Frakturheilung bei beeinträchtigtem Wnt-Signalweg und dadurch hervorgerufener Osteoporose im Mausmodell

Frakturen sind bei Osteoporosepatienten häufig und der verzögerte Heilungsverlauf kann zu Sekundärerkrankungen führen, die zum Verlust der Selbstständigkeit führen und die unter Umständen tödlich enden. Es ist deshalb sehr wichtig Möglichkeiten zu finden, die Frakturheilung bei osteoporotischen Patienten zu verbessern. Gentechnisch veränderte Mausmodelle können zur Untersuchung biologischer Faktoren, die an der Frakturheilung beteiligt sind, einen wertvollen Beitrag leisten. Die vorliegende Arbeit beschäftigte sich mit der Frakturheilung in zwei osteoporotischen Mausmodellen, die aufgrund einer gentechnisch induzierten Störung der Wnt-ß-Catenin-Signaltransduktion einen osteoporotischen Phänotyp aufwiesen. Um die Kallusheilung des diaphysären Knochens in der Maus unter stabiler Fixation sowie unter sehr flexiblen Bedingungen untersuchen zu können, wurde zunächst ein neues, biomechanisch kontrollierbares Osteotomiemodell für das Mausfemur entwickelt. Das Modell bediente sich zweier standardisierter Konfigurationen eines Fixateur externe mit stark unterschiedlich eingestellter axialer Steifigkeit und sehr guter Rotationsstabilität, sowie einer standardisierten Operationsmethode mit einer offenen Osteotomie der Diaphyse. Es konnte in dieser Arbeit gezeigt werden, dass die diaphysäre Frakturheilung unter stabiler und flexibler Fixation im Mausmodell analog zu den Gesetzmäßigkeiten in Großtier-Frakturmodellen abläuft. Es zeigte sich, dass die Beeinträchtigung der Wnt-ß-Catenin-Signaltransduktion in den osteoporotischen Phänotypen die Frakturheilung verzögerte. Daraus ergaben sich neue interessante Ansatzpunkte zur Therapie der Frakturheilungsverzögerung des osteoporotischen Knochens. Die sehr flexible Fixation beeinträchtigte die Heilung des osteoporotischen Knochens noch weitaus stärker als die des gesunden Knochens. Die stabile Fixation osteoporotischer Frakturen hat also höchste Priorität, auch wenn dies im klinischen Alltag oft schwer zu verwirklichen ist. Mit dieser Arbeit konnten wir zum Verständnis der entscheidenden Faktoren der gestörten Regenerationsfähigkeit des osteoporotischen Knochens beitragen

Osteoblast-Specific Krm2 Overexpression and Lrp5 Deficiency Have Different Effects on Fracture Healing in Mice

The canonical Wnt/beta-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5(-/-)) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5(-/-) mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5(-/-) mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active beta-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis

Candidate genes for impaired fracture healing in <i>Col1a1-Krm2</i> and <i>Lrp5^−/−</i> mice.

<p>Differential gene expression in callus tissue was analyzed 10 days post-fracture by microarray analysis (n = 3). Only gene products that fulfilled criteria for significant gene expression changes (see methods) are listed.</p><p>FC: mean value of fold changes of the cross-wise comparisons with wildtype callus (n = 3) as determined by microarray analysis.</p><p>SD: standard deviation.</p><p>― no significantly differential expression when compared to wildtype callus (n = 3) as determined by microarray analysis.</p

<i>Krm2</i> overexpression in osteoblasts has a stronger effect on gene expression than <i>Lrp5</i> deficiency.

<p>(A) The numbers indicate the number of gene products with significantly differential expression in calli of <i>Col1a1-Krm2</i> and <i>Lrp5-/-</i> mice in comparison to WT mice (for gene names see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103250#pone.0103250.s001" target="_blank">Table S1</a>). (B) Relative changes in gene expression of osteoblast associated genes and Wnt target genes. QPCR was performed with samples prepared from callus tissue under semi-rigid fixation 10 days post-fracture. Gene expression in <i>Col1a1-Krm2</i> mice and <i>Lrp5^−/−</i> mice are expressed as fold change relative to wildtype mice. Wildtype (n = 6), <i>Lrp5^−/−</i> (n = 6), <i>Col1a1-Krm2</i> (n = 5) ± SD. * p<0.05, ** p<0.01.</p

<i>Krm2</i> overexpression in osteoblasts and <i>Lrp5</i> deficiency impair regular fracture healing.

