Osteoporoseinduktion am Tiermodell der Ratte : biomechanische Analysen zur Qualitätssicherung am Rattenknochen

Ziel dieser Arbeit war es ein Grundlagenmodell für die weitere Erforschung der Osteoporose zu etablieren. Es sollte eine steroidinduzierte, eine postmenopausale, sowie eine senile Osteoporose an ovarektomierten Ratten induziert werden. Es wurden 80 weibliche Sprague Dawley Ratten im Alter von 14 Wochen (Zeitpunkt 0 Monate) in 4 Gruppen eingeteilt, zwei Kontroll- und zwei Testgruppen. Die Kontrollgruppe wurde unterteilt in 1) eine Ausgangsgruppe (n = 10, 0 Monate) und 2) eine Sham-operierte Gruppe (Sham, 3, 12 und 14 Monate; jeweils n = 10). Die Tiere der Testgruppe wurden ovarektomiert und anschließend entsprechend ihrer unterschiedlichen Behandlung eingeteilt: 1) Kalzium- und Vitamin D-arme Diät (OVX + Diät, 3, 12 und 14 Monate; jeweils n = 10) und 2) eine Dexamethason-21- isonicotinat-Behandlung s.c. (OVX + Steroid, 3 Monate; n = 10). Ex vivo wurden folgende biomechanische Analysen an den Knochen der Tiere durchgeführt: 1) Kompressionstests an den Wirbelkörpern Th8 und Th9 zur Kraftmessung, 2) Torsionsprüfungen der rechten Tibia zur Messung der Steifigkeit und des Bruchmoment und 3) Drei-Punkt-Biegungsprüfungen der rechten Femura zur Testung der Biegesteigung und der Bruchkraft. Nach 3 Monaten wies die Kompressionskraft von Th8 und Th9 zwischen Sham- und Diät- (Th8 p = 0,002 und Th9 p < 0,001) bzw. zwischen Sham- und Steroid-Gruppe (Th8 p = 0,011 und Th9 p = 0,006) signifikante Unterschiede auf. Im weiteren Verlauf der Versuchsreihe verminderte sich die Kraft der Sham-Gruppe jeweils nicht signifikant. In der Diät-Gruppe sank die Kraft bei Th8 nach 12 Monaten signifikant ab (p < 0,001). In der Torsionsprüfung war nach 3 Monaten Standzeit die Steifigkeit tendenziell und das Bruchmoment zwischen Sham- und Diät-Gruppe (p = 0,013) signifikant verringert. Von 3 auf 12 Monate sank die Steifigkeit der Sham-Gruppe gering (12 Monate p = 0,954). Das Bruchmoment verringerte sich bis zum 12. Monat signifikant (p = 0,017), danach nur noch minimal. In der Diät-Gruppe kam es von 3 auf 12 Monate zu signifikanten Differenzen beider Parameter (Steifigkeit p = 0,012 und Bruchkraft p = 0,014). Nach weiteren 2 Monaten war der weitere Unterschied nur noch gering. In der Drei-Punkt-Biegeprüfung kam es nach 3 Monaten bei der Bruchkraft zu signifikant ansteigenden Abweichungen zwischen Sham- und Diät- (p = 0,011) bzw. Sham- und Steroid-Gruppe (p < 0,001). Die Biegesteigung war zwischen Sham- und Steroid-Gruppe (p = 0,029) signifikant erhöht. Beide Parameter der Sham-Gruppe stiegen von 3 auf 12 Monate an (Steigung p < 0,001 und Bruchkraft p = 0,265). Nach weiteren 2 Monaten sanken beide Werte nicht signifikant ab. In der Diät-Gruppe kam es von 3 auf 12 Monate zu signifikanten Differenzen beider mechanischen Parameter (jeweils p < 0,001). Von 12 auf 14 Monate nahmen die mechanischen Parameter nur wenig ab. Die Ovarektomie mit Diät nimmt einen negativen Einfluss auf die mechanischen Eigenschaften der spongiösen Wirbelkörper und der kortikalen Knochen der Ratte. Der osteoporotische Knochenstatus wurde bei den Tieren in den spongiösen Knochen bereits nach 3 Monaten Behandlung induziert (Kompressionstest). Bei den kortikalen langen Röhrenknochen hingegen manifestiert sich die Osteoporose erst 12 Monate nach Ovarektomie und Diätgabe (Torsionstest und Drei-Punkt-Biegeprüfung). Die Steroidgabe erzielte nicht den gewünschten negativen Effekt auf die Knochen. Dementsprechend kann das beschriebene Tiermodell verwendet werden, um weitere Untersuchungen zur osteoporosebedingter Frakturheilung, auch in