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BACKGROUND: Osteoarthritis is one of the most common degenerative diseases, and there are still no drugs to delay or reverse the disease. Only antiinflammatory and analgetic treatments can be performed to relieve symptoms and improve patient’s conditions in the short term. Astragaloside IV is an active component of Astragalus membranaceus with immunomodulatory, ischemic protection, cardiac protection, anti-inflammatory, antiviral, and anti-tumor effects. OBJECTIVE: To investigate the effects of astragaloside IV on interleukin-1β-induced ATDC5 cell injury and its related mechanism. METHODS: ATDC5 cells were randomly divided into blank control group, model group, monomer group, and experimental group. There was no intervention in the blank control group. Cells in the latter three groups were treated with 100 μg/L interleukin-1β, 5 μg/L astragaloside IV, and 100 μg/L interleukin-1β+5 μg/L astragaloside IV, respectively. After 18 hours of treatment, cell counting kit-8 was used to detect cell proliferation. Western blot and qRT-PCR were used to detect the expression of matrix metalloproteinases 3, 9, 13, type II collagen, stromal cell-derived factor 1 (SDF-1), and C-X-C chemokine receptor 4 (CXCR4) at protein and mRNA levels, respectively. Toluidine blue staining was used to detect cell morphological changes and cell numbers, and trypan blue staining was used to detect cell viability. RESULTS AND CONCLUSION: Interleukin-1β (100 μg/L) significantly inhibited the activity of ATDC5 cells, increased the expression of inflammatory markers (matrix metalloproteinases 3, 9, and 13), decreased the expression of type II collagen, increased the expression of SDF-1 and CXCR4, activated the SDF-1/ CXCR4 pathway, and induced inflammatory responses in cells. Compared with the model group, astragaloside IV significantly reduced the expression of matrix metalloproteinases 3, 9, and 13, and increased the expression of type II collagen, indicating that astragaloside IV can alleviate chondrocyte injury induced by interleukin-1β. In addition, the expression of CXCR4 and SDF-1 was decreased in the experimental group compared with the model group, indicating that astragaloside IV alleviates interleukin-1β-induced chondrocyte inflammation through the SDF-1/CXCR4 signaling pathway [ABSTRACT FROM AUTHOR]

BACKGROUND: The pathogenesis of congenital microtia is not clear. Current studies believe that microtia is related to the joint action of environmental and genetic factors, and drug teratogenesis is an important aspect of environmental factors. OBJECTIVE: To observe the embryonic auricle development indexes of pregnant rats exposed to different concentrations of mycophenolate mofetil, in order to establish a drug-induced microtia model and provide a basis for further exploring the pathogenesis of microtia. METHODS: Adult Sprague-Dawley rats were caged according to the ratio of male to female at 2:1, and the female rats were checked the next day. The presence of a vaginal plug was designated as gestational day 0. Thirty-two pregnant rats were randomized into four groups (n=8 per group): control group, 50, 100, and 200 mg/kg mycophenolate mofetil groups. Rats in the four groups were intragastrically given corn oil, and 50, 100, and 200 mg/kg mycophenolate mofetil corn oil respectively at 8 a.m. on gestational days 9 and 10. The pregnant rats in each group were decapitated under anesthesia on gestational day 20.5. The embryonic development and auricle development indexes were generally observed. The development of middle and inner ears was observed by micro-CT imaging. Auricle cartilage and soft tissue changes were histologically observed. RESULTS AND CONCLUSION: (1) Compared with the control group, the numbers of dead fetuses and absorbed fetuses were increased and the number and rate of live fetuses decreased in different mycophenolate mofetil groups. There were significant differences in ear transverse length, ear longitudinal length, ear longitudinal length/ear transverse length, nasal occipital distance/biparietal diameter, eye distance, body length and body mass between different mycophenolate mofetil groups and control group (P < 0.05). The above-mentioned indexes were significantly different in the 50 mg/kg mycophenolate mofetil group from 100 and 200 mg/kg mycophenolate mofetil groups (P < 0.05). There were also significant differences in ear length, body length, body mass, and nasal occipital distance between 100 and 200 mg/kg mycophenolate mofetil groups (P < 0.05). (2) Compared with the control group, the main micro-CT changes in each mycophenolate mofetil group were dysplasia of the skull and incomplete development of auditory vesicle. (3) The results of hematoxylineosin staining, toluidine blue staining and type II collagen immunohistochemistry staining showed that mycophenolate mofetil affected the proliferation and differentiation of chondrocytes and the expression of type II collagen. (4) To conclude, mycophenolate mofetil can cause the dysplasia of rat embryonic auricle in a dose-dependent manner, because mycophenolate mofetil may inhibit the proliferation and differentiation of chondrocytes and the expression of type II collagen. Mycophenolate mofetil can be used to establish an animal model of microtia. [ABSTRACT FROM AUTHOR]

