Proposed diagnostic criteria for the diagnosis of hypophosphatasia in children and adolescents: results from the HPP International Working Group.

Hypophosphatasia (HPP) is a rare inborn error of metabolism that presents variably in both age of onset and severity. HPP is caused by pathogenic variants in the ALPL gene, resulting in low activity of tissue nonspecific alkaline phosphatase (TNSALP). Patients with HPP tend have a similar pattern of elevation of natural substrates that can be used to aid in diagnosis. No formal diagnostic guidelines currently exist for the diagnosis of this condition in children, adolescents, or adults. The International HPP Working Group is a comprised of a multidisciplinary team of experts from Europe and North America who have expertise in the diagnosis and management of patients with HPP. This group reviewed 93 papers through a Medline, Medline In-Process, and Embase search for the terms "HPP" and "hypophosphatasia" between 2005 and 2020 and that explicitly address either the diagnosis of HPP in children, clinical manifestations of HPP in children, or both. Two reviewers independently evaluated each full-text publication for eligibility and studies were included if they were narrative reviews or case series/reports that concerned diagnosis of pediatric HPP or included clinical aspects of patients diagnosed with HPP. This review focused on 15 initial clinical manifestations that were selected by a group of clinical experts.The highest agreement in included literature was for pathogenic or likely pathogenic ALPL variant, elevation of natural substrates, and early loss of primary teeth. The highest prevalence was similar, including these same three parameters and including decreased bone mineral density. Additional parameters had less agreement and were less prevalent. These were organized into three major and six minor criteria, with diagnosis of HPP being made when two major or one major and two minor criteria are present.

Interdisciplinary management of FGF23-related phosphate wasting syndromes: a Consensus Statement on the evaluation, diagnosis and care of patients with X-linked hypophosphataemia.

X-linked hypophosphataemia (XLH) is the most frequent cause of hypophosphataemia-associated rickets of genetic origin and is associated with high levels of the phosphaturic hormone fibroblast growth factor 23 (FGF23). In addition to rickets and osteomalacia, patients with XLH have a heavy disease burden with enthesopathies, osteoarthritis, pseudofractures and dental complications, all of which contribute to reduced quality of life. This Consensus Statement presents the outcomes of a working group of the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases, and provides robust clinical evidence on management in XLH, with an emphasis on patients' experiences and needs. During growth, conventional treatment with phosphate supplements and active vitamin D metabolites (such as calcitriol) improves growth, ameliorates leg deformities and dental manifestations, and reduces pain. The continuation of conventional treatment in symptom-free adults is still debated. A novel therapeutic approach is the monoclonal anti-FGF23 antibody burosumab. Although promising, further studies are required to clarify its long-term efficacy, particularly in adults. Given the diversity of symptoms and complications, an interdisciplinary approach to management is of paramount importance. The focus of treatment should be not only on the physical manifestations and challenges associated with XLH and other FGF23-mediated hypophosphataemia syndromes, but also on the major psychological and social impact of the disease.

Efficacy of Zoledronic Acid in the Treatment of Nonmalignant Painful Bone Marrow Lesions: A Triple-Blind, Randomized, Placebo-Controlled Phase III Clinical Trial (ZoMARS).

