ObjectiveTo analyze the phasic changes of bone mass, bone turnover markers, and estrogen levels at different time points after glucocorticoid (GC) intervention in rat and their correlation. MethodsThirty-four female 3-month-old Sprague Dawley rats were randomly divided into the following 3 groups:baseline group (n=6), dexamethasone (DXM) group (n=14), and control group (n=14). Rats were injected with DXM at the dose of 0.75 mg/kg, twice a week for 12 weeks in DXM group, with salt solution lavage in control group, and no treatment was given in baseline group. The body mass, adrenal weight, and uterus weight were measured. Bone mineral density (BMD), bone mineral content (BMC), and bone area (BA) of lumbar vertebral and femurs were detected by dual energy X-ray absorptiometry. Meanwhile, the serum levels of N-terminal propeptide of type I procollagen (PINP), C-terminal cross-linking telopeptide of type I collagen (β-CTX), and estrogen levels were determined by ELISA before experiment in baseline group and at 4, 8, and 12 weeks after experiment in control and DXM groups. At last, the correlation was analyzed among body weight, BMD, PINP, β-CTX, estrogen levels, and GC intervention duration of DXM group. ResultsThe body mass, adrenal weight, and uterus weight in DXM group were significantly lower than those in baseline group and control group at all the time points (P<0.05). The levels of PINP and β-CTX elevated slowly in DXM group, significant difference was found at 12 weeks (P<0.05), but no significant difference at the 4 and 8 weeks (P>0.05) when compared with those in baseline group and control group. The estrogen level in DXM group was significantly lower than that in baseline group and control group at all the time points (P<0.05). BMD, BMC, and BA of lumbar vertebral and femurs in DXM group were significantly lower than those in control group at all the time points after GC intervention (P<0.05). Loss of bone mass of L2 and femoral trochanteric region in DXM group was the lowest of all ranges of interest (ROIs). BMC and BA of lumbar vertebrae and BA of femoral shaft in DXM group at 4 weeks were significantly lower than those in baseline group (P<0.05). But there was no significant difference in BMD, BMC, and BA of other lumbar vertebrae and femurs' ROIs between DXM group and baseline group at all the time points (P>0.05). After GC intervention, BMD of lumbar vertebrae and femurs had negative correlation with PINP and β-CTX (P<0.05) and positive correlation with estrogen level (P<0.05). ConclusionThe bone mass decreases rapidly at the early stage after GC intervention and then maintains a low level with time, the levels of bone turnover markers show a progressive increase, and the estrogen levels show a decrease trend. In addition, body weight, the levels of bone turnover markers and estrogen are associated with the change of bone mass.
Objective To investigate the preventive effect of carbachol on the formation of postoperative intra-abdominal adhesion. Methods Forty-four Wistar rats were randomly divided into sham operation group (SO group, n=12), operation group (n=16) and carbachol treated group (carbachol group, n=16, carbachol 50 μg/kg). Animal model of abdominal adhesion was established by rubbing the procussus vermiformis of cecum with dry sterile gauze, and by clamping and scuffing abdominal wall. Half of rats were separately killed on day 7 and day 14 after surgery, respectively. The degree of adhesion was evaluated according to Phillips 5-scale grade and the feature of this model. The histopathological changes of adhesive tissues were observed and the content of collagen type Ⅰ in the tissues was detected by immunohistochemistry. Results The scores of intra-abdominal adhesion were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). Mild inflammatory changes and less fibrous proliferation were observed in carbachol group microscopically. The contents of collagen type Ⅰ detected by immunohistochemistry were significantly lower in the carbachol group than those in operation group both on 7 d and 14 d (P＜0.01). There was no significant difference of the score of abdominal adhesion and content of collagen type Ⅰ in the same group between 7 d and 14 d (Pgt;0.05). Conclusion Carbachol may take a significant role in the prevention of postoperative abdominal adhesion in rat.