Copyright of Acta Materiae Compositae Sinica is the property of Acta Materiea Compositae Sinica Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Wool Textile Journal is the property of National Wool Textile Science & Technology Information Center and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective: In order to establish a non-decalcified pathological section preparation method for bone tissue specimens of lumbar vertebral body of miniature pigs implanted with bone repair materials. Methods: In this study, lumbar vertebral bone tissue specimens containing bone repair materials were segmented to expose tissue sections. Dehydration with graded ethanol followed by infiltration with Technovit 7200 VLC photopolymer resin. Photopolymerization embedding of lumbar vertebral body tissue specimens containing bone repair material by co-irradiation with yellow and blue light, Preparation of non-decalcified pathological tissue sections with bone-containing repair materials by means of hard tissue pathological cutting and grinding system. Results: The results show that the pathological tissue sections prepared by the above method, After HE staining and toluidine blue staining, observation under a light microscope can better show various tissue and cell structures of bone, The direction and connection of trabecular bone can be clearly observed. Conclusions: A method for preparing pathological tissue sections of bone tissue specimens containing bone repair materials was established, Realized the preparation of histopathological sections of non-decalcified bone with bone repair material, Histological observation with implants achieved after pathological staining, It provides a new pathological detection method and histological evaluation method for the animal test research of biological materials and medical devices. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Guangxi Normal University - Natural Science Edition is the property of Gai Kan Bian Wei Hui and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

BACKGROUND: Subarachnoid injection is a commonly used intervention method in animal experiments of spinal cord injury. However, at present, there is a lack of methods for one-time large dose of subarachnoid injection that can cooperate with sustained-release/controlled-release dosage forms to reduce the frequency of administration and avoid subarachnoid catheterization. OBJECTIVE: To investigate a method of subarachnoid injection for one-time large dose administration in a rat spinal cord injury model. METHODS: Sprague-Dawley rats were randomly divided into six groups: a spinal cord injury group, a subarachnoid injection group, a spinal cord injury+subarachnoid injection group, a subarachnoid injection sham operation group, a spinal cord injury+subarachnoid injection sham operation group, and a control group. In the spinal cord injury group, New York University spinal cord impactor was used to establish the rat spinal cord injury model. In the subarachnoid injection group, 200 μL of simulated liquid (stroke-physiological saline solution) was injected into the subarachnoid space of rats via the posterior atlanto-occipital interspace. Rats in the spinal cord injury+subarachnoid injection group received subarachnoid injection after modeling. Rats in the control group were given no treatment. In the subarachnoid injection sham operation group, the posterior atlanto-occipital membrane was exposed and the incision was then sutured. In the spinal cord injury+subarachnoid injection sham operation group, the posterior atlanto-occipital membrane was exposed and the incision was then sutured after modeling. Toluidine blue staining was used to detect drug diffusion and distribution in the subarachnoid space at 1.5 hours after injection. Open field test, inclined plane test, hot-plate test, and body mass measurement were used to verify the occurrence of subarachnoid injection-related complications. RESULTS AND CONCLUSION: (1) Subarachnoid injection via the posterior atlanto-occipital interspace did not cause abnormal results of Basso-Beattie- Bresnahan score, hot-plate test, and inclined plane test in each group. (2) Rats in the subarachnoid injection sham operation and the subarachnoid injection groups regained their preoperative body mass at 1 week after operation (P > 0.05). Rats in the spinal cord injury group, the spinal cord injury+subarachnoid injection sham operation group, and the spinal cord injury+subarachnoid injection group regained their preoperative body mass at 2 weeks after operation (P > 0.05). (3) No special signs of central nervous system injury caused by subarachnoid injection were observed intraoperatively and postoperatively. No leakage of cerebrospinal fluid/simulated solution was found. After the operation, all rats could raise their head normally, and no listlessness or self-mutilating behavior occurred. Rats in the subarachnoid injection sham operation and the subarachnoid injection groups did not have dysuria and dysporia. (4) At 1.5 hours after subarachnoid injection, toluidine blue solution was widely diffused in the subarachnoid space and distributed throughout the whole spinal cord. (5) The above data indicate that subarachnoid injection via the posterior atlanto-occipital interspace is safe and reliable. A single dose of 200 μL administrated can reach the injured segment of the spinal cord quickly and has a less risk of serious complications in the rat model of spinal cord injury. Therefore, it is an alternative method of subarachnoid administration in the rat model of spinal cord injury. BACKGROUND: Subarachnoid injection is a commonly used intervention method in animal experiments of spinal cord injury. However, at present, there is a lack of methods for one-time large dose of subarachnoid injection that can cooperate with sustained-release/controlled-release dosage forms to reduce the frequency of administration and avoid subarachnoid catheterization. OBJECTIVE: To investigate a method of subarachnoid injection for one-time large dose administration in a rat spinal cord injury model. METHODS: Sprague-Dawley rats were randomly divided into six groups: a spinal cord injury group, a subarachnoid injection group, a spinal cord injury+subarachnoid injection group, a subarachnoid injection sham operation group, a spinal cord injury+subarachnoid injection sham operation group, and a control group. In the spinal cord injury group, New York University spinal cord impactor was used to establish the rat spinal cord injury model. In the subarachnoid injection group, 200 μL of simulated liquid (stroke-physiological saline solution) was injected into the subarachnoid space of rats via the posterior atlanto-occipital interspace. Rats in the spinal cord injury+subarachnoid injection group received subarachnoid injection after modeling. Rats in the control group were given no treatment. In the subarachnoid injection sham operation group, the posterior atlanto-occipital membrane was exposed and the incision was then sutured. In the spinal cord injury+subarachnoid injection sham operation group, the posterior atlanto-occipital membrane was exposed and the incision was then sutured after modeling. Toluidine blue staining was used to detect drug diffusion and distribution in the subarachnoid space at 1.5 hours after injection. Open field test, inclined plane test, hot-plate test, and body mass measurement were used to verify the occurrence of subarachnoid injection-related complications. RESULTS AND CONCLUSION: (1) Subarachnoid injection via the posterior atlanto-occipital interspace did not cause abnormal results of Basso-Beattie- Bresnahan score, hot-plate test, and inclined plane test in each group. (2) Rats in the subarachnoid injection sham operation and the subarachnoid injection groups regained their preoperative body mass at 1 week after operation (P > 0.05). Rats in the spinal cord injury group, the spinal cord injury+subarachnoid injection sham operation group, and the spinal cord injury+subarachnoid injection group regained their preoperative body mass at 2 weeks after operation (P > 0.05). (3) No special signs of central nervous system injury caused by subarachnoid injection were observed intraoperatively and postoperatively. No leakage of cerebrospinal fluid/simulated solution was found. After the operation, all rats could raise their head normally, and no listlessness or self-mutilating behavior occurred. Rats in the subarachnoid injection sham operation and the subarachnoid injection groups did not have dysuria and dysporia. (4) At 1.5 hours after subarachnoid injection, toluidine blue solution was widely diffused in the subarachnoid space and distributed throughout the whole spinal cord. (5) The above data indicate that subarachnoid injection via the posterior atlanto-occipital interspace is safe and reliable. A single dose of 200 μL administrated can reach the injured segment of the spinal cord quickly and has a less risk of serious complications in the rat model of spinal cord injury. Therefore, it is an alternative method of subarachnoid administration in the rat model of spinal cord injury. [ABSTRACT FROM AUTHOR]

