Correlation Between Osteoporosis And Disc Degeneration In Ovariectomized Goats

Osteoporosis and degenerative changes in the intervertebral disc are common diseases in elderly people, which often co-exist in the same patient and badly affect patients’ quality of life, especially for post-menopausal women. In post-menopausal women, severity of osteoporosis usually reflects the severity of disc degeneration. This phenomenon indicates that there is a certain intrinsic link or a similar pathogenesis for the two diseases. However, researches on estrogen, disc nutrition, inflammatory cytokines, genes have revealed very different conclusions on its relevance. This study attempts to establish female goat models of osteoporosis and disc degeneration by castration and disc puncture in order to explore the pathological processes and the correlation between the two diseases. Meanwhile, the possible resistance role of goat bone marrow-derived mesenchymal stem cell (BMSCs) in degenerative changes of disc was also studied. It aims to provide experimental evidence and new spects for clinical and basic research of the two diseases d for better prevention and treatment.Objective:To establish goat models of osteoporosis by ovariectomy and disc degeneration, then evaluate feasibility of this method, also to explore the possible correlation between the two diseases. BMSCs were separated and cultured in vivo to recognize its biological characteristics, the possible resistance role of BMSCs in degenerative changes of disc was also studied.Methods:A total of183-year-old female goats were divided into group A, B, and C randomly. Group A was sham group, group B underwent bilateral ovariectomy (OVX Ⅰ group), group C was treated with ovariectomy+ntramuscular corticosteroids (OVX Ⅱ group). Then bone densitometry measurement, micro-CT scan reconstruction were conducted, serum calcium and phosphorus, alkaline phosphatase (ALP) as well as osteocalcin (BGP) levels were tested, histological examination and mechanical testing methods were applied to evaluate status of osteoporosis in the goat models. Another20goats were randomly divided into group A:0.1mL saline were injected into L3/4disc with16G needle (experimental group or model group, n=8), group B:L3vertebral body was exposed (sham group, n=8), group C (control group, n=4). Preoperative and postoperative imaging data (1,3,6months after undergoing the operation), histology and type Ⅰ, Ⅱ collagen immunohistochemistry data were used for statistical analysis.24healthy adult female goats were randomly divided into group A (normal control group), group B (castrated group), group C (simple puncture group), group D (composite group:ovariectomy+disc puncture). Lumbar MRI, X ray, bone density, histopathology and immunohistochemistry results were statistically analyzed; BMSCs were extracted, isolated and cultured, growth curve was depicted, surface markers and cell cycle were detected, osteoblasts were induced to differentiate into chondrocytes.15adult goats were selected and randomly divided into group A (control group, n=3), group B (n=12, experimental group) and L2/3, L3/4, L4/5, L5/6(four discs) were exposed, L2/3, L3/4, L4/5of the experimental group (36intervertebral discs) were exposed and treated with puncture+nucleus suction, puncture disc was randomly divided into three sub-groups including B1group (simple-made models), B2group (pure alginate gel transplant group), B3group (mesenchymal alginate composite graft group), postoperative MRI scans, DR examination and histopathological, immunohistochemical staining analysis of stem cells were conducted.Results:Osteoporosis build each model test results show:1bone density testing: Group C after6months of lumbar BMD, BMC compared with preoperative significantly decreased (P<0.05), decreased26.91%and22.95%, respectively; and. with group A and group B were significantly reduced compared to (P<0.05); group B 6and12months after lumbar spine BMD, BMC than before surgery and sham group (Sham group) were significantly lower (P<0.05), decreased by11.96%,18.09%and29.30%,25.50%; Sham group after surgery BMD, there was no significant difference (between BMC change P>0.05).2. Micro-CT detection:Tb.N, Tb.Sp, TBPf, BV/TV, BS/BV, there were significant differences (P<0.001) SMI as a whole; OVX Ⅰ group and OVX Ⅱ group Tb.N, Tb. Sp, TBPf, BV/TV, BS/BV, SMI and Sham group, were statistically significant (P<0.05), while Tb.Th, DA no significant difference (P>0.05). OVX Ⅰ and OVX Ⅱ was no significant difference between the two groups (P>0.05). December castration castration group B and group C on June lumbar spine trabecular thinning significantly higher than the control group, trabecular thinning, breakage, increased porosity, greater local void formation, cortical bone thinning.3serological tests:Compared with sham group, operation group Ca, P, ALP, BGP was significantly decreased (P<0.05), before the sham surgery group BGP levels and other indicators, no significant differences in postoperative (P>0.05).3and6months after the above indicators were higher than group C B (P<0.05); BC group was also statistically significant Self-control (P <0.05).4lumbar spine specimens histopathological observation:preoperative trabecular morphological rules in each group, the large number of neat, uniform thickness, the gap is small and connected arched structure, there is little trabecular fracture. Reduce the number of trabecular bone in ovariectomized group than in the sham surgery group, thinning thinning, or even rupture perforation, canal expansion locally visible bone defect.5. Mechanical testing showed that the maximum expansion pressure vertebral sham