2 groups at 14 days after 200-fold microscopy (t=–2.024, P=0.080), but significant differences were found between 2 groups at different time points under different magnifications (P<0.05). Real-time fluorescent quantitative PCR showed that the mRNA relative expressions of collagen type Ⅰ and OCN in experimental group were significantly higher than those in control group at 7 and 14 days (P<0.05). However, the mRNA relative expression of BMP-2 showing no significant difference between 2 groups (P>0.05). The protein relative expressions of collagen type Ⅰ, BMP-2, and OCN in experimental group were significantly higher than those in control group at 7 and 14 days (P<0.05). Conclusion After dynamic mechanical loading, the expressions of BMP-2, collagen type Ⅰ, and OCN in MC3T3-E1 cells inoculated into 3D biomimetic composite scaffolds are significantly up-regulated, indicating that appropriate mechanical loads favor osteoblast differentiation of MC3T3-E1 cells.
Objective To observe the effect of Melittin on collagen type II (Col-II) expression of rat endplate chondrocytes (EPCs) induced by interleukin 1β (IL-1β). Methods Primary EPCs from the lumbar vertebra of 4-week-old Sprague Dawley rats were culturedin vitro and identified by morphological observation, toluidine blue staining and Col-II immunofluorescence staining. Then, MTT assay was used to determine the optimal concentration of IL-1 and Melittin. Next, EPCs at passage 3 were randomly divided into 4 groups: no treatment was done in group A as control group; the optimal concentration of IL-1β, Melittin, and both IL-1β and Melittin were used in groups B, C, and D respectively. The expression of Col-II was detected by Western blot after 48 hours intervention. Results Under inverted microscope, the first generation EPCs were polygonal; cell proliferation decreased after fifth generation, and cell morphology changed into fusiform. The acidic mucosubstance in the cytoplasm (such as Aggrecan) was stained dark blue by toluidine blue. After marking Col-II by immunofluorescence, the positive expression of cytoskeleton (green fluorescence) could be observed. MTT assay showed that IL-1β and Melittin could inhibit the EPCs in a dose-dependent manner after intervention of 24 and 48 hours, and the optimal concentrations of IL-1β and Melittin intervention were 10 ng/mL and 1.0 μg/mL respectively. Compared with group A, the expression of Col-II was significantly reduced in group B, and was significantly increased in group C by Western blot assay, but there was no significant difference between group D and group A. The Col-II expression levels of groups A, B, C, and D were 0.991±0.024, 0.474±0.127, 1.913±0.350, and 1.159±0.297 respectively, showing significant difference between the other groups (P<0.05) except between group A and group D (P>0.05). Conclusion Melittin has a protective effect on endplate cartilage, and the research results provide experimental basis for the prevention and treatment of spinal degenerative disease.
Objective To review the lately new progress of fish collagen as biomedical materials, and then analyze feasibility and risk management of its application as a substitute of collagen originated from mammals in clinical practice. Methods Based on extensive research on new application and investigation of fish collagen, the paper was prepared to bring comprehensive analysis of its research and application status, and then several key points were focused on. Results Fish collagen has been proved to be a novel collagen of rich source, low risk of virus transmission, low biological risk, less religious barrier, and high biocompatibility. Fish collagen has promising prospect when applied in clinical practice as novel collagen especially as a substitute of collagen derived from mammals. However, very few related translational medicine research of fish collagen has been reported up to now in China. Conclusion As a novel potential substitute of collagen source derived from mammals, fish collagen is concerned to be clinical feasible and necessary in translational medicine. However, massive applied basic researches should be focused on in the further investigations.
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.