BACKGROUND: Knee cartilage loss certainly occurs in aged mice, so it is difficult to extract the articular cartilage from the mice. In particular, it is difficult and rarely reported to extract chondrocytes from the small joint on the articular cartilage surface of the ankle joint. OBJECTIVE: To explore the effect of an improved two-step enzyme digestion method to extract chondrocytes from the ankle joint of aged mice, and to observe and evaluate their biological characteristics in vitro. METHODS: The ankle joints of 46-week-old mice were selected, separated and extracted sequentially by the improved two-step enzymatic digestion method in the laboratory with 0.25% pancreatin EDTA and 2g/L type II collagenase. Extracted cells were cultured in vitro and cell growth morphology was observed with an inverted microscope. After trypan blue staining, the number of primary adherent cells was calculated. Toluidine blue and type II collagen immunofluorescence staining was conducted. Chromogenic method using dimethyl methylene blue was used to determine the content of glycosaminoglycan sulfate in the primary adherent cell culture and digestive fluid. Proliferation of chondrocytes was detected using cell counting kit-8 to identify and evaluate the biological characteristics of chondrocytes. RESULTS AND CONCLUSION: After the two-step enzymatic digestion of the articular cartilage, all cells were dissociated and released. An average of 9×105 primary adherent cells were harvested from each ankle joint of aged mice. Primary cells and the first- and second-generation cells displayed spindle and polygonal morphology. Toluidine blue staining showed blue-purple particles that were easily dyed. Type II collagen immunofluorescence staining revealed the positive expression of red fluorescence. The contents of glycosaminoglycan sulfate in the primary adherent cell culture fluid and digestive fluid were (42.41±15.00) and (65.63±10.00) mg/L, which were (0.35±3.78) and (0.21±2.56) mg/L in the negative control culture fluid and digestive fluid, respectively. The number of primary cells reached the maximum on the 6th day, and the number of the first- and second-generation cells reached the maximum on the 5th day. Meanwhile, the cell viability of the first-generation cells was greater than that of the second-generation cells and the primary cells. To conclude, the improved two-step enzymatic digestion method can successfully isolate chondrocytes from the ankle joint of aged mice. Large amounts of adherent cells with high purity and good biological characteristics can successfully synthesize and secrete type II collagen and proteoglycan. The first-generation chondrocytes have the highest viability, followed by the second-generation cells, which can be used for experimental research. [ABSTRACT FROM AUTHOR]

