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Anna-Karin Gunnarsson,1 Lena Gunningberg,2 Sune Larsson,1 Kenneth B Jonsson1 1Institution of Surgical Sciences, Uppsala University, Uppsala, Sweden; 2Institution of Public Health and Caring Sciences, Uppsala University, Uppsala, Sweden Background: Urinary tract infection (UTI) is a common complication among patients with hip fractures. Receiving an indwelling urinary catheter is a risk factor for developing UTIs. Treatment of symptomatic UTIs with antibiotics is expensive and can result in the development of antimicrobial resistance. Cranberries are thought to prevent UTI. There is no previous research on this potential effect in patients with hip fracture who receive urinary catheters. Aim: The aim of this study is to investigate whether intake of cranberry juice concentrate preoperatively decreases the incidence of postoperative UTIs in hip fracture patients that received a urinary catheter. Design: This study employed a randomized, placebo-controlled double-blind trial. Method: Female patients, aged 60 years and older, with hip fracture (n=227) were randomized to receive cranberry or placebo capsules daily, from admission, until 5 days postoperatively. Urine cultures were obtained at admission, 5 and 14 days postoperatively. In addition, Euro Qual five Dimensions assessments were performed and patients were screened for UTI symptoms. Result: In the intention-to-treat analysis, there was no difference between the groups in the proportion of patients with hospital-acquired postoperative positive urine cultures at any time point. When limiting the analysis to patients that ingested at least 80% of the prescribed capsules, 13 of 33 (39%) in the placebo group and 13 of 47 (28%) in the cranberry group (P=0.270) had a positive urine culture at 5 days postoperatively. However, this difference was not statistically significant (P=0.270). Conclusion: Cranberry concentrate does not seem to effectively prevent UTIs in female patients with hip fracture and indwelling urinary catheter. Keywords: urinary tract infection, elderly, randomized controlled trial, hospital, preventive, urine culture

Background: Operative treatment is widely used for acute proximal hamstring avulsions, but its effectiveness compared with that of nonoperative treatment has not been shown in randomized trials., Methods: In this noninferiority trial at 10 centers in Sweden and Norway, we enrolled patients 30 to 70 years of age with a proximal hamstring avulsion in a randomized trial and a parallel observational cohort. Treatments were operative reinsertion of the tendons or nonoperative management. The primary end point was the Perth Hamstring Assessment Tool (PHAT) at 2 years of follow-up. Secondary outcomes included scores on the Lower Extremity Functional Scale (LEFS)., Results: A total of 119 patients were enrolled in the randomized trial and 97 patients in the observational cohort. In the per-protocol analysis of the randomized trial, the mean (±standard deviation) PHAT scores were 79.9±19.5 and 78.5±19.4 in the operative and nonoperative groups, respectively (PHAT scores range from 0 to 100, with higher scores indicating higher function). The prespecified noninferiority limit of 10 points was not crossed (mean difference, -1.2; 95% confidence interval [CI], -8.6 to 6.2; P=0.009 for noninferiority). Analyses of secondary outcomes, including a mean difference in the LEFS score of -1.6 (95% CI, -5.2 to 2.0), aligned with the primary outcome. The observed numbers of adverse events in the randomized trial were nine in the operative group versus three in the nonoperative group (odds ratio, 0.3; 95% CI, 0.1 to 1.2). In the analysis of the observational cohort, the mean PHAT score difference between the nonoperative and operative treatment groups was -2.6 (95% CI, -9.9 to 4.6)., Conclusions: In patients 30 to 70 years of age with proximal hamstring avulsions, nonoperative treatment was noninferior to operative treatment. (Funded by Afa Försäkring and others; ClinicalTrials.gov number, NCT03311997.).

Background: Proximal humerus fractures (PHFs) are common fractures, especially in older female patients. These fractures are commonly treated surgically, but the consensus on the best treatment is still lacking., Methods and Findings: The primary aim of this multicenter, randomized 3-arm superiority, open-label trial was to assess the results of nonoperative treatment and operative treatment either with locking plate (LP) or hemiarthroplasty (HA) of 3- and 4-part PHF with the primary outcome of Disabilities of the Arm, Shoulder, and Hand (DASH) at 2-year follow-up. Between February 2011 and December 2019, 160 patients 60 years and older with 3- and 4-part PHFs were randomly assigned in 1:1:1 fashion in block size of 10 to undergo nonoperative treatment (control) or operative intervention with LP or HA. In total, 54 patients were assigned to the nonoperative group, 52 to the LP group, and 54 to the HA group. Five patients assigned to the LP group were reassigned to the HA group perioperatively due to high comminution, and all of these patients had 4-part fractures. In the intention-to-treat analysis, there were 42 patients in the nonoperative group, 44 in the LP group, and 37 in the HA group. The outcome assessors were blinded to the study group. The mean DASH score at 2-year follow-up was 30.4 (standard error (SE) 3.25), 31.4 (SE 3.11), and 26.6 (SE 3.23) points for the nonoperative, LP, and HA groups, respectively. At 2 years, the between-group differences were 1.07 points (95% CI [-9.5,11.7]; p = 0.97) between nonoperative and LP, 3.78 points (95% CI [-7.0,14.6]; p = 0.69) between nonoperative and HA, and 4.84 points (95% CI [-5.7,15.4]; p = 0.53) between LP and HA. No significant differences in primary or secondary outcomes were seen in stratified age groups (60 to 70 years and 71 years and over). At 2 years, we found 30 complications (3/52, 5.8% in nonoperative; 22/49, 45% in LP; and 5/49, 10% in HA group, p = 0.0004) and 16 severe pain-related adverse events. There was a revision rate of 22% in the LP group. The limitation of the trial was that the recruitment period was longer than expected due to a high number of exclusions after the assessment of eligibility and a larger exclusion rate than anticipated toward the end of the trial. Therefore, the trial was ended prematurely., Conclusions: In this study, no benefit was observed between operative treatment with LP or HA and nonoperative treatment in displaced 3- and 4-part PHFs in patients aged 60 years and older. Further, we observed a high rate of complications related to operative treatments., Trial Registration: ClinicalTrials.gov NCT01246167., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Launonen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: To compare the recently developed Hansson Pinloc system, which features 3 cylindrical parallel pins with hooks connected through a fixed-angle interlocking plate, with the Hansson Pin System (2 hook pins) for the treatment of femoral neck fractures., Design: One hundred fourteen patients with displaced femoral neck fractures and 325 patients with nondisplaced fractures from 9 orthopaedic centers were randomized to either Hansson Pinloc system or Hansson Pin System and followed for 2 years or until death. Age at inclusion was 50 years or older., Main Outcome Measurements: The primary outcome was failure (defined as early displacement, nonunion, symptomatic avascular necrosis, or deep infection). Secondary outcomes included revision surgery, Timed Up and Go (TUG) test and patient-reported outcome measures (PROMs: EQ-5D and WOMAC)., Results: For nondisplaced fractures, the incidence of failure was 14% (23/169) in the Pinloc group and 16% (25/156) in the Hansson group. For displaced fractures, the analysis was stratified by age. Patients aged 50-69 years with displaced fractures showed a 2-year failure rate of 44% (17/39) in the Pinloc group versus 44% (16/36) in the Hansson group. For patients 70 years or older with displaced fractures, 33% (7/21) in the Pinloc group versus 22% (4/18) in the Hansson group failed. At 3 and 12 months, no clinically significant differences between treatment groups were found for EQ-5D-3L, WOMAC, or for the TUG in any fracture type or age group., Conclusions: There were no advantages for Pinloc in any of the studies aspects., Level of Evidence: 1., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Orthopaedic Trauma Association.)