Objective To investigate the effect of vanadate on prol iferation and collagen type I production of rat medial collateral l igament (MCL)fibroblasts. Methods A total of 12 adult male SD rats were included, weighing 350-375 g. MCL was cut into small pieces (1 mm × 1 mm × 1 mm) in aseptic conditions, and then placed and cultured in culture chambers. Fibroblasts were passaged with 0.25% trypsin. The vanadate (0, 1.0, 2.5, 5.0 ng/mL) was added in the 3rd passage MCL fibroblasts, respectively, and the samples were divided into 4 groups (0, 1.0, 2.5, 5.0 ng/mL groups). MTT was used to measure the cell prol iferation. The production of collagen type I was measured by RT-PCR and ELISA. Twelve samples in each group were measured. Results In fibroblast prol iferation, the absorbency values of the 1.0, 2.5, 5.0 ng/mL groups were significantly different from that of the 0 ng/mL group (P lt; 0.05). The absorbency values of the 0, 1.0, 2.5, and 5.0 ng/mL groups were 0.213 ± 0.016, 0.327 ± 0.023, 0.449 ± 0.137, and 0.561 ± 0.028, respectively. In collagen secretion, vanadate in 1.0, 2.5, 5.0 ng/mL groups could significantly induce the production of collagen type I (P lt; 0.05) ina dose-dependent manner. The expressions of collagen type I of 0-5 ng/mL groups were 0.47 ± 0.02, 0.51 ± 0.03, 0.60 ± 0.01, and 0.72 ± 0.02, respectively. There was significant difference between the 1.0, 2.5, 5.0 ng/mL groups and 0 ng mL group (P lt; 0.05). RT-PCR displayed a dramatic increase of band density. The ratio of band density in the 0-5 ng mLgroups was 1.37 ± 0.76, 1.97 ± 0.53, 2.41 ± 0.94, and 2.73 ± 0.82, respectively. The gene expression of collagen type I in the 1.0, 2.5 and 5.0 ng/mL groups was higher than that in the 0 ng/mL group, and there was significant difference (P lt;0.05). There were statistical significant differences among 1.0, 2.5 and 5.0 ng/mL groups in each index mentioned above.Conclusion Vanadate can effectively induce the prol iferation of fibroblasts and the production of collagen type I in vitro, which may provide a new approach to the treatment of MCL injury.
【Abstract】 Objective To investigate the possibil ity of BMSCs seeded into collagen Ⅰ -glycosaminoglycan (CG)matrices to form the tissue engineered cartilage through chondrocyte inducing culture. Methods Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment (DHT)cross-l inked CG matrices at 1×106 cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen Ⅱ immunohistochemical staining. Results The examinations under the inverted phase contrast microscopeindicated the 2nd and 3nd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-l ike spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen Ⅱ staining appeared around the cells in the experimentalgroup. There was no positive collagen Ⅱ staining appeared in the control group. Conclusion It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.
Objective To investigate the effect of icarin/attapulgite/collagen type Ⅰ/polycaprolactone (ICA/ATP/Col Ⅰ/PCL) composite scaffold in repair of rabbit tibia defect. Methods The ICA/20%ATP/Col Ⅰ/PCL (scaffold 1), ICA/30%ATP/Col Ⅰ/PCL (scaffold 2), 20%ATP/Col Ⅰ/PCL (scaffold 3), and 30%ATP/Col Ⅰ/PCL (scaffold 4) composite scaffolds were constructed by solution casting-particle filtration method. The structure characteristics of the scaffold 2 before and after cross-linking were observed by scanning electron microscopy, and the surface contact angles of the scaffold 2 and the scaffold 4 were used to evaluate the water absorption performance of the material. The in vitro degradation test was used to evaluate the sustained-release effect of the scaffold 2. Thirty male Japanese white rabbits, weighing (2.0±0.1) kg, were randomly divided into groups A, B, C, D, and E, 6 in each group. After making a 1 cm- diameter bilateral tibial defects model, group A was the defect control group without any material implanted. Groups B, C, D, and E were implanted with scaffolds 3, 4, 1, and 2 at the defect sites, respectively. At 4, 8, and 12 weeks after operation, the repairing effects of 4 scaffolds were observed by gross observation, histological observation of HE and Masson staining, and immunohistochemical staining of osteogenic specific transcription factor (runt-related transcription factor 2, RUNX2), osteogenic related transcription factor [Osterix (OSX), Col Ⅰ, osteopontin (OPN)]. Results Scanning electron microscopy observation showed that the scaffolds were all porous. The structure of the material was loose before and after cross-linking. The surface contact angle showed that the scaffold was hydrophobic, and the scaffold 2 was more hydrophobic than scaffold 4. The sustained-release effect in vitro showed that the drug could be released in a micro and long-term manner. In the animal implantation experiment, the gross observation showed that the defects were significantly smaller in groups D and E than in groups A, B, and C at 4 and 12 weeks after operation. HE and Masson staining showed that the defect of group A was full of connective tissue at 4 weeks after operation, a large number of fibers were seen in groups B and C, and the new bone formation was observed in groups D and E. The increase of new bone was observed in each group at 8 weeks after operation. The defect of group A was still dominated by connective tissue at 12 weeks after operation, and a small amount of new bone tissue was observed in groups B and C, and a large number of new bone tissue was observed in groups D and E, especially in group E, and most of the materials degraded. Immunohistochemical staining showed that the expressions of RUNX2 and OSX in the new tissues of groups D and E were significantly higher than those of the other groups at 4 weeks after operation. The expression of RUNX2 decreased at 8 and 12 weeks after operation. After 8 weeks and 12 weeks, the expressions of Col Ⅰand OPN increased than in 4 weeks. And the expressions of Col Ⅰ and OPN in the new tissues of groups D and E were significantly more than those of the other groups. Conclusion ICA/ATP/Col I/PCL composite scaffolds have good porosity and biocompatibility, can promote bone formation, and have good bone regeneration and repair effect.
Objective To investigate the possibility of repairing articular cartilage defects with the mesenchymal stem cells(MSCs) seeded type Ⅰ collagen-glycosaminoglycan(CG) matrices after being cultured with the chondrogenic differentiation medium. Methods The adherent population of MSCs from bone marrow of10 adult dogs were expanded in number to the 3rd passage. MSCs were seeded intothe dehydrothermal treatment (DHT) crosslinked CG matrices; 2×106 cells per 9mm diameter samples were taken. Chondrogenic differentiation was achieved by the induction media for 3 weeks. Cell contractility was evaluated by the measuement of the cell-mediated contraction of the CG matrices with time inculture.The in vitro formation of the cartilage was assessed by an assayemploying immunohistochemical identification of type Ⅱ collagen and by immunohistochemistry to demonstrate smooth muscle actin （SMA）. The cells seededingCGs wereimplanted into cartilage defectsof canine knee joints. Twelve weeks after surgery, the dogs were sacrificed and results were observed. Results There was significant contraction of the MSCsseeded DHT crosslinked CG scaffolds cultured in the cartilage induction medium. After 21 days, the MSCseeded DHT crosslinked matrices were contracted to 64.4%±0.3%; histologically, the pores were found to be compressedandthe contraction coupled with the newly synthesized matrix, transforming the MSCsseeded CG matrix into a solid tissue in most areas. The type Ⅱ collagen staining was positive. The SMA staining was positive when these MSCs were seeded and the contracted CGs were implanted into the cartilage defects of the canine knee joints to repair the cartilage defects. The function of the knee joints recovered and the solid cartilaginous tissue filled the cartilage defects. Conclusion The results demonstrates that MSCs grown in the CG matrices can produce a solid cartilaginous tissuecontaining type Ⅱ collagen after being cultured with the chondrogenic differentiation medium and implanted into cartilage defects. We hypothesize that the following steps can be performed in the chondrogenic process: ①MSCs express SMA, resulting in matrix contraction, thus achieving a required cell density (allowing the cells to operate in a necessary society)； ②Cells interact to form a type Ⅱ collagencontaining extracellular matrix (and cartilaginous tissue)； ③Other factors, suchas an applied mechanical stress, may be required to form a mature cartilage with the normal architecture.