Kombination mit neuen Biomaterialien oder Implantaten, durchzuführen.Aim of this study was to establish a basic model for the further study of osteoporosis. Postmenopausal and senile osteoporosis in ovariectomized rats was established through multi-deficient diet and steroidal induction. The study encompassed four groups, two control and two experimental groups. The two control groups were divided into: 1) an initial control to show bone status before treatment start (control, 0M; n=10); 2) a sham operated group that served as an experimental control (sham, 3M, 12M, 14M; n=10 each). The experimental groups were divided according to their treatment post bilateral ovariectomy into two groups as well: 1) steroid induced osteoporosis, (OVX+Steroid, 3M; n=10); 2) Diet-deficiency induced osteoporosis, (OVX+Diet, 3M, 12M, 14M; n=10 each). The study examined biomechanical competence in several anatomical sites: 1) compression tests of the vertebra body (Th8 und Th9) to asses loading at failure, 2) torsion test of the right tibia to assess torsional stiffness and torque at failure and 3) three-point-bending measurements of the right femur bending and breaking load. At the early phase (after 3M), both treatments reflected a significantly inferior biomechanical competence (compression test) in the vertebral bodies Th8 and Th9 when compared to the sham (OVX+Diet p = 0.002 Th8, p < 0.001 Th9; OVX+Steroid p = 0.011 Th8, p = 0.006 Th8, respectively) showed differences. At the later phase under diet-deficiency this effect intensified (12M, Th8; p < 0.001). However, no further effects were seen at 14M in the vertebrae. Although torsional stiffness showed only a lower trend, the maximum torque at failure was significantly lower in the OVX+Diet at 3M when compared to the sham (p = 0.013). However, torsional stiffness of the sham group was lower at 12M when compared to 3M (p = 0.954) and then higher at 14M compared to 12M (p = 0.025). The maximum torque at failure was significantly lower at 12M compared to sham (p = 0.017). The OVX+Diet group showed lower torsional stiffens and torque at 12M compared to 3M (p = 0.012 and p = 0.014, respectively), nonetheless, minimal changes occurred at 14M. In the three-point bending test significantly higher differences in the breaking load between sham and OVX+Diet (p = 0.011) and sham and OVX+Steroid group (p < 0.001) were observed after 3 months. The bending was significantly increased between sham and OVX+Steroid group (p = 0.029). In the femur, the three-point bending test in the sham group showed higher bending and breaking force at 12M when compared to 3M (p < 0.001 and p = 0.265 respectively), two months later at 14M there was no real change. In the OVX+Diet group the mechanical parameters showed a significant increase (each p < 0.001) between the 3th and the 12th month. From 12 to 14 months it decreased slightly. In conclusion, bilateral ovariectomy combined with multi-deficient diet influence the mechanical properties of cancellous and cortical bone in the rat spine and long bone, negatively. Inferior bone competence in the OVX+Diet group vertebra indicated that. Induction of osteoporotic bone status took place in cancellous bone (compression test) after 3 months of treatment. However in the cortical bone (torsional and three-point- bending tests) the osteoporotic bone status influenced its integrity at later stage after 12 months of treatment. Therefore, the described animal model could be utilized for further investigation of osteoporotic fractures healing aided with novel osteoinductive biomaterials

Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat.