Background and Purpose: Data on incidence and on trends in treatment of hamstring injuries, including proximal hamstring tendon avulsions (PHA), is limited. We aimed to investigate the incidence, trends in operative treatment, age, and sex distribution of hamstring injuries in Sweden between 2001 and 2020., Patients and Methods: We obtained data recorded in the National Patient Register between 2001 and 2020 on patients between 18 and 90 years of age, with the ICD-10 code S76.3, to calculate the incidence of patients treated operatively for hamstring injuries in Sweden. Patients with the NOMESCO classification NFL49 were considered as having been treated operatively. Data on quadriceps and Achilles tendon injuries were obtained for comparison. To calculate incidences, adult population data for every year were obtained from the Statistics Sweden website., Results: The incidence of patients diagnosed with hamstring injuries increased from 2.2 to 7.3 per 100,000 person-years. There was a rising trend of surgical treatment per diagnosed case from 3.0% to 14.2%. Patients diagnosed in units with the highest experience of surgical treatment of hamstring injuries tended to be operated on more often (22.2%) than patients diagnosed in units with limited experience (5.1%), although the fraction of operated patients was increasing in both groups., Conclusion: Between 2001 and 2020 there was an increase in the proportion of operatively treated hamstring injuries.

Background: Proximal hamstring tendon avulsions (PHAs) may be treated nonoperatively or operatively. Little is known about the result of the injury, and its treatment, on the quality and function of the hamstring muscle after healing and rehabilitation. We hypothesized that the injured leg would have greater fatty infiltration and atrophy than the uninjured leg at follow-up and that these findings would correlate to muscle weakness., Methods: In a cross-sectional cohort study, 48 patients treated for PHA, either operatively or nonoperatively, were re-examined 2-11 years post-treatment. We measured muscle strength with isokinetic strength tests, and muscle volume and fatty infiltration with MRI. Primary outcomes were hamstring muscle quality, quantified by outlining the cross-sectional area slice-by-slice, and the degree of fatty infiltration estimated using the Goutallier grading method. Secondary outcome was concentric isokinetic hamstring muscle strength measured using BioDex at 60°/sec and tendon attachment assessed on MRI. Comparisons with the outcomes of the uninjured leg were made., Results: The total hamstring muscle volume was on average reduced by 9% (SD ± 11%, p < 0.001) compared to that of the uninjured leg. Fatty infiltration was significantly more severe in the injured hamstrings than in the uninjured hamstrings (p < 0.001). This was also true when only analyzing operatively treated patients. The reduction in muscle volume and increase in fatty infiltration correlated significantly (r = 0.357, p = 0.013), and there was also a statistically significant correlation with muscle atrophy and reduction in isokinetic strength (r = 494, p < 0.001)., Conclusion: PHA injuries result in fatty infiltration and muscle atrophy and the muscle quality impairment correlates with residual muscle weakness., (© 2023. The Author(s).)

We report a case of a man with a 30-year history of treatment-resistant hypertension, hydropoiesis, tachycardic spells and dysgeusia. Despite repeated visits to the emergency department and work-up in an out-patient clinic, the diagnosis was unknown. Three years prior to remittance to an endocrinologist, the hypertension worsened, and he developed diabetes type-II. Further work-up revealed a 3 cm extra-adrenal pheochromocytoma, a paraganglioma. After surgical removal of the tumor, he is without medication and symptom free. Pheochromocytoma and paraganglioma are rare causes of hypertension, estimated to explain 0.1-0.6% of all cases, but nonetheless an important diagnosis to make, due to serious side effects.

Background and Purpose: Evidence guiding the decision on whether to treat proximal hamstring tendon avulsions (PHA) operatively or non-operatively is very limited. The aim of this study was to identify the current practices and the rationale behind PHA treatment decisions in the Nordic countries., Methods: A survey was sent to orthopaedic surgeons in Sweden, Norway, Finland and Denmark. The study population consisted of responding surgeons with exposure to surgical treatment of PHA (n = 125). The questions covered surgeon and unit characteristics, and surgeons' understanding of the evidence for treatment, and they explored which patient and injury factors influence treatment allocation., Results: Although some surgeons indicated a preference for one of the treatments, 84% stated that the treatment decision was based on patient and injury-related factors. Severe obesity, drug abuse, a sedentary lifestyle, age > 60 years and delayed diagnosis (> 6 weeks) were considered contraindications to surgical treatment. Also, there was agreement that patients expressing a preference for non-operative treatment should not be operated. Complete avulsions with tendon dislocation ≥ 2-3 cm on MRI were relative indications for surgical treatment. The majority of surgeons did not believe that operatively treated patients did better than non-operatively treated patients and experienced that patients, generally, were satisfied with the treatment result, regardless of the type of treatment. Most surgeons had experienced significant complications to operative treatment., Conclusion: Current practices varied among different units, and despite the lack of evidence for their prognostic value, several factors were inconsistently being used as decision modifiers when selecting patients for surgical treatment., (© 2022. The Author(s).)

