| BackgroundAdolescent idiopathic scoliosis (AIS) is a three-dimensional deformity of the spine with lateral curvature and vertebral rotation. AIS affects about (1-3)% of children aged 10-18 years, however, the true etiology of it remains unknown.Recently, the research on the cause of AIS has focused on genetics, growth and development of the spine, nervous system, melatonin, paravertebral muscles, connective tissue, osteoporosis, platelets and so on. However, the particular age of onset and characteristic of growth and development in AIS demonstrate that the emergence and development of scoliosis has affirmative association with the growth and development of the spine. Among several theories about abnormal growth and development in AIS, relative anterior spinal overgrowth seemed receivable. Murray et al thought overgrowth of the anterior column relative to the posterior column could explain the complex biomechanics of scoliosis and could provided a final common pathway by which the multiple aetiological factors could induce AIS. Guo et al found the patients with AIS had longer vertebral bodies between T1 and T12 and attributed the excessive longitudinal growth of the vertebral bodies to the faster endochondral ossification of it. Zhu et al compared the vertebral body growth plate(VBGP) harvested from apical area in AIS patients with those from normal region in congenital scoliosis and found the height and the growth kinetics indexes of VBGP were higher in AIS patients. The mechanisms of abnormal growth of VBGP in AIS patients, however, haven't been studied.VBGP lies between the bone of the vertebral body and the tissues of the intervertebral disc. Just like the long bones, VBGP, which plays important roles in the longitudinal growth of spine, is constituted of hypertrophic zone, proliferative zone and resting zone. Literature indicates that the longitudinal bone growth attributes to endochondral ossification, during which the chondrocytes transformed into bone. Endochondral ossification depends on the numbers of proliferative zone chondrocytes and their rate of proliferation, the volume of hypertrophic chondrocytes and the cartilage matrix turnover. Therefore, to study the abnormal proliferation and differentiation of chondrocytes in VBGP probably is an effective approach to reveal the aetiology of AIS.It is a fact that AIS emerges from adolescent growth spurt during which the level of many hormones change greatly, considering the surprisingly high incidence(1%-3%) of AIS we presume that the hormone disequilibrium may be the radical etiopathogenisis of the disease. The activation of hypothalamus-pituitary-hormone (HPH) axis, as we all know, is the core of endocrine change during puberty. As a part of growth and development, the endochondral ossification of VBGP must be regulated by HPH axis. Obviously, to illuminate the affect of HPH axis on the abnormal proliferation and differentiation of chondrocytes-4n VBGP probably is the key approach to find the aetiology of AIS.Melatonin is a hormone playing extensive roles which secreted by pineal gland. Many investigators have found that melatonin exhibit intense related to the arise of scoliosis in mammals and human beings. Machida and other investigators found that pinealectomy in chickens, bipedal rats, and hamsters could lead to scoliosis, furthermore, Machida also found melatonin supplements could prevent the progression of scoliosis in moderate AIS. As a result, some investigators attributed the generation of scoliosis to decreased melatonin production in those mammals and AIS patients. However, not all pinealectomized animals exhibit scoliosis; AIS patients have an normal ability to form melatonin; patients have melatonin secretion related diseases have no obvious development of scoliosis. Therefore, Weinstein et al speculated scoliosis was unlikely to arise from a simple reduction of melatonin but the consequence of melatonin's effect on other unknown growth mechanisms.It is known that estrogen and growth hormone can affect longitudinal bone growth and the two hermones are important elements of HPH axis, in addition, estrogen has close connections with AIS. As a hormone playing extensive roles, melatonin has close correlation with HPH axis, furthermore, it's known that estrogen can block the activation of estrogen receptor and stimulate the release of growth hormone. Based on these facts, we speculate that melatonin may play an important role in the aetiology of AIS through affecting the HPH axis and the proliferation, differentiation of chondrocytes in VBGP.In 2009, Pei et al. found melatonin could enhance cartilage matrix synthesis of porcine articular chondrocytes, which indicate melatonin can affect chondrocytes. However, the relationship between melatonin and chondrocytes of VBGP hasn't been studied.Considering the restriction of obtaining of VBGP and medical ethics, the study of VBGP on AIS patients and normal people is hard to carry out. Therefore, our pilot study begin with animal and we explore the effect and it's mechanism of melatonin to chondrocytes in rat VBGP firstly. Objectives1. To establish a method for the culture and identification of chondrocytes from rats VBGP in vitro and observe their passage characteristics.2. To compare the histological difference of thoracic VBGPs from rats at different ages and to detect the proliferative capability of them.3. To investigate the expression of melatonin receptors in VBGP of rat.4. To investigate the effct and it's mechanism of melatonin to primary cultured chondrocytes from rats VBGP pilotly.Methods1. Isolation, culture and identification of chondrocytes from rats VBGP.