Bone marrow lesions (BML) represent areas of deteriorated bone structure and metabolism characterized by pronounced water-equivalent signaling within the trabecular bone on magnetic resonance imaging (MRI). BML are associated with repair mechanisms subsequent to various clinical conditions associated with inflammatory and non-inflammatory injury to the bone. There is no approved treatment for this condition. Bisphosphonates are known to improve bone stability in osteoporosis and other bone disorders and have been used off-label to treat BML. A randomized, triple-blind, placebo-controlled phase III trial was conducted to assess efficacy and safety of single-dose zoledronic acid (ZOL) 5 mg iv with vitamin D 1000 IU/d as opposed to placebo with vitamin D 1000 IU/d in 48 patients (randomized 2:1) with BML. Primary efficacy endpoint was reduction of edema volume 6 weeks after treatment as assessed by MRI. After treatment, mean BML volume decreased by 64.53% (±41.92%) in patients receiving zoledronic acid and increased by 14.43% (±150.46%) in the placebo group (p = 0.007). A decrease in BML volume was observed in 76.5% of patients receiving ZOL and in 50% of the patients receiving placebo. Pain level (visual analogue scale [VAS]) and all categories of the pain disability index (PDI) improved with ZOL versus placebo after 6 weeks but reconciled after 6 additional weeks of follow-up. Six serious adverse events occurred in 5 patients, none of which were classified as related to the study drug. No cases of osteonecrosis or fractures occurred. Therefore, single-dose zoledronic acid 5 mg iv together with vitamin D may enhance resolution of bone marrow lesions over 6 weeks along with reduction of pain compared with vitamin D supplementation only. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Percutaneous administration of allogeneic bone-forming cells for the treatment of delayed unions of fractures: a pilot study.

BACKGROUND: Overall, 5-10% of fractures result in delayed unions or non-unions, causing major disabilities and a huge socioeconomic burden. Since rescue surgery with autologous bone grafts can cause additional challenges, alternative treatment options have been developed to stimulate a deficient healing process. This study assessed the technical feasibility, safety and preliminary efficacy of local percutaneous implantation of allogeneic bone-forming cells in delayed unions of long bone fractures. METHODS: In this phase I/IIA open-label pilot trial, 22 adult patients with non-infected delayed unions of long bone fractures, which failed to consolidate after 3 to 7 months, received a percutaneous implantation of allogeneic bone-forming cells derived from bone marrow mesenchymal stem cells (ALLOB; Bone Therapeutics) into the fracture site (50 × 106 to 100 × 106 cells). Patients were monitored for adverse events and need for rescue surgery for 30 months. Fracture healing was monitored by Tomographic Union Score (TUS) and modified Radiographic Union Score. The health status was evaluated using the Global Disease Evaluation (GDE) score and pain at palpation using a visual analogue scale. The presence of reactive anti-human leukocyte antigen (HLA) antibodies was evaluated. RESULTS: During the 6-month follow-up, three serious treatment-emergent adverse events were reported in two patients, of which two were considered as possibly treatment-related. None of the 21 patients in the per-protocol efficacy population needed rescue surgery within 6 months, but 2/21 (9.5%) patients had rescue surgery within 30 months post-treatment. At 6 months post-treatment, an improvement of at least 2 points in TUS was reached in 76.2% of patients, the GDE score improved by a mean of 48%, and pain at palpation at the fracture site was reduced by an average of 61% compared to baseline. The proportion of blood samples containing donor-specific anti-HLA antibodies increased from 8/22 (36.4%) before treatment to 13/22 (59.1%) at 6 months post-treatment, but no treatment-mediated allogeneic immune reactions were observed. CONCLUSION: This pilot study showed that the percutaneous implantation of allogeneic bone-forming cells was technically feasible and well tolerated in patients with delayed unions of long bone fractures. Preliminary efficacy evidence is supporting the further development of this treatment. TRIAL REGISTRATION: NCT02020590 . Registered on 25 December 2013. ALLOB-DU1, A pilot Phase I/IIa, multicentre, open proof-of-concept study on the efficacy and safetyof allogeneic osteoblastic cells (ALLOB®) implantation in non-infected delayed-union fractures.

Challenges in the management of tumor-induced osteomalacia (TIO).