Background: The goal of treatment for a proximal hamstring avulsion (PHA) is an objectively restored muscle and a subjectively satisfied, pain-free patient at follow-up. Different self-reported and performance-based outcome measures have been used to evaluate recovery, but their validity is poorly investigated., Purpose: To investigate (1) the correlation between the commonly used self-reported outcome measurements, the Perth Hamstring Assessment Tool (PHAT) and the Lower Extremity Functional Scale (LEFS); (2) to what extent these scores can be explained by physical dysfunction as measured by performance-based tests; (3) whether performance-based tests can discriminate between the injured and uninjured extremity; and (4) which activity limitations are perceived by patients several years after the injury., Study Design: Cohort study (Diagnosis); Level of evidence, 3., Methods: We included a consecutive series of patients treated for or diagnosed with PHA in our department between 2007 and 2016 having at least 2 tendons avulsed from the ischial tuberosity. Participants attended 2 study visits, answered questionnaires (PHAT, LEFS, and Patient-Specific Functional Scale [PSFS]), and performed physical performance-based tests (single-leg hop tests, single-step down test, and isometric and isokinetic strength tests)., Results: A total of 50 patients were included (26 men [52%], 24 women [48%]; mean age, 50.9 years [SD, 9.8 years]). The mean follow-up time was 5.5 years (SD, 2.7 years), and 74% had been surgically treated. The correlation between PHAT and LEFS was strong ( r = 0.832) and statistically significant ( P < .001). Seven of the performance-based tests exhibited a statistically significant but weak correlation with LEFS (0.340-0.488) and 3 of the tests to PHAT (-0.304 to 0.406). However, only peak torque could significantly discriminate between the extremities. The activity limitation most commonly mentioned in PSFS was running (16 patients [32%])., Conclusion: Although PHAT and LEFS correlated strongly, the correlations between functional tests and the patient-reported outcome scores were weak, and most functional tests failed to discriminate between the injured and uninjured lower extremity in patients with PHA 5 years after injury. In general, patients alleged few activity limitations, but running difficulty was a common sequela after PHA.

Background and purpose - There are few reports on the efficiency of the Hansson Pinloc System (Pinloc) for fixation of femoral neck fractures. We compare Pinloc with the commonly used Hansson Pin System in a randomized clinical trial. The primary outcome measure is non-union or avascular necrosis within 2 years. We now report fracture failures and reoperations within the first year.Patients and methods - Between May 2014 and February 2017, 439 patients were included in the study. They were above 50 years of age and treated for a femoral neck fracture at 9 orthopedic departments in Sweden. They were randomized to either Pinloc or Hansson pins. The fractures were grouped as (a) non-displaced regardless of age, (b) displaced in patients < 70 years, or (c) ≥ 70 years old, but deemed unfit to undergo arthroplasty. Follow-up with radiographs and outpatient visits were at 3 and 12 months. Failure was defined as early displacement/non-union, symptomatic segmental collapse, or deep infection.Results - 1-year mortality was 11%. Of the 325 undisplaced fractures, 12% (21/169) Pinloc and 13% (20/156) Hansson pin patients had a failure during the first year. The reoperation frequencies were 10% (16/169) and 8% (13/156) respectively. For the 75 patients 50-69 years old with displaced fractures, 11/39 failures occurred in the Pinloc group and 11/36 in the Hansson group, and 8/39 versus 9/36 patients were reoperated. Among those 39 patients ≥ 70 years old, 7/21 failures occurred in the Pinloc group and 4/18 in the Hansson group. Reoperation frequencies were 4/21 for Pinloc and 3/18 for the Hansson pin patients. No statistically significant differences were found in any of the outcomes between the Pinloc and Hansson groups.Interpretation - We found no advantages with Pinloc regarding failure or reoperation frequencies in this 1-year follow-up.

Background: Although increasingly used, the benefit of surgical treatment of displaced 2-part proximal humerus fractures has not been proven. This trial evaluates the clinical effectiveness of surgery with locking plate compared with non-operative treatment for these fractures., Methods and Findings: The NITEP group conducted a superiority, assessor-blinded, multicenter randomized trial in 6 hospitals in Finland, Estonia, Sweden, and Denmark. Eighty-eight patients aged 60 years or older with displaced (more than 1 cm or 45 degrees) 2-part surgical or anatomical neck proximal humerus fracture were randomly assigned in a 1:1 ratio to undergo either operative treatment with a locking plate or non-operative treatment. The mean age of patients was 72 years in the non-operative group and 73 years in the operative group, with a female sex distribution of 95% and 87%, respectively. Patients were recruited between February 2011 and April 2016. The primary outcome measure was Disabilities of Arm, Shoulder, and Hand (DASH) score at 2-year follow-up. Secondary outcomes included Constant-Murley score, the visual analogue scale for pain, the quality of life questionnaire 15D, EuroQol Group's 5-dimension self-reported questionnaire EQ-5D, the Oxford Shoulder Score, and complications. The mean DASH score (0 best, 100 worst) at 2 years was 18.5 points for the operative treatment group and 17.4 points for the non-operative group (mean difference 1.1 [95% CI -7.8 to 9.4], p = 0.81). At 2 years, there were no statistically or clinically significant between-group differences in any of the outcome measures. All 3 complications resulting in secondary surgery occurred in the operative group. The lack of blinding in patient-reported outcome assessment is a limitation of the study. Our assessor physiotherapists were, however, blinded., Conclusions: This trial found no significant difference in clinical outcomes at 2 years between surgery and non-operative treatment in patients 60 years of age or older with displaced 2-part fractures of the proximal humerus. These results suggest that the current practice of performing surgery on the majority of displaced proximal 2-part fractures of the humerus in older adults may not be beneficial., Trial Registration: ClinicalTrials.gov NCT01246167., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: OW declared a consultancy for Anatomica, and a paid presentation for DePuy Synthes and Link Sweden. AR declared a paid lecture (Orion Ltd.). The other authors have declared that no competing interests exist.

