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 elucidate the role of the transcription factor liver activator protein (LAP, a member of the C/EBP family) in the expression of α1(I) collagen gene in activated hepatic stellate cells (HSCs). Methods Rat HSCs were prepared from SD rats by in situ perfusion and singlestep density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen α1(I) gene promoter fragments (-804～+1 452 or -804～+222) were constructed. Culture-activated HSCs were co-transfected with the reporter gene contructs and mammalian vector expressing LAP using the cationic-liposome mediated method, and the promoter activity was determined by measuring luciferase activity. Results The luciferase reporter gene construct containing the first intron of α1(I) collagen gene (-804～+1 452, was called as PGL3-col) had a higher level of gene expression, as compared with the construct lacking the first intron 〔was called as PGL3-col (△intron)-in activated HSCs (315±45 U/mg protein vs 220±70 U/mg protein, Ｐ＜0.05). Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL3-col and PGL3-col (△intron) reporter gene vectors (587±62 U/mg protein vs 315±45 U/mg protein and 326±52 U/mg protein vs 220±70 U/mg protein respectively, both Ｐ＜0.05). Conclusion The transcription factor LAP transactivates collagen α1(I) gene in activated HSCs, and the first intron is important for α1(I) collagen gene transcription activity in activated HSCs.
Collagen is a kind of natural biomedical material and collagen based three-dimensional porous scaffolds have been widely used in skin tissue engineering. However, these scaffolds do not meet the requirements for artificial skin substitutes in terms of their poor mechanical properties, short supply, and rejection in the bodies. All of these factors limit their further application in skin tissue engineering. A variety of methods have been chosen to meliorate the situation, such as cross linking and blending other substance for improving mechanical properties. The highly biomimetic scaffolds either in structure or in function can be prepared through culturing cells and loading growth factors. To avoid the drawbacks of unsafety attributing to animals, investigators have fixed their eyes on the recombinant collagen. This paper reviews the the progress of research and application of collagen-based 3-dimensional porous scaffolds in skin tissue engineering.
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 explore the molecular mechanisms involved in the increased collagen synthesis by platelet-derived wound healing factors (PDWHF) during wound healing in alloxan-induced diabetic rats. METHODS: Thirty-three male SD rats were divided into two groups, the normal (n = 9) (group A) and the diabetic group (n = 24). Two pieces of full-thickness skin with diameter of 1.8 cm were removed from the dorsal site of diabetic rats. PDWHF (100 micrograms/wound) was topically applied to one side of the diabetic wounds (group B) on the operation day and then once a day in the next successive 6 days. Meanwhile, bovine serum albumin (100 micrograms/wound) was applied to the other side of diabetic wound as control group (group C) in the same way. Levels of transforming growth factor-beta 1 (TGF-beta 1) and procollagen I mRNA in wound tissue were inspected by dot blotting. RESULTS: TGF-beta 1 mRNA levels in group B were 4 folds and 5.6 folds compared with those in group C after 5 and 7 days (P lt; 0.01), however, still significantly lower than those of group A (P lt; 0.05). There was no significance difference among three groups on the 10th day after wounding. The levels for procollagen I mRNA in group B amounted to 2.1, 1.8 and 2.3 folds of those in group C after 5, 7, and 10 days (P lt; 0.01), respectively. Compared with those in the group A, procollagen I mRNA levels in the group B were significantly lower after 5 and 7 days (P lt; 0.05), and no significant difference was observed between group B and A after 10 days. CONCLUSION: One important way for PDWHF to enhance the collagen synthesis in diabetic wound healing is to increase the gene expression of endogenous TGF-beta 1.
ObjectiveTo evaluate the effect of the combination of collagen scaffold and brain-derived neurotrophic factor (BDNF) on the repair of transected spinal cord injury in rats.MethodsThirty-two Sprague-Dawley rats were randomly divided into 4 groups: group A (sham operation group), T9, T10 segments of the spinal cord was only exposed; group B, 4-mm T9, T10 segments of the spinal cord were resected; group C, 4-mm T9, T10 segments of the spinal cord were resected and linear ordered collagen scaffolds (LOCS) with corresponding length was transplanted into lesion site; group D, 4-mm T9, T10 segments of the spinal cord were resected and LOCS with collagen binding domain (CBD)-BDNF was transplanted into lesion site. During 3 months after operation, Basso-Beattie-Bresnahan (BBB) locomotor score assessment was performed for each rat once a week. At 3 months after operation, electrophysiological test of motor evoked potential (MEP) was performed for rats in each group. Subsequently, retrograde tracing was performed for each rat by injection of fluorogold (FG) at the L2 spinal cord below the injury level. One week later, brains and spinal cord tissues of rats were collected. Morphological observation was performed to spinal cord tissues after dehydration. The thoracic spinal cords including lesion area were collected and sliced horizontally. Thoracic spinal cords 1 cm above lesion area and lumbar spinal cords 1 cm below lesion area were collected and sliced coronally. Coronal spinal cord tissue sections were observed by the laser confocal scanning microscope and calculated the integral absorbance (IA) value of FG-positive cells. Horizontal tissue sections of thoracic spinal cord underwent immunofluorescence staining to observe the building of transected spinal cord injury model, axonal regeneration in damaged area, and synapse formation of regenerated axons.ResultsDuring 3 months after operation, the BBB scores of groups B, C, and D were significantly lower than those of group A (P<0.05). The BBB scores of group D at 2-12 weeks after operation were significantly higher than those of groups B and C (P<0.05). Electrophysiological tests revealed that there was no MEP in group B; the latencies of MEP in groups C and D were significantly longer than that in group A (P<0.05), and in group C than in group D (P<0.05). Morphological observation of spinal cord tissues showed that the injured area of the spinal cord in group B extended to both two ends, and the lesion site was severely damaged. The morphologies of spinal cord tissues in groups C and D recovered well, and the morphology in group D was closer to normal tissue. Results of retrograde tracing showed that the gray matters of lumbar spinal cords below the lesion area in each group were filled with FG-positive cells; in thoracic spinal cords above lesion sites, theIA value of FG-positive cells in coronal section of spinal cord in group A was significantly larger than those in groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), but no significant difference was found between groups C and D (P>0.05). Immunofluorescence staining results of spinal cord tissue sections selected from dorsal to ventral spinal cord showed transected injured areas of spinal cords which were significantly different from normal tissues. The numbers of NF-positive axons in lesion center of group A were significantly larger than those of groups B, C, and D (P<0.05), and in groups C and D than in group B (P<0.05), and in group D than in group C (P<0.05).ConclusionThe combined therapeutic approach containing LOCS and CBD-BDNF can promote axonal regeneration and recovery of hind limb motor function after transected spinal cord injury in rats.