BACKGROUND: Auricle deformity remodeling based on three-dimensional printing scaffold materials has become a focus in recent years, and whether chondrocytes can grow normally on a scaffold is the key issue. OBJECTIVE: To investigate the primary culture methods of rabbit auricular chondrocytes, thereby laying a solid foundation for the application of chondrocytes combined with tissue engineering. METHODS: Rabbit auricular chondrocytes were obtained by trypsin and type II collagenase sequential digestion method. The chondrocytes were cultured and subcultured in vitro. The chondrocytes were identified by morphological observation, growth curve drawing, safranin O-fast green staining, toluidine blue staining, Victoria blue staining, and glycosaminoglycan content determination. RESULTS AND CONCLUSION: The normal morphology of auricular chondrocytes was fusiform and polygonal under inverted phase contrast microscope. After six generations of subculture, the cells became obviously long fusiform, and the proliferation rate slowed down or even stopped. The growth curve showed that the chondrocytes from the 2nd to 5th generations could proliferate in a straight line. Results of safranin O-fast green staining showed clear nucleus and cytoplasm structures of rabbit auricular chondrocytes, with good morphological characteristics. Results of toluidine blue staining indicated that the secretion of cartilage matrix was less at the initial stage of culture and increased gradually with the increase of cell fusion. Results of Victoria blue staining showed the formation of collagen fibers in chondrocytes, suggesting the normal function of chondrocytes. Results of glycosaminoglycans content determination showed the specific secretion of glycosaminoglycans in chondrocytes. Overall, the isolation and identification system of rabbit auricular chondrocytes in primary culture was successfully established, and the auricular chondrocytes with biological activity were obtained, which could be further used in tissue engineering research. [ABSTRACT FROM AUTHOR]

The distribution of the rare perennial fern Adiantum reniforme var. sinense(Pteridaceae)which is endemic to shady cliff environments is limited to small areas of Wanzhou and Fuling, Chongqing, China. In order to reveal the growth characteristics of A. reniforme var. sinense in the germplasm resource nursery,we observed the tissue structure of A. reniforme var. sinense, which including in the roots, rhizomes, leaves grown in the sun and in shade. First of all, the roots, rhizomes, leaves were collected and fixed in formaldehyde-alcohol-acetic acid solution, and we cut the tissue structure of the plant with a double-sided blade to make freehand sections, then the tissue were stained with three kinds of 0.1% Sudan red, 0.1% berberine bisulfate-aniline blue, and 0.05% toluidine blue, at last plant tissue sections were observed under the Lycra optical microscope and the fluorescence microscope. In this study, we used brightfield and epifluorescence microscopy to investigate the anatomical structures and histochemical features that may allow this species to thrive in shady, dry cliff environments. The results were as follows:(1)The A. reniforme var. sinense sporophyte had a primary structure, the roots had an endodermis with Casparian walls, suberin lamellae, a thickened sclerenchyma layer around the endodermis, a cortex, and a rhizodermis.(2)The stems had a dictyostele, an endodermis with Casparian walls and suberin lamellae, a thickened sclerenchyma layer around the endodermis, a cortex, and a thin cuticle.(3)The leaves had an endodermis surrounded the vascular bundles, a peripheral sclerenchyma layer, an isolateral mesophyll, a lignified epidermis and a thin cuticle.(4)The dictyostele, endodermis, sclerenchyma layers, and lignified epidermal walls reflected the drought tolerance of A. reniforme var. sinense, while its thin cuticle and isolateral mesophyll suggested tolerance of shady environments. Thus, according to the anatomical characteristics of A. reniforme var. sinense, it is concluded that A. reniforme var. sinense are consistent with adaptations to shady, dry cliff environments [ABSTRACT FROM AUTHOR]

BACKGROUND: Both in vivo and in vitro studies have shown that platelet-rich plasma plays an important role in promoting chondrocyte proliferation and tissue regeneration and repair. However, little is reported on the repair of cartilage defects with different concentrations of platelet-rich plasma in rabbits with knee osteoarthritis. OBJECTIVE: To compare the different concentrations of platelet rich plasma in the repair of cartilage defects in rabbits with knee osteoarthritis, so as to provide theoretical basis and options for the clinical treatment of cartilage defects in knee osteoarthritis. METHODS: A total of 50 healthy New Zealand white rabbits were randomly divided into blank group, model group, low concentration, medium concentration and high concentration groups of platelet-rich plasma with 10 rabbits in each group. Platelet-rich plasma was prepared by secondary centrifugation. An osteoarthritis model of rabbit knee was made by improved Hulth method in each group except for the blank group. In the model group, 0.3 mL of normal saline was injected into the articular cavity, while in the low, medium and high concentration groups, (1.0-1.2)×1012/L, (1.5-1.7)×1012/L, and (2.0-2.2)×1012/L platelet-rich plasma was injected respectively. The expression of tumor necrosis factor-α, interleukin-1β and interleukin-6 in the synovial fluid was detected by ELISA, and the histological changes of different concentrations of platelet-rich plasma were evaluated by hematoxylin-eosin staining, toluidine blue staining and Mankin’s pathological score. Western blot and RT-PCR were used to detect the protein and mRNA levels of type II collagen and aggrecan. The study protocol was approved by the Experimental Animal Ethics Committee of China Medical University. RESULTS AND CONCLUSION: Compared with the model group, the levels of tumor necrosis factor-α, interleukin-1β and interleukin-6 in the low, middle and high concentration groups decreased to some extent (P < 0.05), the expression levels of these indicators in the middle concentration group were lower than those in the low concentration group and high concentration group (P < 0.05). Hematoxylin-eosin and toluidine blue staining results showed that chondrocytes in the low and high concentration groups were relatively regular in shape and the cell number increased. Chondrocytes in the medium concentration group were arranged neatly, with formation of cartilage-like cells, and a remarkable effect was found in the repair of chondrocytes. Mankin’s pathological scores were highest in the model group, followed by the low and high concentration groups, and lowest in the middle concentration group. Western blot and RT-PCR results showed that compared with the model group, the expression of type II collagen and aggrecan protein and mRNA in cartilage tissues were significantly increased in low, medium, and high concentration groups (P < 0.05). To conclude, different concentrations of platelet-rich plasma can effectively resist the expression of inflammatory factors involved in the process of knee osteoarthritis and has a significant effect on the repair of cartilage defects in knee osteoarthritis, and (1.5- 1.7)×1012/L platelet-rich plasma has better effects. [ABSTRACT FROM AUTHOR]