Here we addressed the potential impact of chymase, a mast-cell restricted protease, on mouse bone phenotype. We show that female mice lacking the chymase Mcpt4 acquired a persistent expansion of diaphyseal bone in comparison with wild type controls, reaching a 15% larger diaphyseal cross sectional area at 12 months of age. Mcpt4-/- mice also showed increased levels of a bone anabolic serum marker and higher periosteal bone formation rate. However, they were not protected from experimental osteoporosis, suggesting that chymase regulates normal bone homeostasis rather than the course of osteoporosis. Further, the absence of Mcpt4 resulted in age-dependent upregulation of numerous genes important for bone formation but no effects on osteoclast activity. In spite of the latter, Mcpt4-/- bones had increased cortical porosity and reduced endocortical mineralization. Mast cells were found periosteally and, notably, bone-proximal mast cells in Mcpt4-/- mice were degranulated to a larger extent than in wild type mice. Hence, chymase regulates degranulation of bone mast cells, which could affect the release of mast cell-derived factors influencing bone remodelling. Together, these findings reveal a functional impact of mast cell chymase on bone. Further studies exploring the possibility of using chymase inhibitors as a strategy to increase bone volume may be warranted., Competing Interests: No competing interest exist. The funding from Uppsala Bio-X does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: Comorbidities and polypharmacy complicate the treatment of geriatric patients with acute orthopedic injuries. A correct medication history and an updated medication list are a prerequisite for safe treatment of these debilitated patients. Published evidence suggests favorable outcomes with comanaged care. The aim of this study was to assess the accuracy of the inpatient medication lists generated at admission and investigate the efficacy of a dedicated ward-based pharmacist to find and correct mistakes in these lists., Methods: A total of 254 patients were enrolled. The ward-based pharmacist performed the assessment regarding the accuracy of the medication list generated at admission by the method of medication reconciliation. Number of discrepancies and types of discrepancy were noted., Results: The 254 patients (176 women) had a mean age of 85 years (standard deviation 7.4 years, range 42-100 years). The most common reason for orthopedic admission was hip fracture. The mean number of discrepancies was 2.1 for all patients (range 0-13). Omission of a prescribed drug was the most common mistake. Fifty-six (22%) of the 254 assessed patients had a correct medication list., Discussion: The many discrepancies in our study may have several explanations but highlight the difficulties in taking a correct medication history of patients in a stressful environment with an extremely high workload. Moreover, electronic medication lists create challenges. Implementing new electronic tools for health care requires feedback, redesign, and adaptation to meet various needs of the users., Conclusion: In conclusion, orthogeriatric patients have an unsatisfactory high number of discrepancies in their medication lists. Clinical pharmacists can accurately identify many of these mistakes.

Introduction: Neurodegeneration is an early component of diabetic retinopathy (DR). It is unclear whether neurodegeneration is an independent factor or a consequence of damaged retinal vasculature. The aims of this study were to review the literature concerning neurodegeneration in diabetic patients without or with early DR, and to examine whether neurodegeneration precedes visible vasculopathy in the pathogenesis of DR., Methods: A systematic literature search was performed to identify studies which used optical coherence tomography (OCT) or multifocal electroretinography (mfERG) to detect neurodegeneration in patients with no or mild DR as compared to healthy controls. Outcome measures were mean retinal thickness (RT), mean retinal nerve fiber layer (RNFL) thickness and ganglion cell layer (GCL) thickness. Also, mfERG amplitude and implicit time were analyzed., Results: Eleven studies which used mfERG and/or OCT to detect neurodegeneration were included. Two OCT studies found significant thinning of RT, 2 found thinning of RNFL, whereas 1 found thickening of RT, RNFL and GCL in patients without DR. Two mfERG studies found a significant delay of implicit time in the same patient group. Retinal thinning and delay of implicit time were also detected in patients with mild DR., Conclusion: Retinal neurodegeneration is an early component of DR, which can precede visible vasculopathy., (© 2016 S. Karger AG, Basel.)

Life history traits such as fecundity are important to evolution because they make up components of lifetime fitness. Due to their polygenic architectures, such traits are difficult to investigate with genetic mapping. Therefore, little is known about their molecular basis. One possible way toward finding the underlying genes is to map intermediary molecular phenotypes, such as gene expression traits. We set out to map candidate quantitative trait genes for egg fecundity in the chicken by combining quantitative trait locus mapping in an advanced intercross of wild by domestic chickens with expression quantitative trait locus mapping in the same birds. We measured individual egg fecundity in 232 intercross chickens in two consecutive trials, the second one aimed at measuring brooding. We found 12 loci for different aspects of egg fecundity. We then combined the genomic confidence intervals of these loci with expression quantitative trait loci from bone and hypothalamus in the same intercross. Overlaps between egg loci and expression loci, and trait-gene expression correlations identify 29 candidates from bone and five from hypothalamus. The candidate quantitative trait genes include fibroblast growth factor 1, and mitochondrial ribosomal proteins L42 and L32. In summary, we found putative quantitative trait genes for egg traits in the chicken that may have been affected by regulatory variants under chicken domestication. These represent, to the best of our knowledge, some of the first candidate genes identified by genome-wide mapping for life history traits in an avian species., (Copyright © 2016 Johnsson et al.)