Tumor-induced osteomalacia (TIO), also known as oncogenic osteomalacia, is a rare acquired paraneoplastic disease, which is challenging to diagnose and treat. TIO is characterized by hypophosphatemia resulting from excess levels of tumor-secreted fibroblast growth factor 23 (FGF23), one of the key physiological regulators of phosphate metabolism. Elevated FGF23 results in renal phosphate wasting and compromised vitamin D activation, ultimately resulting in osteomalacia. Patients typically present with progressive and non-specific symptoms, including bone pain, multiple pathological fractures, and progressive muscle weakness. Diagnosis is often delayed or missed due to the non-specific nature of complaints and lack of disease awareness. Additionally, the disease-causing tumour is often difficult to detect and localize because they are often small, lack localizing symptoms and signs, and dwell in widely variable anatomical locations. Measuring serum/urine phosphate should be an inherent diagnostic component when assessing otherwise unexplained osteomalacia, fractures and weakness. In cases of hypophosphatemia with inappropriate (sustained) phosphaturia and inappropriately normal or frankly low 1,25-dihydroxy vitamin D, differentiation of the potential causes of renal phosphate wasting should include measurement of FGF23, and TIO should be considered. While patients experience severe disability without treatment, complete excision of the tumour is typically curative and results in a dramatic reversal of symptoms. Two additional key current unmet needs in optimizing TIO management are: (1 and 2) the considerable delay in diagnosis and consequent delay between the onset of symptoms and surgical resection; and (2) alternative management. These may be addressed by raising awareness of TIO, and taking into consideration the accessibility and variability of different healthcare infrastructures. By recognizing the challenges associated with the diagnosis and treatment of TIO and by applying a stepwise approach with clear clinical practice guidelines, patient care and outcomes will be improved in the future.

Pharmacodynamics of asfotase alfa in adults with pediatric-onset hypophosphatasia.

BACKGROUND: Hypophosphatasia (HPP) is the rare, inherited, metabolic bone disease characterized by low activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) leading to excess extracellular inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP). Asfotase alfa is the human recombinant enzyme-replacement therapy that replaces deficient TNSALP. However, there is limited information concerning the appropriate dose of asfotase alfa for adult patients with pediatric-onset HPP. Thus, we evaluated the pharmacodynamics and safety/tolerability of different doses of asfotase alfa in such patients. METHODS: This 13-week, Phase 2a, open-label study enrolled adults (aged ≥18 years) with pediatric-onset HPP. They were randomized 1:1:1 to receive a single subcutaneous dose of asfotase alfa (0.5, 2.0, or 3.0 mg/kg) at Week 1, then 3 times per week (ie, 1.5, 6.0, or 9.0 mg/kg/wk) starting at Week 3 for 7 weeks. Key outcome measures included change from Baseline to before the third dose during Week 9 (trough) in plasma PPi (primary outcome measure) and PLP (secondary outcome measure). RESULTS: Twenty-seven adults received asfotase alfa 0.5 (n = 8), 2.0 (n = 10), and 3.0 (n = 9) mg/kg; all completed the study. Median (range) age was 45 (18-77) years; most patients were white (96%) and female (59%). Median plasma PPi and PLP concentrations decreased from Baseline to Week 9 in all 3 cohorts. Differences in least squares mean (LSM) changes in PPi were significant with 2.0 mg/kg (p = 0.0008) and 3.0 mg/kg (p < 0.0001) vs. 0.5 mg/kg. Differences in LSM changes in PLP were also significant for 2.0 mg/kg (p = 0.0239) and 3.0 mg/kg (p = 0.0128) vs. 0.5 mg/kg. Injection site reactions were the most frequent treatment-emergent adverse event (78%), showing increasing frequency with increasing dose. CONCLUSIONS: Adults with pediatric-onset HPP receiving asfotase alfa at 6.0 mg/kg/wk (the recommended dose) or 9.0 mg/kg/wk had greater reductions in circulating PPi and PLP concentrations compared with a lower dose of 1.5 mg/kg/wk. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT02797821.

Impact of whole-body vibration exercise on physical performance and bone turnover in patients with monoclonal gammopathy of undetermined significance.