(1) Isolation and culture of chondrocytes from rats VBGP.VBGP chondrocytes were isolated and cultured in vitro with improved trypsin-collagenase type II digestion method. VBGPs were obtained from 4 Sprague-Dawley (SD) rats aged 1 week. After washed by sterile phosphate-buffered saline (PBS) for 3 times, VBGP samples were digested with 0.25% trypsin for 30 minutes, and then digested with 0.2% collagenase type II for 1 hour. After removing tissue debris VBGPs were minced into about 1 mm3 pieces and then digested with 0.2% collagenase type II for 5 hours. After that, cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin,100 U/ml streptomycin) in a humidified,37℃incubator with 5% CO2. The media were replaced every other day and the cells were subcultured by trypsinization with 0.25% trypsin when reaching a subconfluent state. The first to third passage cells were used in the following studies.(2) Identification of chondrocytes from rats VBGP.The passaged primary chondrocytes were identified by inverted phase contrast microscope, HE and Toluidine Blue staining. Cells growth curve was drawn by MTT colorimetric assay.(3) Observe the characteristics of chondrocytes at different passages.Chondrocytes were subcultured to the sixth passage, the changes of cell morphology were observed by an inverted phase contrast microscope, the changes of expression of collagen II were identified with immunocytochemistry.2. Histological observation and detection of the proliferative capability of thoracic VBGPs from rats at different ages.(1) Histological observation of thoracic VBGPs from rats at different ages.Thoracic VBGPs were obtained from rats aged 1day, 1week,4weeks,8weeks, 16weeks and 28weeks, then the VBGPs were identified by safranin O-fast green staining and the height of hypertrophic zone, proliferative zone, resting zone were measured.(2) Detection of the proliferative capability of thoracic VBGPs from rats at different ages.Chondrocytes obtained from those VBGPs were cultured in vitro with improved trypsin-collagenase typeⅡdigestion method. The gene expression of proliferating cell nuclear antigen(PCNA) was detected by realtime-PCR. The protein expression of PCNA was detected by Western blot.3.Detection of melatonin receptors in VBGP of rat.(1) The expression of melatonin receptors on rats VBGP-chondrocytes were detected by immunocytochemistry.Chondrocytes of VBGPs obtained from SD rats aged 1 week were cultured in vitro. Chondrocytes were passaged and the expression of melatonin receptors (MT1, MT2) of the chondrocytes were detected by immunocytochemistry.(2) The expression of melatonin receptors on rats VBGPs were detected by immunohistochemistry.VBGPs were obtained from SD rats in the age of 1 day, then the expression of melatonin receptors (MT1, MT2) of the VBGPs were detected by immunohistochemistry.(3) Detection of the gene expression of melatonin receptors.The gene expression of melatonin receptors (MT1, MT2) was detected by RT-PCR.(4) Detection of the protein expression of melatonin receptors.The protein expression of melatonin receptors (MT1, MT2) was detected by Western blot.4. Detection of the effect of melatonin to proliferation and differentiation of chondrocytes from rat VBGP.(1) Detection of the effect of melatonin to proliferation of VBGP-chondrocytes.Various concentrations (0,0.1,1,10 and 100μg/ml) of melatonin were used to supplement the medium. The role of melatonin on cell proliferation was examined by the Alamar Blue assay. Brifly, after chondrocyte were inoculated to 96-well plates and cultured for 48 hours, Alamar Blue was added directly to the culture medium at 10% of the medium volume and were incubated in the incubator for 5 hours. Absorbance was measured spectrophotometrically at 590 nm using a microplate reader.(2) Detection of the proliferation and differentiation related factors of melatonin to VBGP-chondrocytes.Various concentrations (0,0.1,1,10 and 100μg/ml) of melatonin were used to supplement the medium. Western blot analyses were applied to examine the protein expression of PCNA (reflecting the proliferation potentiality of cells), Sox9 (a core transcription factor in controlling chondrocyte differentiation) and Smad4 (a common mediator in regulating the proliferation and differentiation of chondrocytes).Results1. Isolation, culture and identification of chondrocytes from rats VBGP.(1) Isolation and culture of chondrocytes from rats VBGP.The size of the isolated vertebral body growth plate of rats aged 1week is about 2mm×1mm×0.5mm. The chondrocytes begin to adhere the wall 6 hours after been inoculated and within 24 hours all the cells become adherent. The packed cells look like "flagstone". MTT colorimetric assay showed that the growth curve of chondrocytes before the third passage presented as "S" shapes. Chondrocytes were found growing logarithmicly from the 4th day, then the growth of chondrocytes reached platform stage from the 9th day and were inhibited from the 11th day.