The development of biomaterial for bone regeneration requires animal models that are reliable and designed to mimic clinically relevant situations. We have previously investigated hydrogels comprised of modified hyaluronic acid and polyvinyl alcohol in models of ectopic bone formation. This hydrogel induces bone regeneration when loaded with bone morphogenetic proteins (BMPs). To allow further optimization of hydrogels, we developed a new, femoral, non-critical-sized cortical defect model. In the rat femur, we drilled standardized, elongated unilateral cortical defects that did not require stabilization and that could be created bilaterally to allow paired comparisons of biomaterials. After optimizing the defect size, subsequent stress fractures occurred in only 8% and the defect healed partially over the 40 day study period. In a time-course experiment, we treated bone defects with the previously studied hyaluronan hydrogel loaded with 10 µg hydroxyapatite and 6 µg BMP-2. The shape of the defect allowed controlled containment of the material within the defect. The defect in the right leg was left untreated, while the left defect was filled with 40 µl of the BMP hydrogel. As determined by pQCT analysis, the treated defects had a higher bone mineral content, bone area and bone density than control defects. The relative difference was greatest between the groups at 10 and 20 days and diminished as the defect healed in the untreated legs. We conclude that this animal model allows facile and rapid screening of biomaterials for bone regeneration in cortical femoral defects without requiring external fixation., (Copyright © 2012 John Wiley & Sons, Ltd.)

Birds have a unique bone physiology, due to the demands placed on them through egg production. In particular their medullary bone serves as a source of calcium for eggshell production during lay and undergoes continuous and rapid remodelling. We take advantage of the fact that bone traits have diverged massively during chicken domestication to map the genetic basis of bone metabolism in the chicken. We performed a quantitative trait locus (QTL) and expression QTL (eQTL) mapping study in an advanced intercross based on Red Junglefowl (the wild progenitor of the modern domestic chicken) and White Leghorn chickens. We measured femoral bone traits in 456 chickens by peripheral computerised tomography and femoral gene expression in a subset of 125 females from the cross with microarrays. This resulted in 25 loci for female bone traits, 26 loci for male bone traits and 6318 local eQTL loci. We then overlapped bone and gene expression loci, before checking for an association between gene expression and trait values to identify candidate quantitative trait genes for bone traits. A handful of our candidates have been previously associated with bone traits in mice, but our results also implicate unexpected and largely unknown genes in bone metabolism. In summary, by utilising the unique bone metabolism of an avian species, we have identified a number of candidate genes affecting bone allocation and metabolism. These findings can have ramifications not only for the understanding of bone metabolism genetics in general, but could also be used as a potential model for osteoporosis as well as revealing new aspects of vertebrate bone regulation or features that distinguish avian and mammalian bone.

Sexual selection and the ornaments that inform such choices have been extensively studied, particularly from a phenotypic perspective. Although more is being revealed about the genetic architecture of sexual ornaments, much still remains to be discovered. The comb of the chicken is one of the most widely recognized sexual ornaments, which has been shown to be correlated with both fecundity and bone allocation. In this study, we use a combination of multiple intercrosses between White Leghorn populations and wild-derived Red Junglefowl to, first, map quantitative trait loci (QTL) for bone allocation and, second, to identify expression QTL that correlate and colocalize with comb mass. These candidate quantitative genes were then assessed for potential pleiotropic effects on bone tissue and fecundity traits. We identify genes that correlate with both relative comb mass and bone traits suggesting a combination of both pleiotropy and linkage mediates gene regulatory variation in these traits., (© 2014 John Wiley & Sons Ltd.)

Fracture healing is a complex interplay between endochondral and intramembranous bone formation processes. The canonical Wnt/β-catenin pathway enhances new bone formation and may play a role in fracture healing. Glycogen synthase kinase 3β (GSK3β) is a key regulator of β-catenin degradation. In this study, we investigate the effects of AZD2858, an orally bioactive GSK3 inhibitor, on fracture healing. Femoral fractures were produced in rats after the insertion of a femoral nail. The rats were treated with oral administration of AZD2858 at a dose of 30 μmol/kg (20mg/kg) daily for up to 3 weeks, while control animals were administered vehicle. At 4days, and at 1, 2 and 3 weeks, histological analysis was performed, and at the 2 and 3 week time points, we performed peripheral quantitative computed tomography (pQCT), X-rays, and four-point bending tests. Peripheral QCT showed an increase in both mineral density (of 28% at 2 weeks and 38% at 3weeks) and mineral content (of 81% at 2 weeks and 93% at 3 weeks) in the calluses from AZD2858 treated animals as compared to vehicle treated animals. Histological analysis demonstrated that rats treated with GSK3 inhibitor healed their fractures rapidly, but without the pre-formation of cartilage tissue. Furthermore, four-point bending tests of fractured femora from animals treated for 2 and 3 weeks showed an increase in strength in treated animals compared to their vehicle-treated controls. In conclusion, AZD2858, a potent GSK3 inhibitor, has a substantial impact on fracture healing. The fractures healed with a bony callus without an obvious endochondral component, suggesting that AZD2858 drives mesenchymal cells into the osteoblastic pathway. This leads to direct bone repair in an unstable fracture milieu., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Transgenic mice overexpressing fibroblast growth factor 23 (FGF23) in osteoblasts have a rachitic bone phenotype. These mice display hypomineralized bones, increased expression of osteoblast markers, but osteoclast numbers are unaltered or slightly reduced. Paradoxically, they show increased serum levels of the bone resorption marker CTX, a type I collagen degradation fragment. Here we analyzed a matrix metalloproteinase- (MMP-) like secreted protease, Adamts1, that has previously been associated with osteoblastic type I collagen breakdown in vitro. Bones from FGF23 transgenic (tg) mice displayed increased Adamts1 protein upon both immunohistological staining and Western blotting. We further found Adamts1 protein together with excessively degraded type I collagen in the non-mineralized bone fraction of FGF23 tg mice. A similar degradation pattern of type I collagen was noticed upon forced expression of Adamts1 in osteoblastic cells in vitro. Importantly, these Adamts1-expressing osteoblastic cells exhibited increased release of CTX fragments when cultured on demineralized bone discs. Together, these results demonstrate for the first time that Adamts1 can be highly induced in bone tissue and that this MMP-like protease can increase osteoblastic release of CTX fragments from non-mineralized bone. Thus, Adamts1 potentially contributes to the increased serum levels of CTX in rickets/osteomalacia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Hyaluronic acid-based hydrogels are proven biocompatible materials and excellent carriers of bone morphogenetic protein-2 (BMP-2) that have been successfully tested for bone generation in vivo. Different formulations, with or without nanohydroxyapatite, have shown promise for craniofacial applications. In this study, 28 rats were used to investigate whether it is possible to achieve mandibular bone augmentation upon injection of novel hyaluronic acid-based hydrogels containing nanohydroxyapatite and different concentrations of BMP-2 (0, 5 and 150 µg/ml). The biomaterials were injected subperiosteally through fine needles into the innate mandibular diastema, imitating a clinical procedure for resorbed mandibles. No incisions, flaps or sutures were necessary. After 8 weeks the mandibles were evaluated by peripheral quantitative computed tomography (pQCT), micro-computed tomography (μCT), histology, immunohistochemistry and fluorochrome labelling. As a result, engineered bone was observed in all treated mandibles, with a statistically significant increase in mandibular bone volume correlated with the amount of BMP-2 loaded in the hydrogel formula. We therefore demonstrated that minimally invasive mandibular bone augmentation is possible upon injection in rats, when using the appropriate injectable scaffolds. This represents an attractive clinical alternative for oral implantology patients., (Copyright © 2012 John Wiley & Sons, Ltd.)