OBJECTIVE: Monoclonal Gammopathy of Undetermined Significance (MGUS) is a risk factor for reduced physical performance, osteoporosis, and fractures due to compromised musculoskeletal metabolism. In this condition it is unknown whether whole-body vibration (WBV) exercise favorably alters physical performance and bone metabolism. METHODS: To evaluate the effect of three-months WBV exercise (30 min; 2x/week) including an optional three-month extension on physical performance, bone metabolism and bone mineral density. Endpoints included functional assessments, bone turnover markers and bone mineral density assessed by peripheral quantitative computed tomography of the tibia. RESULTS: Fifteen MGUS patients (median age 62.0, nine female) completed the first three months of which ten completed the three-month extension. Measures of physical functioning including chair rise test, timed up and go and 6-minute walk test improved (p = 0.007; p = 0.009; p = 0.005) after three and six months of WBV exercise. Total tibial bone mineral density remained unaltered (p > 0.05). WBV exercise tended to increase levels of sclerostin (p = 0.093) with a transient increase in osteoclast resorption markers (N-terminal telopeptide of collagen type 1, tartrate resistant acid phosphatase 5b) after three months while Dickkopf-1 (p = 0.093), procollagen I N-terminal propeptide (p = 0.074) and total alkaline phosphatase (p = 0.016) appeared to decline. No exercise-related adverse events were reported. CONCLUSION: WBV exercise in MGUS patients improves indicators of physical performance. Observed trends in bone turnover markers and changes in distal tibial bone mineral density may indicate a regulatory effect of WBV exercise on bone metabolism and warrants further evaluation by large scale studies.

Penetration of topical diclofenac into synovial tissue and fluid of osteoarthritic knees: a multicenter, randomized, placebo-controlled, pharmacokinetic study.

BACKGROUND: Topical diclofenac, a nonsteroidal anti-inflammatory drug, has proven efficacy and safety in the management of osteoarthritis pain. We investigated penetration of topical diclofenac into knee synovial tissue and fluid (primary objective) and evaluated relative exposure in the knee versus plasma (secondary objective). METHODS: In this phase I, double-blind, multicenter study, patients scheduled for arthroplasty for end-stage knee osteoarthritis were randomly assigned 2:1 to 4 g diclofenac diethylamine 2.32% w/w gel (92.8 mg diclofenac diethylamine, equivalent to 74.4 mg diclofenac, per application) or placebo gel, applied to the affected knee by a trained nurse/designee every 12 h for 7 days before surgery. Diclofenac concentrations were measured in synovial tissue, synovial fluid and plasma from samples obtained during surgery ⩾12 h after last application. Treatment-emergent adverse events (TEAEs) were evaluated. RESULTS: Evaluable synovial tissue or fluid samples were obtained from 45 (diclofenac n = 29; placebo n = 16) of 47 patients. All diclofenac-treated participants had measurable diclofenac concentrations in synovial tissue [geometric mean 1.57 (95% confidence interval (CI) 1.12, 2.20) ng/g] and fluid [geometric mean 2.27 (95% CI 1.87, 2.76) ng/ml] ⩾12 h after the last dose. Geometric mean (95% CI) ratio of diclofenac in synovial tissue:plasma was 0.32 (0.23, 0.45) and in synovial fluid:plasma was 0.46 (0.40, 0.54). TEAE rates were similar for diclofenac (55.2%) and placebo (58.8%); none were treatment related. CONCLUSIONS: Topical diclofenac diethylamine 2.32% w/w gel penetrated into the osteoarthritic knee after repeated application and remained detectable in synovial tissue and fluid at the end of the final 12 h dosing cycle.

Pediatric hypophosphatasia: lessons learned from a retrospective single-center chart review of 50 children.

