Ralf Oheim

Low turnover osteoporosis in sheep induced by hypothalamic-pituitary disconnection

The hypothalamus is of critical importance in regulating bone remodeling. This is underscored by the fact that intracerebroventricular‐application of leptin in ewe leads to osteopenia. As a large animal model of osteoporosis, this approach has some limitations, such as high technical expenditure and running costs. Therefore we asked if a surgical ablation of the leptin signaling axis would have the same effects and would thereby be a more useful model. We analyzed the bone phenotype of ewe after surgical hypothalamo‐pituitary disconnection (HPD + OVX) as compared to control ewe (OVX) after 3 and 12 months. Analyses included histomorphometric characterization, micro‐CT and measurement of bone turnover parameters. Already 3 months after HPD we found osteopenic ewe with a significantly decreased bone formation (69%) and osteoclast activity (49%). After a period of 12 months the HPD group additionally developed an (preclinical) osteoporosis with significant reduction (33%) of femoral cortical thickness, as compared to controls (OVX). Taken together, HPD leads after 12 month to osteoporosis with a reduction in both trabecular and cortical bone caused by a low bone turnover situation, with reduced osteoblast and osteoclast activity, as compared to controls (OVX). The HPD‐sheep is a suitable large animal model of osteoporosis. Furthermore our results indicate that an intact hypothalamo‐pituitary axis is required for activation of bone turnover.

High fluoride and low calcium levels in drinking water is associated with low bone mass, reduced bone quality and fragility fractures in sheep.

SummaryChronic environmental fluoride exposure under calcium stress causes fragility fractures due to osteoporosis and bone quality deterioration, at least in sheep. Proof of skeletal fluorosis, presenting without increased bone density, calls for a review of fracture incidence in areas with fluoridated groundwater, including an analysis of patients with low bone mass.IntroductionUnderstanding the skeletal effects of environmental fluoride exposure especially under calcium stress remains an unmet need of critical importance. Therefore, we studied the skeletal phenotype of sheep chronically exposed to highly fluoridated water in the Kalahari Desert, where livestock is known to present with fragility fractures.MethodsDorper ewes from two flocks in Namibia were studied. Chemical analyses of water, blood and urine were executed for both cohorts. Skeletal phenotyping comprised micro-computer tomography (μCT), histological, histomorphometric, biomechanical, quantitative backscattered electron imaging (qBEI) and energy-dispersive X-ray (EDX) analysis. Analysis was performed in direct comparison with undecalcified human iliac crest bone biopsies of patients with fluoride-induced osteopathy.ResultsThe fluoride content of water, blood and urine was significantly elevated in the Kalahari group compared to the control. Surprisingly, a significant decrease in both cortical and trabecular bones was found in sheep chronically exposed to fluoride. Furthermore, osteoid parameters and the degree and heterogeneity of mineralization were increased. The latter findings are reminiscent of those found in osteoporotic patients with treatment-induced fluorosis. Mechanical testing revealed a significant decrease in the bending strength, concurrent with the clinical observation of fragility fractures in sheep within an area of environmental fluoride exposure.ConclusionsOur data suggest that fluoride exposure with concomitant calcium deficit (i) may aggravate bone loss via reductions in mineralized trabecular and cortical bone mass and (ii) can cause fragility fractures and (iii) that the prevalence of skeletal fluorosis especially due to groundwater exposure should be reviewed in many areas of the world as low bone mass alone does not exclude fluorosis.

Sheep model for osteoporosis: Sustainability and biomechanical relevance of low turnover osteoporosis induced by hypothalamic–pituitary disconnection

Hypothalamo‐pituitary disconnection (HPD) leads to low bone turnover and osteoporosis in sheep. To determine the sustainability of bone loss and its biomechanical relevance, we studied HPD‐sheep 24 months after surgery (HPD + OVX‐24) in comparison to untreated control (Control), ovariectomized sheep (OVX), and sheep 12 months after HPD (HPD + OVX‐12). We performed histomorphometric, HR‐pQCT, and qBEI analyses, as well as biomechanical testing of all ewes studied. Twenty‐four months after HPD, histomorphometric analyses of the iliac crest showed a significant reduction of BV/TV by 60% in comparison to Control. Cortical thickness of the femora measured by HR‐pQCT did not change between 12 and 24 months after HPD but remained decreased by 30%. These structural changes were caused by a persisting depression of osteoblast and osteoclast cellular activity. Biomechanical testing of the femora showed a significant reduction of bending strength, whereas calcium content and distribution was found to be unchanged. In conclusion, HPD surgery leads to a persisting low turnover status with negative turnover balance in sheep followed by dramatic cortical and trabecular bone loss with consequent biomechanical impairment.