ObjectiveTo quantitatively determine the levels of type Ⅰ and type Ⅱ collagen mRNA in the intervertebral disc cartilage endplate of injured animal model, and to clarify the cytological function of intervertebral disc chondrocytes during fibrosis repair after intervertebral disc injury.MethodsForty healthy New Zealand rabbits were randomly divided into fibrosus puncture group, upper cartilage endplate single puncture group, upper and lower cartilage endplate multiple puncture group, and sham group. Two experimental animals were randomly selected from each group on the 2nd day, and the 2nd, 8th, 12th, and 24th week after the animal modeling operation to obtain intervertebral disc specimens. The levels of type Ⅰ collagen and type Ⅱ collagen in cartilage endplate cells of the intervertebral disc were determined by reverse transcriptase polymerase chain reaction (PCR). RNA was extracted from the endplate of the intervertebral disc, and the RNA concentration and the ratio of RNA concentration to protein concentration were determined by nucleic acid analyzer. Reverse transcription was performed by Revertaid M-Mulv reverse transcriptase, type Ⅰ and type Ⅱ collagen primers were designed to establish a PCR reaction system, 2% agarose gel electrophoresis (120 V, 40 min) was prepared by using 0.5×TBE electrophoresis buffer. The amplification results were observed under ultraviolet light, and the gray values of different electrophoresis bands were determined.ResultsThe level of type Ⅰ collagen mRNA in each operation group showed a progressive increase after 8 weeks, and the magnitude of the increase was related to the degree of injury. The level of type Ⅱ collagen mRNA showed a transient increase in the fibrosis puncture group and the upper endplate single-puncture group in the first two weeks after the endplate punctures were completed, and then began to decline progressively; in the multiple puncture group, it showed a downward trend from the beginning of the operation. ConclusionsThe synthesis of type Ⅰ collagen in chondrocytes of the injured nucleus pulposus tissue continues to increase with time, while the synthesis of type Ⅱ collagen begin to decrease progressively after a small increase. The formation and change of type Ⅰ and type Ⅱ collagen in injured intervertebral disc chondrocytes are different from natural degeneration.
ObjectiveTo investigate the bone repair and regeneration abilities of biomimetic mineralized collagen bone graft material and autologous bone marrow in rabbit posterolateral spinal fusion model.MethodsTwenty-seven 20-week-old male New Zealand white rabbits were used to establish the posterolateral spinal fusion model of L5 and L6 segments by stripping the transverse process and exposing cancellous bone with electric burr. The rabbits were randomly divided into 3 groups, 9 in each group. Groups A, B, and C were implanted 1.5 mL autologous iliac bone, 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material, and 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material and autologous bone marrow in each bone defect. At 4, 8, and 12 weeks after operation, the apparent hardness of the bone grafting area was observed by manipulation method, in order to evaluate bone graft fusion effects. Three animals were sacrificed in each group at each time point, the vertebral body specimens were excised and the bone defect repair and fusion were observed by X-ray films, and three-dimensional CT examination was performed to evaluate whether new bone was formed in the body. HE staining was performed at each time point to observe the formation of new bone and the repair and fusion of bone defects.ResultsThe manipulation test showed that bone graft fusion was not found in all groups at 4 weeks after operation; 3 (50.0%), 2 (33.3%), and 4 (66.7%) of groups A, B, and C reached bone graft fusion at 8 weeks after operation; 5 (83.3%), 4 (66.7%), and 5 (83.3%) of groups A, B, and C reached bone graft fusion at 12 weeks after operation; the fusion rate of group C was similar to that of group A, and all higher than that of group B. X-ray film observation showed that the fusion rate of group C at 8 and 12 weeks after operation was higher than that of group B, which was similar to group A. Three-dimensional CT observation showed that the degree of bone fusion in group C was better than that in group B, which was close to group A. HE staining observation showed that large area of mature lamellar bone coverage appeared in the bone graft area of groups A, B, and C at 12 weeks after operation, the material was completely degraded, and the marginal boundary of the host bone disappeared and tightly combined.ConclusionBiomimetic mineralized collagen bone graft material mixed with autologous bone marrow has good osteoinduction and osteogenesis guidance. Compared with biomimetic mineralized collagen bone graft material, it has better and faster osteogenesis effect, which is close to autologous bone transplantation.