BACKGROUND: Hypophosphatasia (HPP) is a rare, inherited metabolic disorder caused by loss-of-function mutations in the ALPL gene that encodes the tissue-nonspecific alkaline phosphatase TNAP (ORPHA 436). Its clinical presentation is highly heterogeneous with a remarkably wide-ranging severity. HPP affects patients of all ages. In children HPP-related musculoskeletal symptoms may mimic rheumatologic conditions and diagnosis is often difficult and delayed. To improve the understanding of HPP in children and in order to shorten the diagnostic time span in the future we studied the natural history of the disease in our large cohort of pediatric patients. This single centre retrospective chart review included longitudinal data from 50 patients with HPP diagnosed and followed at the University Children's Hospital Wuerzburg, Germany over the last 25 years. RESULTS: The cohort comprises 4 (8%) perinatal, 17 (34%) infantile and 29 (58%) childhood onset HPP patients. Two patients were deceased at the time of data collection. Diagnosis was based on available characteristic clinical symptoms (in 88%), low alkaline phosphatase (AP) activity (in 96%), accumulating substrates of AP (in 58%) and X-ray findings (in 48%). Genetic analysis was performed in 48 patients (31 compound heterozygous, 15 heterozygous, 2 homozygous mutations per patient), allowing investigations on genotype-phenotype correlations. Based on anamnestic data, median age at first clinical symptoms was 3.5 months (min. 0, max. 107), while median time to diagnosis was 13 months (min. 0, max. 103). Common symptoms included: impairment of motor skills (78%), impairment of mineralization (72%), premature loss of teeth (64%), musculoskeletal pain and craniosynostosis (each 64%) and failure to thrive (62%). Up to now 20 patients started medical treatment with Asfotase alfa. CONCLUSIONS: Reported findings support the clinical perception of HPP being a chronic multi-systemic disease with often delayed diagnosis. Our natural history information provides detailed insights into the prevalence of different symptoms, which can help to improve and shorten diagnostics and thereby lead to an optimised medical care, especially with promising therapeutic options such as enzyme-replacement-therapy with Asfotase alfa in mind.

Physical contact between mesenchymal stem cells and endothelial precursors induces distinct signatures with relevance to the very early phase of regeneration.

Multipotent adult stem cells/precursor cells, especially of the mesenchymal and endothelial lineage, may have great potential for bone tissue engineering. Although their potential is highly recognized, not much is known about the underlying molecular mechanisms that initiate the regeneration process, connect osteogenesis, and angiogenesis and, finally, orchestrate renewal of bone tissue. Our study addressed these questions by generating two in vitro cell culture models to examine the changes in the global gene expression patterns of endothelial precursor cells and mesenchymal stem cells after 24 hours of either humoral (conditioned medium) or direct cell-cell interaction (co-culture). Endothelial precursor cells were isolated from human buffy coat and mesenchymal stem cells from the bone marrow of the femoral head. The comparison of the treated and control cells by microarray analyses revealed in total more than 1500 regulated genes, which were analyzed for their affiliation to angiogenesis and osteogenesis. Expression array analyses at the RNA and protein level revealed data with respect to regulated genes, pathways and targets that may represent a valid basis for further dissection of the systems biology of regeneration processes. It may also be helpful for the reconstitution of the natural composition of a regenerative microenvironment when targeting tissue regeneration both in vitro and in situ.

Cellular pharmacodynamic effects of Pycnogenol® in patients with severe osteoarthritis: a randomized controlled pilot study.

BACKGROUND: The standardized maritime pine bark extract (Pycnogenol®) has previously shown symptom alleviating effects in patients suffering from moderate forms of knee osteoarthritis (OA). The cellular mechanisms for this positive impact are so far unknown. The purpose of the present randomized pilot controlled study was to span the knowledge gap between the reported clinical effects of Pycnogenol® and its in vivo mechanism of action in OA patients. METHODS: Thirty three patients with severe OA scheduled for a knee arthroplasty either received 100 mg of Pycnogenol® twice daily or no treatment (control group) three weeks before surgery. Cartilage, synovial fluid and serum samples were collected during surgical intervention. Relative gene expression of cartilage homeostasis markers were analyzed in the patients' chondrocytes. Inflammatory and cartilage metabolism mediators were investigated in serum and synovial fluid samples. RESULTS: The oral intake of Pycnogenol® downregulated the gene expression of various cartilage degradation markers in the patients' chondrocytes, the decrease of MMP3, MMP13 and the pro-inflammatory cytokine IL1B were statistically significant (p ≤ 0.05). Additionally, protein concentrations of ADAMTS-5 in serum were reduced significantly (p ≤ 0.05) after three weeks intake of the pine bark extract. CONCLUSIONS: This is the first report about positive cellular effects of a dietary supplement on key catabolic and inflammatory markers in patients with severe OA. The results provide a rational basis for understanding previously reported clinical effects of Pycnogenol® on symptom scores of patients suffering from OA. TRIAL REGISTRATION: ISRCTN10754119 . Retrospectively registered 08/10/2015.