BACKGROUND: The microporous structure of a porous titanium alloy can not only provide sufficient space for the proliferation and migration of osteoblasts, but also promote the angiogenesis of mesenchymal stem cells, affect the expression of osteogenic genes and the differentiation of osteoblasts, which is an important factor for bone integration. OBJECTIVE: To analyze the osseointegration ability of three-dimensional printed porous titanium alloy scaffolds with different apertures in repairing rabbit femoral condyle defect. METHODS: Totally 40 hind legs of 20 New Zealand rabbits were randomly divided into group A, group B, group C, and group D. A 5 mm×5 mm bone defect model was established. Groups A, B, and C were implanted with porous titanium alloy scaffolds with a diameter of 200, 600, and 1 000 μm, while group D was not implanted. X-ray films were taken at 4 and 8 weeks after operation to observe whether there was displacement, prolapse and osteolysis, and osteonecrosis around the implants. At 8 weeks after operation, the trabecular thickness, trabecular number, trabecular volume, bone surface area density, and trabecular separation were detected by micro CT. The osseointegration was observed by histology. The experimental protocol was approved by the Animal Experiment Ethics Committee of Soochow University. RESULTS AND CONCLUSION: (1) X-ray films showed that the implants were in good position, without obvious displacement or falling off, and the bone around the materials was good, without osteolysis, osteonecrosis or other bad phenomena. (2) Micro-CT examination showed that bone tissue existed in the pores and around the implants in three groups. The number of trabeculae and bone surface area density in group B were significantly higher than those in the groups A and C (P < 0.05). The trabecular thickness of micropores in the group A was significantly higher than that in the groups B and C (P < 0.05). (3) Toluidine blue staining showed that the new bone tissue grew into almost all the surface micropores in group B, and some of the new bone extended into the deep pores of the experimental scaffold, closely combined with the porous structure, forming mechanical interlocking. Groups A and C had only a small amount of new bone tissues in surface micropores, and there was not new bone tissues in deep pores. The combination of new bone and implant was poor. (4) The results showed that the three-dimensional printed porous titanium alloy scaffold with 600 μm aperture was more conducive to new bone formation compared with 200 μm and 1 000 μm aperture. [ABSTRACT FROM AUTHOR]

BACKGROUND: Common methods for obtaining chondrocytes include mechanical-enzymatic digestion, sequential digestion of pronase and collagenase, sequential digestion of trypsin and type II collagenase, and simple type II collagenase digestion. OBJECTIVE: To investigate the isolation, extraction and identification of knee joint chondrocytes from Sprague-Dawley rats, and to observe the morphological characteristics of chondrocytes from primary to passages 3. METHODS: We used single-step digestive approach (type II collagenase digestion) to isolate and obtain chondrocytes from the knee joints of SpragueDawley rats in vitro, and then established a culture system in vitro. Cell proliferation assay and inverted phase contrast microscope were used to observe morphohistology of the cells. Toluidine blue staining, type II collagen as well as proteoglycan immunofluorescence staining were used to identify the characteristics of chondrocytes. The experimental protocol was approved by the Ethics Committee of Zhongshan Hospital of Dalian University. RESULTS AND CONCLUSION: Microscopic observation and cell proliferation assay both showed satisfactory cell morphology and proliferation at passages 2-3, and the capacity of the cells decreased gradually. Moreover, toluidine blue staining, type II collagen and proteoglycan immunofluorescence staining revealed superior characteristics of passage 3 chondrocytes cultured in vitro. These findings indicate that chondrocytes can successfully be isolated by the singlestep type II collagenase digestion. Cell proliferation and passage cultivation can also be achieved. And dedifferentiation capacity of chondrocytes is improved gradually after the passages 3 cultured in vitro. Passage 3 cells cultured in vitro have superior characteristics for the use in experimental researches. [ABSTRACT FROM AUTHOR]

