Einfluss der Hypochlorhydrie-induzierten Calcium-Malabsorption auf die Frakturheilung

Calcium is an essential mineral in the human body, which is consumed with food and mainly embedded in bone. Gastric acidification plays a major role in the absorption of calcium and interruptions such as gastritis can lead to calcium malabsorption. In recent years, proton pump inhibitors (PPI), which are a class of pharmaceuticals commonly used against gastric diseases, were shown to increase fracture risk. PPI irreversibly bind to the proton pump of gastric parietal cells and inhibit gastric acidification leading to an elevated gastric pH and hypochlorhydria. Calcium carbonate, which is a calcium compound commonly found in calcium supplementations, is hardly soluble under neutral conditions and poorly absorbed in individuals with hypochlorhydria. Thus, the effect of calcium carbonate supplementation on bone metabolism and fracture repair is doubtful if hypochlorhydria is present. Because little is known about the influence of hypochlorhydria and calcium malabsorption on bone repair, fracture healing was analyzed in cholezystokinin-B/gastrin-receptor deficient mice (Cckbr-/-) having hypochlorhydria and an elevated gastric pH. After osteotomy of the femur, Cckbr-/- and wildtype (WT) mice received either a standard diet, a calcium carbonate diet or a diet containing calcium gluconate. Results of the present study showed that hypochlorhydria induced calcium malabsorption did not influence fracture healing. However, the malabsorption led to negative effects on non osteotomized bone such as a decreased bending stiffness. Calcium carbonate supplementation neither improve fracture healing, nor prevented the negative effects on intact bone. Calcium gluconate improved fracture healing in WT mice as well as in Cckbr-/- because it has a good solubility at higher pH in contrast to calcium carbonate. Positive effects on intact bone of Cckbr-/- were also found after administration of the calcium gluconate diet

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 G: peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (-81%) and bone formation (-80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations

Midkine-deficiency delays chondrogenesis during the early phase of fracture healing in mice.

The growth and differentiation factor midkine (Mdk) plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process

Hypochlorhydria-induced calcium malabsorption does not affect fracture healing but increases post-traumatic bone loss in the intact skeleton

Efficient calcium absorption is essential for skeletal health. Patients with impaired gastric acidification display low bone mass and increased fracture risk because calcium absorption is dependent on gastric pH. We investigated fracture healing and post‐traumatic bone turnover in mice deficient in Cckbr, encoding a gastrin receptor that affects acid secretion by parietal cells. Cckbr−/− mice display hypochlorhydria, calcium malabsorption, and osteopenia. Cckbr−/− and wildtype (WT) mice received a femur osteotomy and were fed either a standard or calcium‐enriched diet. Healed and intact bones were assessed by biomechanical testing, histomorphometry, micro‐computed tomography, and quantitative backscattering. Parathyroid hormone (PTH) serum levels were determined by enzyme‐linked immunosorbent assay. Fracture healing was unaffected in Cckbr−/− mice. However, Cckbr−/− mice displayed increased calcium mobilization from the intact skeleton during bone healing, confirmed by significantly elevated PTH levels and osteoclast numbers compared to WT mice. Calcium supplementation significantly reduced secondary hyperparathyroidism and bone resorption in the intact skeleton in both genotypes, but more efficiently in WT mice. Furthermore, calcium administration improved bone healing in WT mice, indicated by significantly increased mechanical properties and bone mineral density of the fracture callus, whereas it had no significant effect in Cckbr−/− mice. Therefore, under conditions of hypochlorhydria‐induced calcium malabsorption, calcium, which is essential for callus mineralization, appears to be increasingly mobilized from the intact skeleton in favor of fracture healing. Calcium supplementation during fracture healing prevented systemic calcium mobilization, thereby maintaining bone mass and improving fracture healing in healthy individuals whereas the effect was limited by gastric hypochlorhydria. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1914–1921, 2016

The Wnt serpentine receptor Frizzled-9 regulates new bone formation in fracture healing.

Wnt signaling is a key regulator of bone metabolism and fracture healing. The canonical Wnt/β-catenin pathway is regarded as the dominant mechanism, and targeting this pathway has emerged as a promising strategy for the treatment of osteoporosis and poorly healing fractures. In contrast, little is known about the role of non-canonical Wnt signaling in bone. Recently, it was demonstrated that the serpentine receptor Fzd9, a Wnt receptor of the Frizzled family, is essential for osteoblast function and positively regulates bone remodeling via the non-canonical Wnt pathway without involving β-catenin-dependent signaling. Here we investigated whether the Fzd9 receptor is essential for fracture healing using a femur osteotomy model in Fzd9(-/-) mice. After 10, 24 and 32 days the fracture calli were analyzed using biomechanical testing, histomorphometry, immunohistochemistry, and micro-computed tomography. Our results demonstrated significantly reduced amounts of newly formed bone at all investigated healing time points in the absence of Fzd9 and, accordingly, a decreased mechanical competence of the callus tissue in the late phase of fracture healing. In contrast, cartilage formation and numbers of osteoclasts degrading mineralized matrix were unaltered. β-Catenin immunolocalization showed that canonical Wnt-signaling was not affected in the absence of Fzd9 in osteoblasts as well as in proliferating and mature chondrocytes within the fracture callus. The expression of established differentiation markers was not altered in the absence of Fzd9, whereas chemokines Ccl2 and Cxcl5 seemed to be reduced. Collectively, our results suggest that non-canonical signaling via the Fzd9 receptor positively regulates intramembranous and endochondral bone formation during fracture healing, whereas it does not participate in the formation of cartilage or in the osteoclastic degradation of mineralized matrix. The finding that Fzd9, in addition to its role in physiological bone remodeling, regulates bone repair may have implications for the development of treatments for poorly or non-healing fractures