Epidermal growth factor as a mechanosensitizer in human bone marrow stromal cells.

Epidermal growth factors (EGFs) e.g. EGF, heparin-binding EGF and transforming growth factor alpha and their receptors e.g. EGFR and ErbB2 control proinflammatory signaling and modulate proliferation in bone marrow stromal cells (BMSC). Interleukin-6 and interleukin-8 are EGF targets and participate in the inflammatory phase of bone regeneration via non-canonical wnt signaling. BMSC differentiation is also influenced by mechanical strain-related activation of ERK1/2 and AP-1, but the role of EGFR signaling in mechanotransduction is unclear. We investigated the effects of EGFR signaling in telomerase-immortalized BMSC, transfected with a luciferase reporter, comprising a mechanoresponsive AP1 element, using ligands, neutralizing antibodies and EGFR inhibitors on mechanotransduction and we found that EGF via EGFR increased the response to mechanical strain. Results were confirmed by qPCR analysis of mechanoresponsive genes. EGF-responsive interleukin-6 and interleukin-8 were synergistically enhanced by EGF stimulation and mechanical strain. We show here in immortalized and primary BMSC that EGFR signaling enhances mechanotransduction, indicating that the EGF system is a mechanosensitizer in BMSC. Alterations in mechanosensitivity and -adaptation are contributors to age-related diseases like osteoporosis and the identification of a suitable mechanosensitizer could be beneficial. The role of the synergism of these signaling cascades in physiology and disease remains to be unraveled.

Distribution of Constituents and Metabolites of Maritime Pine Bark Extract (Pycnogenol®) into Serum, Blood Cells, and Synovial Fluid of Patients with Severe Osteoarthritis: A Randomized Controlled Trial.

The present randomized controlled study aimed to investigate the in vivo distribution of constituents or metabolites of the standardized maritime pine bark extract Pycnogenol®. Thirty-three patients with severe osteoarthritis scheduled for a knee arthroplasty were randomized to receive either 200 mg per day Pycnogenol® (P+) or no treatment (Co) over three weeks before surgery. Serum, blood cells, and synovial fluid samples were analyzed using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization (LC-ESI/MS/MS). Considerable interindividual differences were observed indicating pronounced variability of the polyphenol pharmacokinetics. Notably, the highest polyphenol concentrations were not detected in serum. Catechin and taxifolin primarily resided within the blood cells while the microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone, ferulic, and caffeic acid were mainly present in synovial fluid samples. Taxifolin was detected in serum and synovial fluid exclusively in the P+ group. Likewise, no ferulic acid was found in serum samples of the Co group. Calculating ratios of analyte distribution in individual patients revealed a simultaneous presence of some polyphenols in serum, blood cells, and/or synovial fluid only in the P+ group. This is the first evidence that polyphenols distribute into the synovial fluid of patients with osteoarthritis which supports rationalizing the results of clinical efficacy studies.

High Prevalence of Vitamin D Deficiency in Patients With Bone Marrow Edema Syndrome of the Foot and Ankle.