group was significantly higher than castration group (P<0.05), and also a significant difference (P<0.05) between the BC group.Construction of disc degeneration model results show:1.DR piece:A month after a group of disc space narrowing appears, and gradually decline. Disc height change (%DHI) in each group after3and6months ABC was85.45±2.48,68.90±4.07,56.77±4.43,94.37±2.42,94.50±1.85,93.87±2.64and95.89±2.60,94.82±1.98,95.13±2.81. control group at each time point the difference was not statistically significant (P>0.05), the experimental group and the control group at each time point difference was statistically significant (P<0.05).2MRI examination:two control groups L3/4disc T2-weighted images showed high signal strength always, the nucleus pulposus region signal uniform, bright, clear boundaries nucleus pulposus and the annulus, front disc height consistent disc that did not happen sham group degeneration; while the A group L3/4disc nucleus T2-weighted image signal intensity decreased, after a month the disc is gray, grayish black after3months,6months after surgery was black, disc space narrowing gradually. L3/4disc nucleus sagittal MRI T2W1signal value (gray value) cues change comparing differences in group A after surgery was statistically significant (P<0.05), and modeling the longer the signal strength decreases, the more obvious. A group compared with the control group were statistically significant gray values (P<0.05).(3) histological evaluation: eye view of the normal control group nucleus pulposus white jelly, clear water full of rich, fibrous ring structure clearly neatly arranged in concentric circles; puncture-made modules disc thickness decreased, the color becomes dark nucleus, less, the annulus structure rough. HE staining of normal intervertebral disc distinct, clear lines and the nucleus, there is a grid-like structure of cells and small vacuoles rich class cartilage-like cells, collagen arranged rules within the nucleus; degenerated intervertebral disc arrangement disorders and even rupture, the nucleus shrink, reducing the number of cells, the nucleus within the grid-like structure disappeared, moisture reduction, the nucleus pulposus and the annulus boundaries unclear.4.Ⅱ collagen immunohistochemical detection:the control group in the central nucleus staining mainly extracellular matrix was strongly positive brown staining; made module after a month to see yellow dye and stain gradually fades, almost no expression after6months. Three months after A, group B grayscale values were178.87±4.44,159.07±4.77;6months after ABC group were217.84±10.33,160.76±7.58,163.34±5.90. Nucleus between the average gray value at each time point after group A and BC There were significant differences (P<0.05), before and after surgery itself A comparison group were statistically significant (P<0.05), each control point no significant difference (P>0.05).5I collagen immunohistochemistry:. BC group outer fibrous ring of type Ⅰ collagen were strongly positive staining, the annulus inner weak staining, non-staining nuclei marrow, A group of type I collagen in the annulus and the inner marrow nuclear staining was gradually increasing trend. A group at each time point after the average gray value of statistical difference between the BC group (P<0.05), A Self-control group were significantly different (P<0.05), no statistically significant difference between the control group (P>0.05).The results suggest that the correlation between the two:a lumbar MRI:no between group B and group A before and after surgery and6months was statistically significant, P>0.05; Group B at12months, group C and D. L3/4disc than in group A group of6,12months after surgery were degenerative changes, P<0.05; where D group after6and12months compared with the other groups, a statistically disc degeneration significance, P<0.05;6,12months after surgery, CD disc T2WI signal values group and B group was statistically significant, P<0.05; D group C itself contrast, the value of the signal at each time point Rooms were statistically significant, P<0.05; while group B, there was significant after12months between T2WI signal value and6months postoperatively, P<0.052lumbar spine X-ray:CD group.3months after the beginning of the disc space narrowing and gradually declined.3,6,12months after the group C, D group compared with the other groups and its own front and rear%DHI, were statistically significant, P<0.05; D group after6and12months compared with group C, disc height changes%DHI was statistically significant, P<0.05; B group before surgery and6months after surgery and between group A and meaningless, P>0.05,12month when compared with group A statistically significant P<0.05. Between the normal control group from0to12months%DHI no statistically significant P>0.05.3BMD examination:6and12months postoperatively, group B and D group compared with the preoperative BMD was significantly lower (P<0.05), compared with the AC group were significantly lower (P<0.05), but there was no significant difference between the two groups (P>0.05); former group C BMD compared with group A patients and no significant lower (P>0.05); was no significant difference (P>0.05) between the A group.4vertebrae histological HE staining:before castration, A group of trabecular bone morphology group C than the rule, neat, uniform thickness, a smaller gap. The BD group to reduce the number of trabecular thinning thinning, breaking perforation, expand the canal, and even bleeding and bone defects.5disc histological HE staining:A group of lamellar structure of the annulus clear fibers arranged in neat rows, nucleus-like structure full of lacunae rich more than the