ADAMTS1, a secreted multifunctional metalloproteinase with disintegrin and thrombospondin motifs, is an early response gene of parathyroid hormone (PTH) in osteoblasts. Mice engineered to lack Adamts1 are smaller compared to wild-type (WT) mice and ADAMTS1 metalloproteinase activity has been shown to increase osteoblastic growth in collagen gels. However, there are no reports investigating the consequence of Adamts1 over-expression on bone tissue in vivo. Here, we analyze bones of female and male transgenic (TG) mice over-expressing mouse Adamts1 using peripheral quantitative computed tomography to evaluate its effect on bone shape and mineral density. Western blotting of protein extracts and immunohistochemistry of bone sections reveal increased presence of Adamts1 protein in TG bones compared to WT bones. Phenotypic analyses of femur show that female TG mice have reduced metaphyseal total density, trabecular bone mineral density and trabecular mineral content. In contrast, male TG mice which were without changes in the metaphysis showed increased total density and cortical density at the mid-diaphysis cortical site. Female TG mice showed no significant changes at the cortical site compared to WT mice. Furthermore, diaphyseal endosteal compartment was only affected in male TG mice. Along these lines, Adamts1 increased blood levels of PTH only in females whereas it reduced osteocalcin levels only in males. These results reveal that Adamts1 has an impact on bone mineral density and thus further confirm Adamts1 as a potent regulator of bone remodeling.

Glycogen synthase kinase 3β (GSK-3β) actions are central in the canonical Wnt pathway, important in many biological processes and a potential drug target for treating several diseases. It is appreciated that a balanced Wnt canonical signaling is crucial for the maintenance of normal bone mass. In this study we investigated the effects of a potent orally active GSK-3 inhibitor, AZD2858, on bone mass in rats. Treatment (1 μM) of human osteoblast cells with AZD2858 in vitro increased β-catenin levels after a short period of time. In rats, oral AZD2858 treatment caused a dose-dependent increase in trabecular bone mass compared to control after a two-week treatment with a maximum effect at a dose of 20 mg/kg once daily (total BMC: 172% of control; p<0.001). A small but significant effect was also seen at cortical sites (total BMC: 111% of control; p<0.001). Biomechanical testing demonstrated an increase in both vertebral compression strength at a dose of 20 mg/kg once daily (Load at failure: 370% of control, p<0.001) and diaphyseal strength of femora subjected to a three point bending test (Load at failure: 115% of control; p<0.01). Furthermore, histomorphometry showed a dramatic increase in bone formation indices, and serum markers of both bone formation (Osteocalcin, 146% of control; p<0.001) and resorption (CTX, 189% of control; p<0.001) were elevated. Our conclusion is that a GSK-3 inhibitor drug may prove effective as an anabolic strategy in the treatment of diseases characterized by low bone mass, since AZD2858 has extensive bone building effects at predominantly trabecular sites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Domestication is one of the strongest forms of short-term, directional selection. Although selection is typically only exerted on one or a few target traits, domestication can lead to numerous changes in many seemingly unrelated phenotypes. It is unknown whether such correlated responses are due to pleiotropy or linkage between separate genetic architectures. Using three separate intercrosses between wild and domestic chickens, a locus affecting comb mass (a sexual ornament in the chicken) and several fitness traits (primarily medullary bone allocation and fecundity) was identified. This locus contains two tightly-linked genes, BMP2 and HAO1, which together produce the range of pleiotropic effects seen. This study demonstrates the importance of pleiotropy (or extremely close linkage) in domestication. The nature of this pleiotropy also provides insights into how this sexual ornament could be maintained in wild populations., Competing Interests: The authors have declared that no competing interests exist.