BACKGROUND: Bone marrow edema syndrome (BMOS) is a phenomenon primarily affecting the lower extremity. It is characterized by a sudden onset of pain and an ill-defined osseous hyperintense signal in magnetic resonance imaging. The main cause of BMOS is still largely unknown. Its pathophysiology is presumably multifactorial and it has recently been demonstrated that it usually involves an increase in bone turnover and alterations within the bone microenvironment. Vitamin D plays a pivotal role in maintaining a healthy and well-balanced bone microenvironment. However, to date only limited information has been reported on vitamin D status in patients with BMOS. Moreover, it is still uncertain whether hypovitaminosis D is associated with the etiology and course of the disease. For this reason, the aim of this study was to determine serum vitamin D levels (25(OH)D) of patients diagnosed with BMOS of the foot and ankle. METHODS: Patients were identified and laboratory results collected by retrospective review of the medical records between year 2011 and 2015. Diagnosis was based on clinical examination, the existence of prolonged foot pain, the presence of abnormal bone marrow signal intensity in T1- and T2-weighted magnetic resonance imaging, and the patient's medical history. All patients who demonstrated other concomitant diagnoses were excluded from the study. RESULTS: Overall, 31 patients were affected by BMOS with a mean age of 44.4 (range, 18-76) years. Notably, 84% of patients (26/31) had low vitamin D levels with a mean 25(OH)D level of 19.03 ng/mL. Specifically, 61% of patients (19/31) were vitamin D deficient, 23% (7/31) vitamin D insufficient, and only 5 patients (16%) had sufficient vitamin D levels. Statistical analysis showed no significant difference comparing vitamin D levels with patient age, sex, and time of diagnosis. Moreover, there was no correlation between vitamin D status and the number of bony foci or location of BMOS. CONCLUSION: We found a widespread rate of vitamin D deficiency in patients presenting with BMOS of the foot and ankle. Comparing these data to the vitamin D status of the general population in Germany and to patients living in comparable latitudes, this raises the possibility that BMOS might be associated with low vitamin D status. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Dissection of mechanoresponse elements in promoter sites of the mechanoresponsive CYR61 gene.

Mechanotransduction is important for mesenchymal regeneration and differentiation. Exaggerated high or very low impact yields pathological outcome resulting in fracture or tissue atrophy. Pathological strain in animal models was described but tools to dissect the respective stimuli and downstream pathways are limited. We expand the analytical tools to describe DNA strain response elements in a reporter gene approach. Deletion constructs of the human cysteine-rich protein 61 (CYR61) promoter were cloned into luciferase vectors and stably transfected into human telomerase-immortalised mesenchymal stem cells (hMSC-TERT). Cells were mechanically stimulated with variable frequencies, amplitudes and durations. Promoter activity was determined as well as CYR61 mRNA and protein expression. In silico promoter analysis identified putative transcription factor binding sites, one of which was a cAMP response element, verified by electrophoretic mobility shift assay. We demonstrate for the first time that the activity of promoter regions is inhibited in low, but stimulated in high frequency stimulations. We conclude that by varying conditions of mechanical strain it is possible to characterize stimulatory versus inhibitory strain on cellular levels. Our work may be helpful in future studies to dissect the molecular pathways of physiological versus pathological strain and may have implications for clinical exercise based treatment strategies.

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 ∼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 small scale cell culture system to analyze mechanobiology using reporter gene constructs and polyurethane dishes.

Mechanical forces are translated into biochemical signals and contribute to cell differentiation and phenotype maintenance. Mesenchymal stem cells and their tissue-specific offspring, as osteoblasts and chondrocytes, cells of cardiovascular tissues and lung cells are sensitive to mechanical loading but molecules and mechanisms involved have to be unraveled. It is well established that cellular mechanotransduction is mediated e.g. by activation of the transcription factor SP1 and by kinase signaling cascades resulting in the activation of the AP1 complex. To investigate cellular mechanisms involved in mechanotransduction and to analyze substances, which modulate cellular mechanosensitivity reporter gene constructs, which can be transfected into cells of interest might be helpful. Suitable small-scale bioreactor systems and mechanosensitive reporter gene constructs are lacking. To analyze the molecular mechanisms of mechanotransduction and its crosstalk with biochemically induced signal transduction, AP1 and SP1 luciferase reporter gene constructs were cloned and transfected into various cell lines and primary cells. A newly developed bioreactor and small-scale 24-well polyurethane dishes were used to apply cyclic stretching to the transfected cells. 1 Hz cyclic stretching for 30 min in this system resulted in a significant stimulation of AP1 and SP1 mediated luciferase activity compared to unstimulated cells. In summary we describe a small-scale cell culture/bioreactor system capable of analyzing subcellular crosstalk mechanisms in mechanotransduction, mechanosensitivity of primary cells and of screening the activity of putative mechanosensitizers as new targets, e.g. for the treatment of bone loss caused by both disuse and signal transduction related alterations of mechanotransduction.