number of cells, nuclei stained nucleus cytoplasm lightly stained; CD set of the annulus board layer boundaries blur, irregular arrangement of fibers, the nucleus lacuna-like structures due to the rules, reduce or smaller cells decreased matrix density low light staining; D group rarely nucleus pulposus cells, the nucleus reduced fibrosis, degeneration re-the group C. While Group B has little lamellar structure damage, reduce the number of nucleus pulposus cells.6type Ⅱ collagen immunohistochemistry:AB group dominated central nucleus staining, extracellular matrix stained brown; Group C yellow dye, D group almost no expression. There collagen Ⅱ average gray value statistically significant difference between groups (P<0.05).Stem cells were isolated and cultured in vitro results:1primary cells adherent growth was short fusiform or with a plurality of projections, and a large, clear nucleolus, split-phase, turning into a uniform shape, radially arranged. And more elongated spindle after passage proliferation significantly faster, fused into a sheet.2growth curve:. P3S-shaped growth curve,1to3days of relatively stagnant, pp.4-9logarithmic growth phase, and then gradually into the growth plateau. Osteogenic and chondrogenic cell growth curve is similar.3display surface antigen markers: CD29(76.8%),(93.6%) positive CD44, do not express CD34(4.3%), CD45(5.9%).4third generation BMSCs cell cycle analysis showed that:G1phase of the cell was68.33%, G2phase cells was21.42%, S phase cells was10.25%, that most of the cells in the stationary phase.5alkaline phosphatase staining:osteogenic ALP staining2-3weeks, most of the cytoplasm was uniform light gray, brown cytoplasm have positive particles, cells clear boundaries, showed ALP staining.6Alizarin red staining:red-brown calcified nodules in the extracellular matrix, Alizarin red staining was positive.7HE staining:see chondrogenic14d chondrocyte cell pericellular nodule mass less round, nuclear small part of the cell cytoplasm and the nucleus has been lost shell formation, extracellular matrix blue stained cells was similar to hyaline cartilage-like appearance.8type II collagen immunohistochemical staining:the visible yellow granular cytoplasmic positive cells were positive cell rate of more than70%.9Alcian blue staining:Alcian blue staining cytoplasm of most cells and the extracellular matrix is dyed clear blueUsing bone mesenchymal stem cells to treat osteoporosis goat intervertebral disc degeneration results:1X-ray examination:. B1and B2group after group that began Jan. disc space narrowing, progressive loss of disc height.1,3,6month B1, B2group and comparison group A vertebral%DHI statistically significant change, P<0.05; among B2and B1group after group also statistically significant, P<0.05; B3group3and6months after surgery compared with B1, B2group, disc height has been restored, the difference was statistically significant, P<0.05; B3group after a month with the normal control group was statistically significant, P<0.05, and B1B2comparison group had no significant difference in P>0.05; A group of0to12months in each group was not statistically significant comparison P>0.052MRI examination showed:no significant difference in each group before surgery, P>0.05;1,3,6month B1, B2groups, with group A T2WI signal intensity differences were statistically significant, P<0.05, between B1B2but no significant difference between groups, P>0.05; B3group3,6months after surgery and showed no significant difference between group A, P>0.05, and B1B2groups were statistically significant, P<0.053HE staining:.. B3group after six months lamellar structure of the annulus relatively clear, fibers arranged in neat rows, increasing the number of cells, stromal staining high density deep, deeply stained nuclei nucleus cytoplasm lightly stained; while the B1group after six months of lamellar boundaries blur, nucleus narrow, irregular arrangement of fibers, nucleus-like structure due to the rules lacunae, decrease, cells decreased matrix density low light staining.4type Ⅱ collagen immunohistochemical staining analysis:1,3,6months B1group gray value of160.13±1.66,177.44±3.24,192.69±4.24; B2group was168.93±3.76,188.81±3.47,201.29±4.85; B3was159.20±5.67,147.80±5.00,140.69±1.58; A group was136.36±3.80,135.69±2.65,134.36±3.14, statistics are between3and6months postoperatively B3group and other groups significance P<0.05; B2group and B1 group also had statistically significant, P<0.05, and with A control group were significantly different, P<0.05; addition to the normal control group, but also the rest of the group set of statistics significance, P<0.05.Conclusions:(1). Goat models of osteoporosis were successfully established1year after ovariectomy, bilateral ovariectomy+hormone injection+low calcium diet can quickly establish goat model of osteoporosis.(2) Goat models of disc degeneration can be established half a year after saline annulus puncture with larger number (14G) needle, besides, saline annulus puncture puncture+nucleus aspiration method can establish goat model of disc degeneration.(3). Osteoporosis may inevitably cause mild disc degeneration, disc degeneration may aggravate the process of disc degeneration while there are other risk factors (or disc degeneration has already occurred). But whether the increased disc degeneration aggravate osteoporosis progress can not determined.(4). BMSCs cultured with whole bone marrow method can provide an ideal seed cells for the intervertebral disc tissue engineering. BMSCs that were treated with alginate gel encapsulation transplanted into goat degenerative disc can effectively inhibit further degeneration of the intervertebral disc.