Current treatment of fractures often involves the use of bone graft or bone morphogenetic proteins (BMP) to induce fracture healing, especially in patients with a compromised healing capacity. BMP has to be delivered in conjunction with a carrier. Unfortunately, there are drawbacks and limitations with current carriers, including their bovine origin which carries the risk of an immunological response. The physical properties also limit the use to open surgical procedures, as it cannot be injected. New carriers with improved properties are therefore needed. The aim of this study was to assess the ectopic bone forming capability of various calcium phosphate compounds when used in conjunction with a hydrogel as the carrier for BMP-2. Five different ceramic additives were tested, including β-tricalcium phosphate and four types of hydroxyapatite (HAP) (nanoHAP, HAP, clods of HAP >100 μm, and the biomimetic HAP Ostim35®). The compounds were injected into the thigh muscle of rats, where it formed a gel in situ. After 4 weeks bone formation was evaluated by peripheral quantitative computed tomography and histology. The major finding was that the 20 nm nanoHAP yielded a higher bone density than the other additives (P=0.0008, ANOVA with Tukey's multiple comparison test). We hypothesize that the higher bone density induced by nanoHAP might be due to nanocrystals of calcium phosphate acting as direct building blocks for biomineralization., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

A strategy has been designed to develop hyaluronic acid (HA) hydrogel for in vivo bone augmentation using minimal invasive technique. A mild synthetic procedure was developed to prepare aldehyde modified HA by incorporating an amino-glycerol side chain via amidation reaction and selective oxidation of the pendent group. This modification, upon mixing with hydrazide modified HA formed hydrazone-crosslinked hydrogel within 30s that was stable at physiological pH. In vitro experiments showed no cytotoxicity of hydrogel with the controlled release of active bone morphogenic protein-2 (BMP-2). In vivo evaluation of this gel as a BMP-2 carrier was performed by injecting gels over the rat calvarium and showed bone formation in 8 weeks in correlation with the amount of BMP-2 loaded (0, 1 and 30μg) within the gel. Furthermore, hydrogels with 30μg of BMP-2 induced less bone formation upon subcutaneous injection in comparison with subperiosteal implantation. Histological examination showed newly formed bone with a high expression of osteocalcin, osteopontin and with angiogenic bone marrow when higher BMP-2 concentration was employed. Our result suggests that novel HA hydrogels could be used as a BMP-2 carrier and can promote bone augmentation for potential orthopedic applications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Over the last decade, the regulation of phosphate (Pi) homeostasis has been under intense investigation. By utilizing modern biochemical and genetic tools, the pathophysiological mechanisms behind several known hereditary and acquired hypo- and hyperphosphatemic diseases have been clarified. The results of these efforts have opened new insights into the causes of Pi dysregulation and hereby also the physiological mechanisms determining Pi homeostasis. Although several potential Pi-regulating proteins have been discovered and investigated, current data strongly argues for fibroblast growth factor-23 (FGF23), a hormonal factor produced in bone, as a particularly important regulator of Pi homeostasis. In this article, we review the discovery of the FGF23 protein, as well as its biochemistry, localization of production, receptor specificity and mechanisms of action.

Summary: We evaluated the relation between serum FGF23 and bone mineral density (BMD) in a community-based cohort of elderly men. There was a weak correlation between FGF23 and BMD, which was primarily dependent on body weight., Introduction: FGF23 is a hormonal factor produced in bone and regulates serum levels of phosphate (Pi) and vitamin D. FGF23 over-expression is associated with skeletal abnormalities, including rickets/osteomalacia. The relation between FGF23 and Bone Mineral Density (BMD) in the community remains unexplored., Methods: We employed a large, population-based cohort of 3014 Swedish men aged 69-80 years, without known renal disease. BMD was measured with dual X-ray absorptiometry (DXA) in the hip and lumbar spine. Serum intact FGF23 was analyzed with a two-site monoclonal ELISA., Results: There was a weak but significant correlation between FGF23 and BMD in femoral neck (r = 0.04, p < 0.05), femoral trochanter (r = 0.05, p = 0.004), total hip (r = 0.06, p = 0.0015) and lumbar spine (r = 0.07, p = 0.0004). The correlations remained significant when adjusting for biochemical covariates (Pi, calcium, PTH, 25(OH)D and renal function). However, the association became insignificant in all regions when adjusting for established confounding variables including age, height, weight and smoking. Further analysis confirmed a significant correlation between FGF23 and body weight (r = 0.13, p < 0.0001)., Conclusions: The weak correlation between FGF23 and BMD in elderly male subjects is mainly due to an association between FGF23 and body weight. Therefore, FGF23 may not play a significant role in the hormonal regulation of BMD.

Hypophosphatemic transgenic (tg) mice overexpressing FGF23 in osteoblasts display disorganized growth plates and reduced bone mineral density characteristic of rickets/osteomalacia. These FGF23 tg mice were used as an in vivo model to examine the relation between osteoclast polarization, secretion of proteolytic enzymes and resorptive activity. Tg mice had increased mRNA expression levels of the osteoblast differentiation marker Runx2 and mineralization-promoting proteins alkaline phosphatase and bone sialoprotein in the long bones compared to wild type (wt) mice. In contrast, expression of alpha1(I) collagen, osteocalcin, dentin matrix protein 1 and osteopontin was unchanged, indicating selective activation of osteoblasts promoting mineralization. The number of osteoclasts was unchanged in tg compared to wt mice, as determined by histomorphometry, serum levels of TRAP 5b activity as well as mRNA expression levels of TRAP and cathepsin K. However, tg mice displayed elevated serum concentrations of C-terminal telopeptide of collagen I (CTX) indicative of increased bone matrix degradation. The majority of osteoclasts in FGF23 tg mice lacked ultrastructural morphological signs of proper polarization. However, they secreted both cathepsin K and MMP-9 at levels comparable to osteoclasts with ruffled borders. Mineralization of bone matrix thus appears essential for inducing osteoclast polarization but not for secretion of osteoclast proteases. Finally, release of CTX by freshly isolated osteoclasts was increased on demineralized compared to mineralized bovine bone slices, indicating that the mineral component limits collagen degradation. We conclude that ruffled borders are implicated in acidification and subsequent demineralization of the bone matrix, however not required for matrix degradation. The data collectively provide evidence that osteoclasts, despite absence of ruffled borders, effectively participate in the degradation of hypomineralized bone matrix in rachitic FGF23 tg mice.

