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

The Transcriptional Profile of Mesenchymal Stem Cell Populations in Primary Osteoporosis Is Distinct and Shows Overexpression of Osteogenic Inhibitors

Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC) are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79-94 years old) suffering from osteoporosis (hMSC-OP). In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1) and of genes involved in osteoclastogenesis (CSF1, PTH1R), but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of similar to 30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP. Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L2, a novel repressor of BMP-induced transcription. Such transcriptional changes may reflect epigenetic changes, which are part of a specific osteoporosis-associated aging process

A retrospective analysis of bone mineral status in patients requiring spinal surgery

Abstract Background Impaired bone quality is associated with poor outcome of spinal surgery. The aim of the study was to assess the bone mineral status of patients scheduled to undergo spinal surgery and to report frequencies of bone mineral disorders. Methods We retrospectively analyzed the bone mineral status of 144 patients requiring spinal surgery including bone mineral density by dual-energy X-ray absorptiometry (DXA) as well as laboratory data with serum levels of 25-hydroxyvitamin D (25-OH-D), parathyroid hormone, calcium, bone specific alkaline phosphate, osteocalcin, and gastrin. High-resolution peripheral quantitative computed tomography (HR-pQCT) was additionally performed in a subgroup of 67 patients with T-Score below − 1.5 or history of vertebral fracture. Results Among 144 patients, 126 patients (87.5%) were older than 60 years. Mean age was 70.1 years. 42 patients (29.1%) had suffered from a vertebral compression fracture. 12 previously undiagnosed vertebral deformities were detected in 12 patients by vertebral fracture assessment (VFA). Osteoporosis was present in 39 patients (27.1%) and osteopenia in 63 patients (43.8%). Only 16 patients (11.1%) had received anti-osteoporotic therapy, while 54 patients (37.5%) had an indication for specific anti-osteoporotic therapy but had not received it yet. The majority of patients had inadequate vitamin D status (73.6%) and 34.7% of patients showed secondary hyperparathyroidism as a sign for a significant disturbed calcium homeostasis. In a subgroup of 67 patients, severe vertebral deformities were associated with stronger deficits in bone microarchitecture at the distal radius compared to the distal tibia. Conclusions This study shows that bone metabolism disorders are highly prevalent in elderly patients scheduled for spinal surgery. Vertebral deformities are associated with a predominant deterioration of bone microstructure at the distal radius. As impaired bone quality can compromise surgical outcome, we strongly recommend an evaluation of bone mineral status prior to operation and anti-osteoporotic therapy if necessary

<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

<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

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 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