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies

Biomechanical testing of tibia and femur of the ovariectomized rat after three months of multi-deficient diet treatment.

<p>(A) Ultimate torque at failure shows no significance between the groups at 3 M. (B) Tibia of treated rats showed lower torsional stiffness compared with the Sham operated rats. (C) Breaking load of the femur shows an increased needed load to break the femur of treated animals at 3 M. (D) Bending stiffness of the femur showed no significant difference between the treatment group and the Sham group. (* = p≤0.05, Mann Whitney U with bonferroni correction, n = 8 per group).</p

µCT analysis of cortical bone parameters show affected porosity and thickness in the OVX+Diet treatment after 3 months.

<p>(A) Porosity measurement showed that both groups; the 0 M control and the 3 M Sham have significantly lower porosity than the 3 MOVX+Diet. (B) Cortical thickness was significantly higher in the 3 M Sham group when compared to either 3 M OVX+Diet or 0 M control group. (* = p≤0.05, Mann Whitney U with bonferroni correction, n = 8 per group).</p

Qualitative analysis of trabecular bone shows inferior bone quality resulting from multi-deficiencies diet combined with bilateral ovariectomy in rats after 3

<p> <b>M.</b> (A) BV/TV showed how bone tissue is affected within the total volume; these results suggest that at 3 M the treatment results in less mineralized tissue in OVX+Diet when compared to the 3 M Sham. (B) Structure Model Index (SMI) indicated the trabecular shape change at 3 M in the OVX+Diet group. (C) Tb. N was lower in the 3 M OVX+Diet group compared to the Sham group. (D) Higher trabecular separation in the OVX+Diet group when compared to the Sham after 3 M of treatment. (* = p≤0.05, one-way ANOVA with bonferroni correction, 0 M, n = 8; 1 M, n = 3 per group; 3 M n = 8 per group).</p

Biochemical markers reflect the bone metabolism.

<p>(A–D) Opn, BGP, RANKL, and PTH concentration in serum was higher in the 3 M OVX+Diet than the 3 M Sham. (D -F) lower concentrations of TRAP5b and leptin were seen in the OVX+Diet at 3 M compared to the Sham group. (* = (p≤0.05), Mann Whitney U with bonferroni correction, n = 8 per group).</p

Unbalanced cellular populations contribute in 3 M OVX+Diet bone alteration.

<p>(A+B) 3 M OVX+Diet showed less activity of osteoblasts at the trabecular area when compared with 3 M Sham animals suggesting a lower bone formation. (C+D) Osteoclasts number at trabecular surface of OVX+Diet showed a higher trend than in the Sham group at 3 M indicating an increased bone resorption caused by the treatment. (* = (p≤0.05), Mann Whitney U with bonferroni correction, n = 3 per group 15 microscopic fields, scale bar = 5 mm, Tb = trabecular bone, OB = osteoblasts, OC = osteoclasts).</p

Weight and bone length of different groups at all time points.

<p>Weight and bone length of different groups at all time points.</p

A chart depicting the work plan and experimental design.

<p>Female Sprague-Dawley rats were utilized for this experiment, 10 of which were sacrificed, analysis on these animals were carried out to obtain an initial bone status. Eleven animals were then ovariectomized (OVX), and another eleven rats were Sham operated (Sham) and animals were left to recover for two weeks. After recovery OVX animals were given a multi-deficiencies-diet where Sham animals received a standard diet. One month (1 M) and three months (3 M) after dietary treatment begun, 3 and 8 animals were sacrificed, respectively. Rats were scanned by DEXA at every time point and DEXA results were reported elsewhere. Besides DEXA, at 1 M only µCT analysis was performed on the left tibia. At both 0 M and 3 M Left tibia was analyzed in µCT before performing undecalcified histology; right femur and right tibia were tested biomechanically, and right femur was used for decalcified histology.</p