Flexural rigidity and moment of inertia was significantly reduced in <i>Mdk</i>-deficient mice after 21 days of the healing period.

<p>Biomechanical and μCT analyses of fractured femur after 21 and 28 days of healing. The volume of interest was determined as the whole periosteal callus between the two inner pin holes. A) Relative flexural rigidity of fractured femur in comparison to intact femur at day 21 and B) at day 28. C) Moment of inertia of the periosteal fracture callus in bending axis x at day 21 and D) at day 28. E) Bone volume to total volume fraction of the periosteal fracture callus at day 21 and F) at day 28. *Significantly different from wildtype (p<0.05). (n = 6–7 per group).</p

<i>Mdk</i> is expressed during ATDC5 cell differentiation and <i>Mdk</i> knockdown significantly delayed early chondrogenic differentiation via suppression of Wnt-target genes.

<p>ATDC5 cells were differentiated and gene expression was evaluated using real-time RT-PCR. <i>B2M</i> was used as the housekeeping gene and gene expression values were normalized to the pre-differentiation values (dotted line). ATDC5 cells were incubated in differentiation medium for 5, 7 and 10 days and A) <i>Mdk</i> and B) <i>collagen2a1</i> gene expression was evaluated using real time RT-PCR. C) ATDC5 cells were incubated with control siRNA or Mdk siRNA for 24 h and subsequently differentiated for 5 days. <i>Mdk</i> knockdown was verified by analyzing <i>Mdk</i> gene expression. Differentiation was analyzed by evaluation of D) <i>aggrecan</i> or E) <i>collagen2a1</i> gene expression. Beta-catenin signaling was analyzed by evaluation of F) <i>lef1</i> and G) <i>axin2</i> gene expression. H) Western blot analysis of Mdk, collagen type 2 and beta-catenin protein expression at days 0 and 5 of differentiation. GAPDH was used as control. *Significantly different from the control values (p<0.05). (n = 6 per group).</p

Immunohistochemical staining showing reduced beta-catenin levels in chondrocytes of <i>Mdk</i>-deficient mice.

<p>Sections of fractured femurs from four mice of each time point and genotype group were stained for each antigen and counterstained using hematoxylin. Representative images are shown; C = cortex; PC = proliferating chondrocyte; Ob = osteoblast; V = vessel; OT = osteocyte; scale bar 50 µm; 200-fold magnification. Beta-catenin staining of the periosteal callus at day 10. PECAM staining of the periosteal fracture callus bridging the osteotomy gap showing the endothelial cells of the newly formed vessels in an area of proliferating chondrocytes at day 10. Enpp1 staining of the osteotomy gap at day 10 showing positively stained proliferating chondrocytes. Dmp1 staining of the periosteal callus at day 10 showing positively stained cortex, osteocytes and areas of new bone formation. Osteoblasts were Dmp1 negative. (n = 4 per group).</p

<i>Mdk</i>-deficient mice aged 9 months displayed increased trabecular number and decreased cortical thickness in the femur.

<p>A) Trabecular bone volume to tissue volume ratio assessed by μCT analysis of volume of interest (VOI) 1 in the intact femur. B) Trabecular number assessed by μCT analysis of the VOI 1 in the intact femur. C) Trabecular thickness. D) Representative μCT images of the intact femurs. E) Cortical thickness assessed by μCT analysis of VOI 2 in the intact femur. *Significantly different from wildtype (p<0.05). (n = 6–7 per group).</p

Immunostaining of β-catenin at day 10 and histochemical TRAP staining indicating osteoclasts at day 24 in the fracture calli.

<p>WT: upper panel, <i>Fdz9</i>^−/−: lower panel. β-catenin was expressed in osteoblasts (OB) and proliferating chondroblasts (CB) but to a lesser extend in hypertrophic chondrocytes (HC). C: cortex. There were no differences between both genotypes. Only TRAP positive cells with ≥2 nuclei were identified as osteoclasts (OC). TRAP-staining either revealed no significant differences between both genotypes.</p