ObjectiveTo investigate the effect of repairing radial bone defect with scaffold material of attapulgite/collagen type I/poly (caprolactone) (ATP/Col I/PCL) in rabbits and the possibility as bone graft substitutes. MethodsATP/Col I/PCL materials were prepared via adding ATP to hexafluoroisopropanol after dissolved Col I/PCL (3∶2), and Col I/PCL materials via dissolving Col I/PCL (3∶2) in hexafluoroisopropanol served as control. The structure of scaffolds was observed under scanning electron microscope (SEM). Twenty-four Japanese white rabbits (male, 2 months old) were used to establish the bilateral radius defect model of 15 mm in length, and randomly divided into group A (6 rabbits, 12 defects), group B (9 rabbits, 18 defects), and group C (9 rabbits, 18 defects); then the Col I/PCL scaffold was implanted in the bone defect area in group B, the ATP/Col I/PCL scaffold in group C, no treatment was done in group A as control. The general condition of rabbits was observed after operation, and bone defect repair was evaluated by X-ray at 4, 8, and 12 weeks. At 12 weeks, the tissue of defect area was harvested for the general, SEM, Micro-CT, histological, and immunohistochemical staining to observe defect repair and material degradation. ResultsSEM observation showed that two kinds of materials were porous structure, ATP/Col I/PCL structure was more dense than Col I/PCL. All animals survived to the end of experiment, and no incision infection occurred during repair process.X-ray films showed that the bone marrow cavity was re-opened in defect area of group C with time, the repair effect was superior to that of groups A and B. At 12 weeks after operation, general observation showed that scaffold material had good fusion with the surrounding tissue in groups B and C, defect was filled with connective tissue in group A. SEM indicated that the surface and pore of the scaffold were covered with a large number of cells and tissues in groups B and C. Micro-CT demonstrated that the new bone volume, bone mineral content, tissue mineral content, and connectivity density of group C were significantly higher than those of groups A and B (P<0.05). The observation of histology and immunohistochemical staining indicated that there were lots of connective tissues in defect area of group A, and ALP, Col I, and OPN were weakly expressed; there were many collagen fibers in scaffold degradation area in group B, and the expression levels of ALP, Col I, and OPN were higher than those of group A; there was few new bone in group C, the degradation rate of the scaffold was slower than that of group B, and the expression of Col I and OPN were enhanced, while ALP was weakened when compared with groups A and B. ConclusionATP/Col I/PCL composite scaffold material can degrade in vivo, and has dense three-dimensional porous structure, good biocompatibility, and high potentiality of bone repair, so it can be used as bone substitute material.
ObjectiveElectrospinning technique was used to manufacture polycaprolactone (PCL)/collagen typeⅠ nanofibers orientated patches and to study their physical and chemical characterization, discussing their feasibility as synthetic patches for rotator cuff repairing.MethodsPCL patches were prepared by electrospinning with 10% PCL electrospinning solution (control group) and PCL/collagen typeⅠorientated nanofibers patches were prepared by electrospinning with PCL electrospinning solution with 25% collagen type Ⅰ(experimental group). The morphology and microstructure of the two patches were observed by gross and scanning electron microscopy, and the diameter and porosity of the fibers were measured; the mechanical properties of the patches were tested by uniaxial tensile test; the composition of the patches was analyzed by Fourier transform infrared spectroscopy; and the contact angle of the patch surface was measured. Two kinds of patch extracts were co-cultured with the third generation of rabbit tendon stem cells. Cell counting kit 8 (CCK-8) was used to detect the toxicity and cell proliferation of the materials. Normal cultured cells were used as blank control group. Rabbit tendon stem cells were co-cultured with the two patches and stained with dead/living cells after 3 days of in vitro culture, and laser confocal scanning microscopy was used to observe the cell adhesion and activity on the patch.ResultsGross and scanning electron microscopy showed that the two patch fibers were arranged in orientation. The diameter of patch fibers in the experimental group was significantly smaller than that in the control group (t=26.907, P=0.000), while the porosity in the experimental group was significantly larger than that in the control group (t=2.506, P=0.032). The tensile strength and Young’s modulus of the patch in the experimental group were significantly higher than those in the control group (t=3.705, P=0.029; t=4.064, P=0.034). Infrared spectrum analysis showed that PCL and collagen type Ⅰ were successfully mixed in the experimental group. The surface contact angle of the patch in the experimental group was (73.88±4.97)°, which was hydrophilic, while that in the control group was (128.46±5.10) °, which was hydrophobic. There was a significant difference in the surface contact angle between the two groups (t=21.705, P=0.002). CCK-8 test showed that with the prolongation of culture time, the cell absorbance (A) value increased gradually in each group, and there was no significant difference between the experimental group and the control group at each time point (P>0.05). Laser confocal scanning microscopy showed that rabbit tendon stem cells could adhere and grow on the surface of both patches after 3 days of culture. The number of cells adhered to the surface of the patches in the experimental group was more than that in the control group, and the activity was better.ConclusionPCL/ collagen type Ⅰ nanofibers orientated patch prepared by electrospinning technology has excellent physical and chemical properties, cell adhesion, and no cytotoxicity. It can be used as an ideal scaffold material in tendon tissue engineering for rotator cuff repair in the future.