BACKGROUND: Lumbar disc degeneration is the main pathological change of lumbar disc herniation, which is closely related to the programmed death of intervertebral disc annulus fibrosus cells. Previous clinical studies have shown that Tongdu Huoxue Decoction has a significant effect in the treatment of lumbar disc herniation, and it is worth further studying whether it can play a role by inhibiting the pyrolysis of annulus fibrosus cells. OBJECTIVE: To investigate the inhibitory effect of Tongdu Huoxue Decoction-medicated serum on the pyroptosis of annulus fibrosus cells in the intervertebral disc induced by lipopolysaccharide/adenosine triphosphate (ATP), so as to explore the possible mechanism of Tongdu Huoxue Decoction in the treatment of lumbar disc herniation. METHODS: Thirty-six 10-week-old Spraue-Dawley rats were gavaged with Tongdu Huoxue Decoction, and blood samples were taken from the abdominal aorta after 7-day continuous gavage to prepare drug-containing serum. Four-week-old Sprague-Dawley rats were taken to extract their lumbar intervertebral discs, and then the annulus fibrosus cells were extracted from the intervertebral disc tissues by mechanical-enzymatic digestion. The cells cultured to the second generation were identified by toluidine blue staining and type II collagen immunohistochemical staining. After that, the second-generation cells were randomly divided into blank group, model group, and low-, middle-, and high-dose serum-containing groups. Except for the blank group, the remaining four groups were given lipopolysaccharide/ATP to make the cell pyroptosis model. After modeling, 5%, 10%, and 20% of Tongdu Huoxue Decoction medicated serum were administrated in the low-, middle-, and high-dose serum-containing groups, respectively, and no intervention measures were taken in the blank group. We then used the MTT method to find the best concentration and time for the intervention of pyroptosis of annulus fibrosus cells with the drug-containing serum. Flow cytometry and Annexin V FIFT/PI double staining were used to measure the rate of cell pyroptosis. ELISA method was used to detect the expression of interleukin-1β and interleukin-18 in cell supernatant. qRT-PCR was used to detect the mRNA expression of Caspase-1, Gasdermin D, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). RESULTS AND CONCLUSION: MTT showed that the best intervention concentration of Tongdu Huoxue Decoction medicated serum for the pyroptosis of annulus fibrosus cells was 10%, and the best intervention time was 24 hours. Flow cytometry results showed that compared with the blank group, the pyroptosis rate in the other four groups was significantly increased (P < 0.05). Compared with the model group, the pyroptosis rate in the three drug-containing serum groups was significantly decreased (P < 0.05). Compared with the low- and high-dose groups, the pyroptosis rate of the middle-dose group was significantly decreased (P < 0.05). Results from ELISA, qRT-PCR, and western blot assay indicated that compared with the blank group, the expression levels of interleukin-1β, interleukin-18, Caspase-1, Gasdermin D, NLRP3 mRNAs and proteins were significantly increased (P < 0.05), while compared with the model group, these expression levels were significantly decreased in the three drug-containing serum groups (P < 0.05). Moreover, the expression levels of interleukin1β, interleukin-18, Caspase-1, Gasdermin D, NLRP3 in the middle-dose group were significantly lower than those in the low- and high-dose groups (P < 0.05). Therefore, Tongdu Huoxue Decoction-medicated serum can effectively relieve lipopolysaccharide/ATP induced pyroptosis of intervertebral disc annulus fibrosus cells and reduce inflammation. The middle-dose group has the best effect in delaying cell pyroptosis and reducing inflammation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Bisphosphonates are a novel inhibitor of bone resorption that can inhibit the activity and function of osteoclasts. OBJECTIVE: To observe the effects of sodium ibandronate on the expression of dentin matrix protein 1, Caspace3, Bcl-2 and Bax in condylar cartilage in osteoporosis rats. METHODS: Thirty-six female rats were randomly divided into sham group, osteoporosis group and sodium ibandronate group, twelve in each group. The sham group did not excise ovaries during surgery. Bilateral ovaries of rats were removed in the osteoporosis and sodium ibandronate groups. On the 7th day after operation, rats in the sodium ibandronate group were intraperitoneally given sodium ibandronate 10 μg/kg, once every 7 days. After 90 days, the rat ovaries were taken. Bone mineral density was measured in each group. The changes of condylar cartilage were observed by toluidine blue staining and TUNEL staining. The expression of dentin matrix protein 1 protein was detected by immunohistochemistry. The levels of Caspase3, Bcl-2 and Bax were detected by western blot assay. The study protocol was approved by the Animal Ethics Committee of Nanhua Hospital in China with the approval No. SLXD_201804010. RESULTS AND CONCLUSION: Compared with the sham group or sodium ibandronate group, the bone mineral density in the osteoporosis group was significantly decreased (P < 0.05). The results of toluidine blue staining showed that the hypertrophic layer of condylar cartilage in the sodium ibandronate group was significantly thicker than that in the osteoporosis group. Compared with the sham group or sodium ibandronate group, the number of apoptotic cells in condylar cartilage and subchondral bone increased significantly in the osteoporosis group (P < 0.05). The expression of dentin matrix protein 1 protein was significantly lower in the osteoporosis group than the sham group, but it increased after treatment with sodium ibandronate (P < 0.05). Compared with the sham group, the expression of Caspase 3 and Bax in the osteoporosis group increased significantly, and the expression of Bcl-2 decreased. However, treatment with sodium ibandronate decreased the expression of Caspase 3 and Bax and increased the expression of Bcl-2 significantly. Overall, our findings reveal that sodium ibandronate can inhibit the apoptosis of condylar chondrocytes and the number of osteoclasts in osteoporotic state, which may be related to the regulation of Caspase 3, Bcl-2, Bax and dentin matrix protein 1 expression. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recent studies have shown that the occurrence of Wallerian degeneration is closely related to autophagy in Schwann cells. The regulation of autophagy in Schwann cells can significantly affect the occurrence and development of Wallerian degeneration, subsequently altering axon regeneration and myelination. OBJECTIVE: To clarify whether sciatic nerve allograft can achieve higher efficiency when the cell autophagy is inhibited by 3-methyladenine. METHODS: We harvested 16 sciatic nerve segments from 8 female C57BL/6J mice that were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. in China. All the segments were equally divided into experimental and control groups and cultured in 3-methyladenine culture medium and nonnal culture medium for 72 hours, respectively. Another 16 female C57BL/6J mice were taken to make animal models of left sciatic nerve defects. After modeling, the sciatic nerve segments were grafted to repair sciatic nerve defect through microsurgery: 3-methyladenine-treated nerve segments in the experimental group and normally treated nerve segments in the control group. Sciatic nerve index in each mouse was recorded at 2, 4, 6, and 8 weeks after modeling. At 8 weeks after modeling, the regenerated nerve segments were histologically analyzed using hematoxylin-eosin staining, immunofluorescent staining, toluidine blue staining, and transmission electron. The animal experiment was approved by the Animal Ethics Committee of Peking Union Medical College. RESULTS AND CONCLUSION: There were no differences in the sciatic nerve index between the two groups (P > 0.05) except at 8 weeks after modeling (P < 0.05). Hematoxylin-eosin staining revealed an intact nerve structure in the experimental group but a large area of voids in the control group. Immunofluorescent staining indicated that there were nerve tracts with more complete structures in the experimental group than the control group. Toluidine blue staining revealed some myelinated and unmyelinated nerve fibers regenerated in the experimental group and only a few of myelinated nerve fibers and unmyelinated axons newly formed in the control group. Under the transmission electron microscope, myelin sheath thickness and myelinated fiber diameter were significantly higher in the experimental group than the control group (P < 0.05). Therefore, 3-methyladenine-treated nerve allografts could inhibit autophagy in Schwann cells, maintain the myelin sheath structure of the allograft, and promote axonal regeneration and functional recovery. [ABSTRACT FROM AUTHOR]