Background: Fibroblast growth factor-23 (FGF23), a circulating protein produced in bone, causes decreased renal inorganic phosphate (Pi) reabsorption by reducing the expression of the sodium phosphate cotransporter type 2a (Npt2a). We have previously generated transgenic mice expressing human wild-type (WT) FGF23 under the control of the alpha1 (I) collagen promoter., Methods: In this study, we performed a large-scale gene expression study of kidneys from FGF23 transgenic mice and WT littermates. Microarray expression data of key transcripts were verified by real-time RT-PCR analysis., Results: Several genes that play a role in Pi regulation revealed decreased expression levels in the transgenic mice, such as Npt2a and Pdzk1, a scaffolding protein known to interact with Npt2a. Importantly, Klotho, a suggested FGF23 receptor cofactor, was the most significantly decreased transcript and alpha2-Na(+)/K(+)-ATPase (Atp1a2), a gene isoform of alpha1-Na(+)/K(+)-ATPase (Atp1a1) which has recently been shown to interact with Klotho and regulate calcium metabolism, was the most increased transcript. In contrast, other genes proposed to regulate Pi levels, such as secreted frizzled-related protein-4 (sFrp4) and Na(+)/H(+) exchanger regulatory factor-1 (Nherf1) revealed no changes., Conclusions: FGF23 transgenic mice display differentially expressed transcript levels of several genes essential in renal Pi regulation. These findings may lead to further understanding of how FGF23 mediates its actions on renal Pi regulation.

Objective: Fibroblast growth factor-23 (FGF23) is a circulating factor involved in phosphate (Pi) and vitamin D metabolism. Serum FGF23 is increased at later stages of chronic kidney disease due to chronic hyperphosphatemia and decreased renal clearance. Recent studies also indicate that FGF23 may directly regulate the expression of parathyroid hormone (PTH) in vitro. Therefore, the objective of the current study was to determine the relationship between FGF23, PTH, and other biochemistries in vivo in subjects with no history of renal disease., Design: Serum biochemistries were measured in a subsample of the population-based Swedish part of the MrOS study. In total, 1000 Caucasian men aged 70-80 years were randomly selected from the population., Methods: Intact FGF23, Pi, calcium, albumin, estimated glomerular filtration rate (eGFR, calculated from cystatin C), PTH, and 25(OH)D3 were measured. Association studies were performed using linear univariate and multivariate regression analyses., Results: The median FGF23 level was 36.6 pg/ml, ranging from 0.63 to 957 pg/ml. There was a significant correlation between log FGF23 and eGFR (r=-0.21; P<0.00001) and log PTH (r=0.13; P<0.001). These variables remained as independent predictors of FGF23 in multivariate analysis. In addition, log PTH (beta=0.082; P<0.05) and eGFR (beta=-0.090; P<0.05) were associated with log FGF23 in subjects with eGFR>60 ml/min. Only eGFR (beta=-0.35; P<0.0001) remained as a predictor of log FGF23 in subjects with eGFR<60 ml/min., Conclusions: Serum FGF23 and PTH are associated in vivo, supporting recent findings that FGF23 directly regulates PTH expression in vitro. Additionally, eGFR is associated with FGF23 in subjects with normal or mildly impaired renal function, indicating that GFR may modulate FGF23 levels independent of serum Pi.

Fibroblast growth factor-23 (FGF23) is a circulating factor that decreases serum levels of inorganic phosphate (Pi) as well as 1,25-dihydroxyvitamin D(3). Recent studies also suggest a correlation between serum levels of FGF23 and parathyroid hormone (PTH) in patients with chronic kidney disease. It is, however, unknown whether FGF23 directly modulates PTH expression, or whether the correlation is secondary to abnormalities in Pi and vitamin D metabolism. The objective of the current study was therefore to elucidate possible direct effects of FGF23 on bovine parathyroid cells in vitro. Treatment of parathyroid cells with a stabilized form of recombinant FGF23 (FGF23(R176Q)) induced a rise in early response gene-1 mRNA transcripts, a marker of FGF23 signaling. FGF23(R176Q) potently and dose-dependently decreased the PTH mRNA level within 12 h. In agreement, FGF23(R176Q) also decreased PTH secretion into conditioned media. In contrast, FGF23(R176Q) dose-dependently increased 1alpha-hydroxylase expression within 3 h. FGF23 (R176Q) did not affect cell viability nor induce apoptosis, whereas a small but significant increase in cell proliferation was found. We conclude that FGF23 is a negative regulator of PTH mRNA expression and secretion in vitro. Our data suggest that FGF23 may be a physiologically relevant regulator of PTH. This defines a novel function of FGF23 in addition to the previously established roles in controlling vitamin D and Pi metabolism.

Background: The parathyroid hormone-/parathyroid hormone-related protein (PTH/PTHrP) receptor plays a crucial role in endochondral bone formation and possibly also in fracture healing. Patients with Jansen's metaphysial chondrodysplasia (JMC) have a gain-of-function mutation in the PTH/PTHrP receptor. Transgenic mice expressing JMC PTH/PTHrP receptor mutants in osteoblasts are characterized by increased trabecular bone formation and reduced osteoblastic activity at periosteal sites. We have analyzed the bone phenotype and studied the fracture healing process in this model., Methods: We performed bone density analysis of tibiae from 17-week-old transgenic mice and controls. Also, tibial fractures were produced in 14-week-old mice. Fracture healing was examined by radiographic and histological analysis., Results: Transgenic mice had a lower total bone mineral content (BMC), by a factor of one-third. The changes were bone compartment-specific with an increase in trabecular bone volume and a decrease in cortical thickness. The calluses in the transgenic mice were smaller, with a reduction in BMC and mean cross-sectional area by a factor of one-half. Despite the smaller size, however, the morphology and progression through the healing process were similar in both transgenic and wild-type littermates., Interpretation: We conclude that the constitutively active PTH/PTHrP receptor has compartment-specific effects on bone formation when expressed in osteoblasts. During fracture healing, however, both the periosteal and the endochondral processes are activated, leading to fracture healing that is temporally and morphologically normal, although the callus tissue is less prominent.