Estrogen receptor and Wnt signaling interact to regulate early gene expression in response to mechanical strain in osteoblastic cells.

Bone mass homeostasis is regulated by an interaction of various factors, including growth factors, systemic hormones and mechanical loading. Two signal transduction pathways, the estrogen receptor (ER) and the Wnt/beta-catenin signal transduction pathway, have been shown to have an important role in regulating osteoblast and osteoclast function and to be involved in mechanotransduction. Therefore, dysfunction of these pathways can lead to osteoporotic bone loss. However, less is known about the modulation of gene expression by the interaction of these pathways in response to mechanical strain. We performed in vitro stretch experiments using osteoblastic MC3T3-E1 cells to study the effect of both pathways and mechanical strain on the expression of cyclooxygenase-2 (Cox-2), which is involved in the synthesis of prostaglandins, modulators of bone formation and resorption. Using specific agonists and antagonists, we demonstrated a regulation by an interaction of these pathways in mechantransduction. Estradiol (E2) had a sensitizing effect on mechanically induced Cox-2 expression, which seemed to be ligand-specific as it could be abolished using the antiestrogen ICI182,780. However, mechanical strain in the presence of Wnt signaling activators diminished both the E2 sensitizing effect and the stimulatory effect of Wnt signaling in the absence of strain. This interaction might be one regulatory mechanism by which mechanical loading exerts its role in bone mass homeostasis.

BMP12 and BMP13 gene transfer induce ligamentogenic differentiation in mesenchymal progenitor and anterior cruciate ligament cells.

BACKGROUND AIMS: To date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts. METHODS: We describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively. RESULTS: Transgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin. CONCLUSIONS: It appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.

Pulse treatment with zoledronic acid causes sustained commitment of bone marrow derived mesenchymal stem cells for osteogenic differentiation.

The aminobisphosphonate zoledronic acid (ZA) is a bone seeking specific inhibitor of protein farnesylation and geranylgeranylation, which causes inhibition of osteoclast function and apoptosis. It is widely used as an osteoclast targeted antiresorptive treatment of metastatic bone disease, Paget's disease and osteoporosis. Mesenchymal stem cells (MSC) and osteoblast precursors can also be targets of bisphosphonates, but the clinical relevance of these effects is under debate. We show here that ZA in vitro causes inhibition of proliferation and induction of apoptosis in hMSC, when applied in concentrations of 20 and 50 microM for more than 24 h which can be rescued by treatment with 10 microM geranylgeranyl pyrophosphate (GGPP). However, pulse stimulation for 3 and 6 h with these concentrations and subsequent culture for up to 2 weeks under osteogenic conditions exerts sustained regulation of osteogenic marker genes in hMSC. The effect on gene regulation translates into marked enhancement of mineralization, as shown by alizarin red and alkaline phosphatase staining after 4 weeks of osteogenic culture. ZA, when applied as a pulse stimulus, might therefore also stimulate osteogenic differentiation in vivo, since muM plasma concentrations can be achieved by intravenous application of 5 mg in patients. These data set the stage for the future dissection of the effects of ZA and other aminobisphosphonates on cells beyond osteoclasts, with respect to cell differentiation in benign metabolic and to antitumor efficacy in metastatic bone diseases, as well as adverse events due to putative substance accumulation in bone during long-term treatment.