Metaphyseal fracture healing in a sheep model of low turnover osteoporosis induced by hypothalamic–pituitary disconnection (HPD)

We recently established a large animal model of osteoporosis in sheep using hypothalamic–pituitary disconnection (HPD). As central regulation is important for bone metabolism, HPD‐sheep develop severe osteoporosis because of low bone turnover. In this study we investigated metaphyseal fracture healing in HPD‐sheep. To elucidate potential pathomechanisms, we included a treatment group receiving thyroxine T4 and 17β‐estradiol. Because clinically osteoporotic fractures often occur in the bone metaphysis, HPD‐sheep and healthy controls received an osteotomy in the distal femoral condyle. Half of the HPD‐sheep were systemically treated with thyroxine T4 and 17β‐estradiol during the healing period. Fracture healing was evaluated after 8 weeks using pQCT, µCT, and histomorphometrical analysis. Bone mineral density (BMD) and bone volume/total volume (BV/TV) were considerably reduced by 30% and 36%, respectively, in the osteotomy gap of the HPD‐sheep compared to healthy sheep. Histomorphometry also revealed a decreased amount of newly formed bone (−29%) and some remaining cartilage in the HPD‐group, suggesting that HPD disturbed fracture healing. Thyroxine T4 and 17β‐estradiol substitution considerably improved bone healing in the HPD‐sheep. Our results indicate that fracture healing requires central regulation and that thyroxine T4 and 17β‐estradiol contribute to the complex pathomechanisms of delayed metaphyseal bone healing in HPD‐sheep.

A Novel ANO5 Mutation Causing Gnathodiaphyseal Dysplasia with High Bone Turnover Osteosclerosis

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome that involves an osteopetrosis‐like sclerosis of the long bones and fibrous dysplasia–like cemento‐osseous lesions of the jawbone. Although the genetic analysis of the respective patients has revealed mutations in the ANO5 gene as an underlying cause, there is still no established consensus regarding the bone status of GDD patients. We report a new case of GDD in a 13‐year‐old boy with recurrent diaphyseal fractures of the femur, in whom we identified a novel de novo missense mutation in the ANO5 gene, causing a p.Ser500Phe substitution at the protein level. After confirming the presence of GDD‐characteristic abnormalities within the jaw bones, we focused on a full osteologic assessment using dual‐energy X‐ray absorptiometry (DXA), high‐resolution peripheral quantitative computed tomography (HR‐pQCT), and serum analyses. We thereby identified increased trabecular bone mass accompanied by elevated serum markers of bone formation and bone resorption. The high turnover bone pathology was further confirmed through the analysis of an iliac crest biopsy, where osteoblast and osteoclast indices were remarkably increased. Taken together, our findings provide evidence for a critical and generalized role of anoctamin‐5 (the protein encoded by the ANO5 gene) in skeletal biology. As it is reasonable to speculate that modifying the function of anoctamin‐5 might be useful for therapeutically activating bone remodeling, it is now required to analyze its function at a molecular level, for instance in mouse models.

Can we induce osteoporosis in animals comparable to the human situation

Osteoporosis is a chronic systemic bone disease of growing relevance due to the on-going demographic change. Since the underlying regulatory mechanisms of this critical illness are still not fully understood and treatment options are not satisfactorily resolved, there is still a great need for osteoporosis research in general and animal models in particular. Ovariectomized rodents are standard animal models for postmenopausal osteoporosis and highly attractive due to the possibility to specifically modify their genetic background. However, some aspects can only be addressed in large animal models; such as metaphyseal fracture healing and advancement of orthopedic implants. Among other large animal models sheep in particular have been proven invaluable for osteoporosis research in this context. In conclusion, today we are able to influence the bone metabolism in animals causing a more or less pronounced systemic bone loss and structural deterioration comparable to the situation found in patients suffering from osteoporosis. However, there is no perfect model for osteoporosis, but a variety of models appropriate for answering specific questions. Though, the appropriateness of an animal model is not only defined in regard to the similarity to human physiology and the disease itself, but also in regard to acquisition, housing requirements, handling, costs, and particularly ethical concerns and animal welfare.

Matrix-Associated Autologous Chondrocyte Implantation A Clinical Follow-Up at 15 Years

Introduction A prospective clinical investigation was carried out in order to clarify whether Matrix-associated autologous chondrocyte implantation (MACI) results in clinical improvement at long-term follow-up. Hypothesis MACI will result in clinical improvement at long-term follow-up. Study Design Case series; level of evidence, 4. Methods Thirty-eight patients were treated with MACI. These patients were evaluated for up to a mean of 16 years (range 15-17 years) after the intervention. Three different scores (Lysholm-Gilquist score, International Cartilage Repair Society score, and Tegner score) formed the basis of this study. Overall, we were able to obtain valid preoperative and postoperative results from 18 (47%) of 38 patients. In 1 patient, both knees were treated. In 4 patients, an arthroplasty was implanted over the course of time; thus they were excluded from this case series. In conclusion, follow-up of 15 knees was performed in the recent series. Results In subjective rating, 12 out of 14 patients (86%) rated the function of their knee as much better or better than before the index procedure. All numerical outcome scores showed significant improvement compared to the preoperative value (preoperative/postoperative at 5 years/postoperative at 15 years): Lysholm score 59.6 (±24.6)/78.6 (±21.5)/82.7 (±11.3), International Knee Documentation Committee score 50.6 (±22.7)/64.7 (±21.6)/69.7 (±18.7), Tegner score 3.0 (±2.2)/3.6 (±1.5)/5.2 (±1.7). Conclusion The significantly improved results on 3 scores after 15 years suggest that MACI represents a suitable treatment of local cartilage defects in the knee.