(2) Identification of chondrocytes from rats VBGP.Inverted phase microscope and HE staining show the cells are polygonal with affluent endochylema and buninoid nucleus. Toluidine Blue staining show the nucleus of the cells are stained into blue. All the description above consistent with the characteristics of chondrocyte.(3) Observe the characteristics of chondrocytes at different passages.With the passages of the primary cultured chondrocytes, the shape of chondrocytes would change from polygonal into long shuttle and the cells show the tendency of changing into fibroblast from 4th passege. The collagen type II immunocytochemical staining was extensive positive in chondrocytes before the third passage and the expression of collagen II on cells decreases gradually with the passages.2. Histological observation and detection of the proliferative capability of thoracic VBGPs from rats at different ages.(1) Safranin O-fast green staining.Safranin O-fast green staining of vertebral body growth plates of rats aged 1 day, 1 week,4weeks,8weeks,16weeks and 28weeks show the cartilage tissue of growth plate was stained into red. The ossification phenomenon can be seen in the resting zone of rats from age 16weeks. The VBGP can be seen till 28 weeks but most of the resting zone change into ossified.(2) Histological observation of 3 zones of thoracic VBGPs from rats at different ages.The hypertrophic zone of rats aged 1 day and 1 week is obviously higher than that in other groups (P<0.01). The proliferative zone in rats aged 1 day and 1 week is higher than that in other groups (P<0.05) and this zone in rats aged 4 weeks is higher than that in rats aged 28 weeks (P<0.05). The resting zone in rats aged 1 day and 1 week is higher than that in other groups (P<0.05) and this zone in rats aged 4 weeks is higher than that in rats aged 16 and 28 weeks (P<0.05).(3) Detection of the proliferative capability of thoracic VBGPs from rats at different ages.The relative expression amount of PCNA gene decreases with the increasing ages. The gene expression of rats aged 1 week,4 weeks,8weeks,16weeks and 28 weeks is notably lower than that aged 1 day and the difference has notable statistical significance (P<0.01). The relative expression amount of PCNA protein decreases with the increasing ages. The protein expression of rats aged 1 week,4 weeks, 8weeks,16weeks and 28 weeks is notably lower than that aged 1 day and-the difference has notable statistical significance (P<0.01).3. Detection of melatonin receptors in VBGP of rat.(1) The expression of melatonin receptors on rats VBGP-chondrocytes were detected by immunocytochemistry.Immunocytochemistry staining of melatonin receptor on chondrocytes cultured from rats vertebral body growth plate show the chondrocytes were stained into buffy.(2) The expression of melatonin receptors on rats VBGPs were detected by immunohistochemistry.Immunohistochemistry staining of melatonin receptor on vertebral body growth plate of a rat aged 1 week show the chondrocytes in all the 3 zones of rats VBGP were stained into buffy.(3) Detection of the gene expression of melatonin receptors.Comparing with DAPDH, the relative expression amount of MT1 and MT2 gene are 0.11±0.02 and 0.05±0.01 respectively.(4) Detection of the protein expression of melatonin receptors.Comparing withβ-actin, the relative expression amount of MT1 and MT2 protein are 0.28±0.11 and 0.22±0.09 respectively.4. Detection of the effect of melatonin to proliferation and differentiation of chondrocytes from rat VBGP.(1) Detection of the effect of melatonin to proliferation of VBGP-chondrocytes:Melatonin significantly inhibited the proliferation of passaged VBGP-chondrocytes at 10 and 100μg/ml. The vehicle of melatonin (alcohol) has no effect on the proliferation of the cells. The proliferation-inhibition effect of melatonin on chondrocytes was inhibited by luzindole, a melatonin receptors antagonist.(2) Detection of the proliferation and differentiation related factors of melatonin to VBGP-chondrocytes.The expression of PCNA, Sox9 and Smad4 decreases when the concentration of melatonin increases. Conclusions1. The improved trypsin-collagenase typeⅡdigestion method can isolate and culture chondrocytes successfully. This method can acquire pure chondrocytes in short time. The cells before passage 3 maintain phenotype of chondrocyte and grow quickly.2. The 3 zones of VBGPs in rats aged 1 day and 1 week are higher than in the older ones. The ossification phenomenon can be seen in the resting zone of rats from age 16weeks. The VBGP can be seen till 28 weeks but most of the resting zone change into ossified. The proliferation ability of VBGP-chondrocytes decrease with the increase of age.3. There exist melatonin receptors (MT1, MT2) in VBGPs of rats. It suggests that melatonin may act on the longitudinal growth of spine.4. High concentrations of melatonin could inhibit the proliferation and differentiation of VBGP-chondrocytes.
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