Objective: To optimization the method of isolating and culturing human degenerative nucleus pulposus cells in vitro, and to provide seed cells for the prevention and cure investigation of intervertebral disc degeneration. Methods: Nucleus pulposus tissue was collected in a sterile environment. Multiple enzymatic digestions were adopted to extract primary human nucleus pulposus cells. The cells were cultured in thermostat incubator with 5%CO2 under the condition of 37℃. Cell morphous was observed under inverted phase contrast microscope. The cell growth curve was plotted by MTT assay. The expression of proteoglycan and type Ⅱ collagen were detected by toluidine blue staining and cellular immunofluorescence staining respectively. Results: The cells had spindle or polygonal shapes. The primary cells had an average adherence time of 48 hours and took nearly 8 days to reach 90%confluence. Cells at passage 3 had an average adherence time of 12 hours and took nearly 5 days to reach 90% confluence. The results of toluidine blue staining and cellular immunofluorescence staining showed that the cultured cells secreted proteoglycan and type Ⅱ collagen. Conclusion: Multiple enzymatic digestions of nucleus pulposus tissue could release a large amount of pure human nucleus pulposus cells. The culture efficiency was improved. The primary and passage cells could maintain stable chondrocyte-like phenotype and could be used as seed cells for intervertebal disc tissue engineering. [ABSTRACT FROM AUTHOR]

Objective: We want to provide a simple and feasible cell culture method for the future study of injury repair of rat meniscus by isolation, culture and identification of rat meniscus fibrochondrocytes in vitro. Methods: The lateral meniscus were mechanical separation and digestion by 0.1% II collagenase, cultured in the 10% FBS medium. The morphology and growth situation of meniscal cells were observed by the inverted microscope dynamic at different time points, the identification of meniscus fibrochondrocytes by using toluidine blue staining and II type collagen immune cell chemical dyeing method. The proliferation capacity of fibrochondrocytes was detected by CCK-8 method. Results: At different time points, the cells showed different physiological morphology, which was transformed from polygonal or short shuttle-shaped to fusiform, and finally appeared triangular or ellipsoidal, and the proliferation capacity of the cells increased gradually with the extension of time. The OD value at 48 h and 72 h in cell culture was significantly higher than that at 24 h (P<0.05), and the difference was statistically significant. The OD value of cell culture at 48 h and 72 h was increased, but there was no significant difference (P>0.05). By toluidine blue staining and collagen type II immunofluorescence stain is positive. Conclusions: Cultured cells by this method are stable in morphology, strong in proliferation, and have the basic biological characteristics of fibrochondrocytes in vivo, which can provide a simple and reliable method for rat meniscus fibrochondrocytes culture. [ABSTRACT FROM AUTHOR]