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 Astragalus polysaccharide (APS) has promoting angiogenesis function. To explore the effects of APS collagen sponge on enhancing angiogenesis and collagen synthesis so as to provide evidence for the future tissue engineering appl ication as a kind of angiogenic scaffold. Methods APS collagen sponges were prepared by covalent binding with collagen polypeptides by using of crossl inking agents at the ratio of 1 ∶ 1 (W/W). Twenty 10-week-old SpragueDawley rats (10 males and 10 females, and weighing 200-250 g) were selected. Longitudinal incision was made at both sides of the back to form subcutaneous pockets. APS collagen sponges of 5 mm × 5 mm × 5 mm at size were implanted into the left pockets as the experimental group, collagen sponges without APS of the same size into the right pockets as the control group. The general conditions were observed after operation. At 3, 7, 14，and 21 days, 5 rats were sacrificed and the samples were harvested to count the number of microvessels, to measure the contents of the hydroxyprol ine (Hyp), and to detect the mRNA expressions of angiopoetin 1 (Ang1), matrix metalloproteinases 9 (MMP-9), and tissue inhibitors of metalloproteinases 1 (TIMP-1). Results All rats were al ive during experiment period. The number of microvessels increased gradually, and reached the peak at 14 days in 2 groups; the expermental group was significantly higher than the control group (P lt; 0.05). The contents of Hyp increased gradually in 2 groups, and the experimental group was significantly higher than the control group (P lt; 0.05). The mRNA expressions of Ang1 and MMP-9 in the experimental group were significantly higher than those in the control group at 3, 7, and 14 days (P lt; 0.05); the mRNA expression of TIMP-1 in the experimental group was significantly lower than that in the control group at 3 days and was significantly higher at 14 and 21 days (P lt; 0.05). Conclusion The APS collagen sponges can improve angiogenesis and collagen synthesis in wound heal ing by regulating the expressions of Ang1, MMP-9, and TIMP-1.
The aim of this article is to study how andrographolide-releasing collagen scaffolds influence rabbit articular chondrocytes in maintaining their specific phenotype under inflammatory environment. Physical blending combined with vacuum freeze-drying method was utilized to prepare the andrographolide-releasing collagen scaffold. The characteristics of scaffold including its surface morphology and porosity were detected with environmental scanning electron microscope (ESEM) and a density instrument. Then, the release of andrographolide from prepared scaffolds was measured by UV-visible spectroscopy. Rabbit chondrocytes were isolated and cultured in vitro and seeded on andrographolide-releasing collagen scaffolds. Following culture with normal medium for 3 d, seeded chondrocytes were cultured with medium containing interleukin-1 beta (IL-1β) to stimulate inflammation in vitro for 7 d. The proliferation, morphology and gene transcription of tested chondrocytes were detected with Alamar Blue assay, fluorescein diacetate (FDA) staining and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) test respectively. The results showed that the collagen scaffolds prepared by vacuum freeze-dry possess a high porosity close to 96%, and well-interconnected chambers around (120.7±17.8) μm. The andrographolide-releasing collagen scaffold continuously released andrographolide to the PBS solution within 15 d, and collagen scaffolds containing 2.22% andrographolide significantly inhibit the proliferation of chondrocytes. Compared with collagen scaffolds, 0.44% andrographolide-containing collagen scaffolds facilitate chondrocytes to keep specific normal morphologies following 7 d IL-1β induction. The results obtained by RT-qPCR confirmed this effect by enhancing the transcription of tissue inhibitor of metalloproteinase-1 (TIMP-1), collagen II (COL II), aggrecan (Aggrecan) and the ratio of COL II/ collagen I(COL I), meanwhile, reversing the promoted transcription of matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-13 (MMP-13). In conclusion, our research reveals that andrographolide-releasing (0.44%) collagen scaffolds enhance the ability of chondrocytes to maintain their specific morphologies by up-regulating the transcription of genes like COL II, Aggrecan and TIMP-1, while down-regulating the transcription of genes like MMP-1 and MMP-13 which are bad for phenotypic maintenance under IL-1β simulated inflammatory environment. These results implied the potential use of andrographolide-releasing collagen scaffold in osteoarthritic cartilage repair.
Objective To review the research progress of C terminal propeptide of collagen type II (CTX-II), a osteoarthritis (OA) biomarker. Methods Domestic and international l iterature about CTX-II was reviewed extensively and summarized. Results CTX-II is investigated broadly and has the best performance of all currently available biomarkers. CTX-II is a truly useful biomarker for early diagnosis, prognosis, and measurement of treatment response in OA. Conclusion Single CTX-II may be not sufficient for early diagnosis and prognosis of OA, so a combination of CTX-II and other biomarkers or diagnosis methods is needed.