ObjectiveTo explore the interaction between immune cell infiltration and extracellular matrix (ECM) in diffuse gastric cancer (DGC), and to identify novel diagnostic biomarkers and therapeutic targets. MethodsTranscriptomic data of DGC patients from The Cancer Genome Atlas (TCGA) database were analyzed to screen potential regulator factor of immune-related and ECM receptor-related signaling pathways. Differential expression of the identified regulator was assessed between the DGC tissues and the adjacent gastric tissues. Bioinformatics analysis was utilized to evaluate the relation between the regulator factor and immune cell infiltration and ECM, as well as prognosis. The clinical validation was performed using 90 paraffin-embedded DGC tissues and adjacent gastric tissues from the patients treated at The Lanzhou University Second Hospital (hereafter “our hospital”) from January 2017 to December 2019. The immunohistochemical staining was employed to examine the expression of regulator factor, followed by analysis of its association with immune cell infiltration, clinicopathologic features, and prognosis. Additionally, 10 paired DGC tissues and adjacent gastric tissues from the patients treated in our hospital in 2024 were collected for validation using real-time quantitative PCR to assess mRNA expression. The significance level was set at α=0.05. ResultsThe collagen type I alpha 1 chain (COL1A1), a potential regulator factor linked to immune and ECM receptor signaling pathways, was identified from the TCGA database. The COL1A1 was significantly overexpressed in the DGC tissues compared to the adjacent gastric tissues (P<0.001), and its high expression correlated with poorer prognosis [HR(95%CI)=2.98(1.21, 7.30), P=0.017]. The COL1A1 gene expression negatively correlated with CD8+ T cell enrichment score (CIBERSORT: r=−0.17, P<0.001; xCELL: r=−0.32, P<0.001) but positively correlated with M2 tumor-associated macrophage enrichment score (CIBERSORT: r=0.32, P<0.001; xCELL: r=0.24, P<0.001). The clinical validation confirmed that the COL1A1 protein and mRNA were both overexpressed in the DGC tissues (P<0.001). The patients with high COL1A1 protein expression had worse overall survival (P<0.001), and high expression (vs. low) was an independent risk factor for postoperative overall survival [HR(95%CI)=6.607(3.374, 12.940), P<0.001]. The COL1A1 protein expression positively correlated with CD163 (an M2 macrophage marker; r=0.76, P<0.001) and negatively with CD8+ (T cell marker, r=−0.84, P<0.001). ConclusionThis study demonstrates that COL1A1 is a potential therapeutic target for immune suppression and ECM interaction in DGC and a critical prognostic factor for long-term survival in patients with DGC.
Objective To investigate the influence of collagen on the biomechanics strength of tissue engineering tendon. Methods All of 75 nude mice were madethe defect models of calcaneous tendons, and were divided into 5 groups randomly. Five different materials including human hair, carbon fibre (CF), polyglycolic acid (PGA), human hair and PGA, and CF and PGA with exogenous collagen were cocultured with exogenous tenocytes to construct the tissue engineering tendons.These tendons were implanted to repair defect of calcaneous tendons of right hind limb in nude mice as experimental groups, while the materials without collagenwere implanted to repair the contralateral calcaneous tendons as control groups. In the 2nd, 4th, 6th, 8th and 12th weeks after implantation, the biomechanicalcharacteristics of the tissue engineering tendon was measured, meanwhile, the changes of the biomechanics strength were observed and compared. Results From the 2nd week to the 4th week after implantation, the experimental groups were ber than the control groups in biomechanics, there was statistically significantdifference (Plt;0.05). From the 6th to 12th weeks, there was no statisticallysignificant difference between the experiment and control groups (Pgt;0.05). Positivecorrelation existed between time and intensity, there was statistically significant difference (Plt;0.05). The strength of materials was good in human hair,followed by CF, and PGA was poor. Conclusion Exogenous collagen can enhance the mechanics strength of tissue engineering tendon, and is of a certain effect on affected limb rehabilitation in early repair stages.