Metaphyseal bone formation induced by a new injectable β-TCP-based bone substitute: a controlled study in rabbits.

Purpose Adequate filling of bone defects still poses a challenge in every day clinical work. As many bone defects are irregularly shaped the need for appropriate scaffolds reaching the complete defect surface are great. The purpose of this pre-clinical pilot study was to investigate the handling, biocompatibility, biodegradation and osteoconductivity of a new pasty bone substitute (pure phase β-TCP, hyaluronic acid, methylcellulose) in bone tissue. Methods In an unilateral tibial defect model the peri-implant and bone tissue response to the new pasty bone substitute was tested in New Zealand white rabbits for up to 24 weeks compared to empty controls. Analysis included HR-pQCT scans, histomorphometric evaluation and quantification of vascularization of un-decalcified histological slices. Results After 1 week the experimental group presented significantly higher new bone volume fraction (p = 0.021) primarily consisting of immature bone matrix and higher vessel density compared to controls (p = 0.013). After 4 weeks bone formation was not significantly different to controls but was distributed more evenly throughout the defect. Bone matrix was now mineralized and trabeculae were thicker than in controls (p = 0.002) indicating faster intramedullary bone maturation. Controls presented extensive periosteal bone formation, major fibrous tissue influx and high vascularization. After 12 and 24 weeks there was no new bone detectable. There were no severe signs of inflammation at all time points. Conclusion The substitute showed an early induction of bone formation. It promoted accelerated intramedullary bone repair and maturation and prevented periosteal bone formation indicating its potential use for reconstructive surgery of bone defects.

Targeting the lateral but not the third ventricle induces bone loss in ewe: an experimental approach to generate an improved large animal model of osteoporosis.

BACKGROUND: Osteoporosis is a chronic disease characterized by bone loss and increased skeletal fragility. Large animal models are required for preclinical testing of new therapeutic approaches. We have recently demonstrated that continuous intracerebroventricular (ICV) application of leptin into the lateral ventricle (LV) induces bone loss in ewe. On the basis of these findings, we reasoned that the third ventricle (TV) is an even better target because of its closer location to the hypothalamus that mediates leptin effects on bone. METHODS: Corriedale sheep were randomly mixed to four groups of four ewe each: control entire (control), ovarectomy plus ICV application of cerebrospinal fluid (OVX), OVX plus ICV application of leptin into the LV (leptin-LV); and ICV application of leptin into the TV (leptin-TV). After 3 months, histomorphometric characterization and bone turnover parameters were analyzed. RESULTS: Highly significant loss of trabecular bone was observed only in leptin-LV group. Increased osteoclast indices and urinary cross-lap excretion were observed in OVX and leptin-TV group. In contrast, serum parameters of osteoblast activity were only significantly decreased in leptin-LV group. Autopsy of ewe brain showed fibrosis around the stainless steel cannula in leptin-TV group. CONCLUSIONS: ICV application of leptin into the LV strongly reduces bone formation and leads to a highly significant trabecular bone loss in ewe. In contrast, ICV application of leptin into the TV is technically more demanding and results are unpredictable, because the required use of stainless steel cannula induces peri-implant fibrosis that might prevent leptin to enter the cerebrospinal fluid.

High bone turnover in mice carrying a pathogenic Notch2-mutation causing Hajdu-Cheney syndrome†

Hajdu‐Cheney syndrome (HCS) is a rare autosomal‐dominant disorder primarily characterized by acro‐osteolysis and early‐onset osteoporosis. Genetically, HCS is caused by nonsense or deletion mutations within exon 34 of the NOTCH2 gene, resulting in premature translational termination and production of C‐terminally truncated NOTCH2 proteins that are predicted to activate NOTCH2‐dependent signaling. To understand the role of Notch2 in bone remodeling, we developed a mouse model of HCS by introducing a pathogenic mutation (6272delT) into the murine Notch2 gene. By μCT and undecalcified histology, we observed generalized osteopenia in two independent mouse lines derived by injection of different targeted embryonic stem (ES) cell clones, yet acro‐osteolysis did not occur until the age of 52 weeks. Cellular and dynamic histomorphometry revealed a high bone turnover situation in Notch2+/HCS mice, since osteoblast and osteoclast indices were significantly increased compared with wild‐type littermates. Whereas ex vivo cultures failed to uncover cell‐autonomous gain‐of‐functions within the osteoclast or osteoblast lineage, an unbiased RNA sequencing approach identified Tnfsf11 and Il6 as Notch‐signaling target genes in bone marrow cells cultured under osteogenic conditions. Because we further observed that the high‐turnover pathology of Notch2+/HCS mice was fully normalized by alendronate treatment, our results demonstrate that mutational activation of Notch2 does not directly control osteoblast activity but favors a pro‐osteoclastic gene expression pattern, which in turn triggers high bone turnover.