<p>The fractured femurs of wildtype, <i>Col1a1-Krm2</i> and <i>Lrp5^−/−</i> mice were stabilized using a semi-rigid fixator in order to provide better mechanical healing conditions as described in the Material and Methods section. (A) In order determine the mechanical competence of the femurs the stiffness of both intact and fractured femurs was measured. Wildtype (n = 8), <i>Lrp5^−/−</i> (n = 9), <i>Col1a1-Krm2</i> (n = 7) ± SD * p<0.05. (B) µCT analysis was performed to measure callus volume (TV), maximum moment of inertia (Imax) and bone volume fraction (BV/TV). Wildtype (n = 8), <i>Lrp5^−/−</i> (n = 7), <i>Col1a1-Krm2</i> (n = 7) ± SD. * p<0.05 versus wildtype. (C) For histological analysis, the paraffin sections of the calli were stained with Giemsa at day 10 post fracture. Sections of methacrylate embedded calli were stained with Paragon at day 21 post fracture. The black arrows indicate crude and less branched bone trabeculae in the callus of <i>Col1a1-Krm2 mice</i> (D) Histomorphometric evaluation was performed to quantify total osseous tissue, cartilage, and fibrous tissue in the callus at day 10 and 21 post fracture. Wildtype (n = 8), <i>Lrp5^−/−</i> (n = 5), <i>Col1a1-Krm2</i> (n = 5) ± SD. * p<0.05 versus wildtype.</p

Bony bridging of the fracture callus.

<p>Bony bridging of the fracture callus evaluated at 4 locations in the fracture callus: anterior and posterior callus peripherally (periosteal callus) and in between the cortices (intracortical callus). The quality of bone bridging was described by a scoring system (4 =  complete bony bridging at all locations; 3 =  at 3 locations; at 2 locations; 1 =  at 1 location; 0 =  no bony bridging). The table displays the number of mice with a distinct bridging score within the groups. n = 4–7 mice per group.</p

<i>Krm2</i> overexpression and <i>Lrp5</i> deficiency result in increased osteoclast number.

<p>(A) Osteoclasts were identified by histochemical staining of tartrat-resistant acid phosphatase (TRAP) at day 10 post fracture. (B) Osteoclast number was determined in the peripheral callus at day 10 post fracture. Wildtype (n = 6), <i>Lrp5^−/−</i> (n = 4), <i>Col1a1-Krm2</i> (n = 5) ± SD. * p<0.05 versus wildtype. Representative images of (C) Smpd3 expression and (D) active β-catenin levels detected by immunostaining at day 10 post fracture.</p

The different genotypes do not affect limb loading during fracture healing.

<p>(A) Motion of the mice was measured pre- and postoperatively using an infrared beam detection system. (B) Peak vertical ground reaction forces of the operated limb were recorded during movement of the mice using a force plate. n = 3–7 randomly selected mice per group (wildtype: dashed line (n = 7), <i>Lrp5^−/−</i>: without line (n = 3), <i>Col1a1-Krm2</i>: black line (n = 6)). Postoperative values were related to preoperative measurements. Data are expressed as means ± SD.</p

<i>Krm2</i> overexpression in osteoblasts lead to a more strongly impaired healing than Lrp5 deficiency.

<p>The fractured femurs of wildtype, <i>Col1a1-Krm2</i> and <i>Lrp5^−/−</i> mice were stabilized using a flexible fixator in order to induce delayed healing as described in the Material and Methods section. (A) In order determine the mechanical competence of the femurs the stiffness of the fractured femurs was measured. (B) µCT analysis was performed to measure callus volume (TV), maximum moment of inertia (Imax) and bone volume fraction (BV/TV). Wildtype (n = 5), <i>Lrp5^−/−</i> (n = 4), <i>Col1a1-Krm2</i> (n = 7) ± SD. * p<0.05 versus wildtype. § p<0.05 versus rigidly fixated group. (C) For histological analysis, sections of methacrylate embedded calli were stained with Paragon at day 21 post fracture. (D) Histomorphometric evaluation was performed to quantify total osseous tissue, cartilage, and fibrous tissue in the calli at day 21 post fracture. Wildtype (n = 5), <i>Lrp5^−/−</i> (n = 4), <i>Col1a1-Krm2</i> (n = 5) ± SD. * p<0.05 versus wildtype. (E) Representative images of collagen type II expression detected by immunostaining at day 10 post fracture.</p