Objective: To compare the chondrogenic differentiation potential of bone marrow mesenchymal stem cells(BMSCs), adipose-derived mesenchymal stem cells(AMSCs) Methods: and synovi-al mesenchymal stem cells(SMSCs). The rabbit BMSCs, AMSCs and SMSCs were respectively separated with the adherence screening method, and were continually subcultured. The growth curves of the passage 3 BMSCs, AMSCs and SMSCs were drawn, and the doubling times of three kinds of cells were compared. Then the passage 3 BMSCs, AMSCs and SMSCs were treated with chondrogenic induction. After induced for 14 days, the BMSCs, AMSCs and SMSCs were treated with toluidine blue staining and type II immunohistochemistry staining respectively to evaluate the ability of the chondrogenic differentiation potential of them. Methods: The doubling time of AMSCs was shorter than that of the BMSCs. The doubling time of SMSCs was the shortest of the three. After induced for 14 days, all of them could express glycosaminoglycans and type II collagen, and there was significant difference in the expression of collagen type II between any two groups. The expression level of type II collagen in SMSCs after chondrogenic induction was higher than that in the BMSCs P＜0.01, and the expression level of type II collagen in BMSCs after chondrogenic induction was higher than that in the AMSCs P＜0.01 .Conclusions: BMSCs, AMSCs and SMSCs all could transform into chondrocytes under certain conditions, but SMSCs seemed to be the fastest proliferation andthe most potential of the three. [ABSTRACT FROM AUTHOR]

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As an important part of China’s sustainable development strategy, wood’s quality is mainly destroyed by wood decay.White-rot fungus is the most serious damage to wood cell wall in various wood rot fungi. In this paper, the inhibition of white-rot fungi was studied by using low toxicity and low pollution toluidine blue( TB) mediated photodynamic therapy to inhibit pathogenic bacteria, improve wood utilization. When the different concentrations( 0. 01, 0. 02, 0. 05 mg/m L) of TB were applied to white-rot fungi by the Oxford Cup method, no bacteriostatic effect was observed. However, when TB was excited by low-power 630 nm laser, it was found that the three groups of photosensitizer TB inhibited white-rot fungi, and the inhibition rates with TB concentration were 69.67%, 91. 21%, and 99. 44%, respectively. The experimental results show that toluidine blue-mediated photodynamic therapy has a good antibacterial effect on white-rot fungi, and the concentration of photosensitizer is positively correlated with its antibacterial effect, which reflects the good anti-corrosion effect of wood. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to identify the morphology of mast cells by using a modified toluidine blue staining scheme, so as to provide a powerful reference for the experimental basis research of mast cells. Methods: Bone marrow-derived mast cells were induced in vitro. After 4 weeks, the cells were collected, fixed, and stained. Mast cells were fixed at different temperature during different time. The optimum condition was determined by comparing the effects of toluidine blue staining. Results: Bone marrow cells were induced to differentiate into mast cells by SCF and IL-3 in vitro. When mast cells were stained with modified toluidine blue staining,the staining effect was better. Mast cells were round or oval and the cell membrane was complete and the cytoplasm was filled with a large number of purple particles. Conclusion: In this study, we successfully applied a modified toluidine blue staining method to mast cells cultured in vitro. The results showed that the condition at 37 ℃ full fixation with staining could reduce the degeneration of mast cells. This method was easy to operate with good stability. It was suitable for the morphological observation of mast cells cultured in vitro. [ABSTRACT FROM AUTHOR]

PURPOSE: To set up differential protein expression profiling of rat condylar chondrocytes during their development, search and identify their characteristics and functions. METHODS: The 1,7,14 and 28 day-old SD rat condylar chondrocytes were incubated in vitro, identified by toluidine blue stain and type II collagen immunohistochemistry reaction. After extracting from rat condylar chondrocytes of each group, total proteins were labeled with iTRAQ reagents and resolved using 2D nano-high performance liquid chromatograph (HPLC),protein spots were then identified by mass spectrometry, using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology (MALDI-TOF/TOF), to quantitatively analyze the differential protein expression of rat condylar chondrocytes during their development. All data were normalized by MASCOT software to search useful differentially expressed proteins, which were then classified by Gene Ontology. RESULTS: We identified 500 differential proteins, corresponding to 137 reliability proteins, 15 high reliability proteins. Their molecular mass values ranged from 7806.24 to 608459.2. The biological functions of a significant proportion of protein were involved in metabolic and developmental processes, as well as participating in different biological functions including structural constituent of cytoskeleton, signal transduction and molecular signaling, response to stimulus, immune response, cell communication and transport and so on. CONCLUSIONS: This study provided the first differential protein expression profiling of rat developmental condylar chondrocytes, which was identified successfully by mass spectrometry. This would lay a foundation to study the protein expression changes and molecular mechanism of chondrocytes in normal condyle or TMJ diseases in the future. Supported by Medical Leading Project of Science and Technology Commission of Shanghai Municipality(114119a3500). [ABSTRACT FROM AUTHOR]