Erosive Progression by High-Resolution Peripheral Quantitative Computed Tomography and Conventional Radiography in Rheumatoid Arthritis.

OBJECTIVES: To investigate the diagnostic accuracy of high-resolution peripheral quantitative computed tomography (HR-pQCT) to assess erosive progression during one year compared to conventional radiography (CR) in rheumatoid arthritis (RA). METHODS: This prospective study included 359 patients with RA (disease duration ≥ 5 years) between March 2018 and October 2020. HR-pQCT and CR were obtained at inclusion and after one year. Erosive assessment was performed at two metacarpophalangeal joints of the dominant hand using HR-pQCT and progression was defined as an increase in erosion number ≥ 1 or an increase in erosive volume > least significant change. CR of hands, wrists, and feet were evaluated using Sharp/van der Heijde scores and erosive progression was defined as a 1.1-point increase in erosion score according to the smallest detectable change. RESULTS: In paired analyses (n = 310), erosive progression was identified in 30 patients using CR and in 40 patients using HR-pQCT. In the 40 patients with erosive progression on HR-pQCT, progression was not identified by CR in 33 patients. Adding HR-pQCT to CR doubled the proportion of patients identified with progression from 30 (10%) to 63 (20%) patients. Using CR as the reference, the sensitivity (% (95% CI)) of HR-pQCT for identifying erosive progression was 23.3 (9.9-42.3) and the specificity was 88.2 (83.8-91.7). CONCLUSION: A substantial proportion of patients with erosive progression are overlooked using CR only to monitor erosive progression. Adding high-resolution peripheral CT to CR doubles the proportion of patients, who may benefit from individualised therapy targeting erosive progression in RA.

Loss of Adgra3 causes obstructive azoospermia with high penetrance in male mice.

The adhesion receptor ADGRA3 (GPR125) is a known spermatogonial stem cell marker, but its impact on male reproduction and fertility has not been examined. Using a mouse model lacking Adgra3 (Adgra3-/- ), we show that 55% of the male mice are infertile from puberty despite having normal spermatogenesis and epididymal sperm count. Instead, male mice lacking Adgra3 exhibited decreased estrogen receptor alpha expression and transient dilation of the epididymis. Combined with an increased estradiol production, this indicates a post-pubertal hormonal imbalance and fluid retention. Dye injection revealed a blockage between the ejaculatory duct and the urethra, which is rare in mice suffering from infertility, thereby mimicking the etiologies of obstructive azoospermia found in human male infertility. To summarize, male reproductive tract development is dependent on ADGRA3 function that in concert with estrogen signaling may influence fluid handling during sperm maturation and storage.

A well-developed endolysosomal system reflects protein reabsorption in segment 1 and 2 of rat proximal tubules.

Proteinuria is a well-established marker and predictor of kidney disease. The receptors megalin and cubilin reabsorb filtered proteins and thereby proteinuria is avoided. It is unknown if all segments of the proximal tubule are involved in clearing the filtrate or if there exists a reserve capacity in case of increased glomerular protein filtration. To determine this, we performed serial sectioning of rat kidney and used stereology to quantify the endolysosomal system of the three segments of cortical and juxtamedullary nephrons by electron microscopy. Immunohistochemistry was applied to analyze the adaptor protein Dab2, which assists in megalin mediated endocytosis, megalin, and endocytic uptake of two endogenous megalin ligands; retinol binding protein and ß2-microglobulin at exact tubular positions. Proteinuric rats (puromycin-treated) and mice (podocin knock-out) were analyzed to clarify the response of the tubule to increased protein filtration. We found that the endolysosomal system was most prominent in segment 1 and 2, whereas segment 3 was less developed. The depth of ligand uptake varied among nephrons, but it descended into segment 2 although uptake was lower than in segment 1 and it was never observed in segment 3. This was supported by prominent expression of Dab2 in segment 1 and 2. When protein filtration increased, segment 3 was included in the reabsorption process in proteinuric animals. Thus, segment 1 and 2 are responsible for clearing the filtrate for protein during normal physiological conditions, but the tubule exhibits plasticity and is able to include segment 3 under proteinuric stress.

Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

Spatial organization of the vascular bundle and the interbundle region: three-dimensional reconstruction at the inner stripe of the outer medulla in the mouse kidney.

The vascular bundle (VB) is a complex structure that resides in the inner stripe of the outer medulla. At present, the tubulovascular spatial organization of the VB, which is crucial for the formation of the osmolarity gradient and for solute transport, is still under debate. In this study, we used computer-assisted digital tracing combined with aquaporin-1 immunohistochemistry to reconstruct all tubules and vessels in the VB of the mouse kidney. We found, first, that the descending and ascending vasa recta travelled exclusively through the VB. The ascending vasa recta received no tributaries (no branches) along their entire path in the medulla and were not connected with the capillary plexus in the interbundle region. Second, a specific group of the descending vasa recta were closely accompanied by the longest ascending vasa recta, which connected only to the capillary plexus at the tip of the papilla. Third, the descending thin limbs of all short-looped nephrons travelled exclusively through the outer part of the VB. The loops of these nephrons (both descending and ascending parts) were distributed in a regular pattern based on their length. Finally, the thick ascending limbs of all long-looped nephrons were located at the margin of the VB (except a few within the VB), which formed a layer separating the VB from the interbundle region. In conclusion, our three-dimensional analysis of the VB strongly suggest a lateral osmolarity heterogeneity across the inner stripe of the outer medulla, which might work as a driving force for water and solute transport.

Three-dimensional reconstruction of the rat nephron.

This study gives a three-dimensional (3D) structural analysis of rat nephrons and their connections to collecting ducts. Approximately 4,500 2.5-µm-thick serial sections from the renal surface to the papillary tip were obtained from each of 3 kidneys of Wistar rats. Digital images were recorded and aligned into three image stacks and traced from image to image. Short-loop nephrons (SLNs), long-loop nephrons (LLNs), and collecting ducts (CDs) were reconstructed in 3D. We identified a well-defined boundary between the outer stripe and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between the inner stripe and the inner medulla, i.e., the transition of ascending thin limbs into ascending thick limbs of LLNs. In all nephrons, a mosaic pattern of proximal tubule (PT) cells and descending thin limb (DTL) cells was observed at the transition between the PT and the DTL. The course of the LLNs revealed tortuous proximal "straight" tubules and winding of the DTLs within the outer half of the inner stripe. The localization of loop bends of SLNs in the inner stripe of the outer medulla and the bends of LLNs in the inner medulla reflected the localization of their glomeruli; i.e., the deeper the glomerulus, the deeper the bend. Each CD drained approximately three to six nephrons with a different pattern than previously established in mice. This information will provide a basis for evaluation of structural changes within nephrons as a result of physiological or pharmaceutical intervention.

A comparison of osteoclast-rich and osteoclast-poor osteopetrosis in adult mice sheds light on the role of the osteoclast in coupling bone resorption and bone formation.

Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts.

Mapping RANKL- and OPG-expressing cells in bone tissue: the bone surface cells as activators of osteoclastogenesis and promoters of the denosumab rebound effect.

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11+ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Osteoclasts are not crucial for hematopoietic stem cell maintenance in adult mice.

The osteoclast is vital for establishment of normal hematopoiesis in the developing animal. However, its role for maintenance of hematopoiesis in adulthood is more controversial. To shed more light on this process, we transplanted hematopoietic stem cells from two osteopetrotic mouse models, with lack of osteoclasts or defective osteoclast function, to normal adult mice and examined the bone phenotype and hematopoiesis in the recipients. B6SJL mice were lethally irradiated and subsequently transplanted with oc/oc, Receptor Activator of Nuclear Factor Kappa B knockout or control fetal liver cells. Osteoclasts derived from the recipient animals were tested in vitro for osteoclastogenesis and resorptive function. Bone remodeling changes were assessed using biomarkers of bone turnover and micro-CT. Hematopoiesis was assessed by flow cytometry and colony formation, and hematopoietic stem cell function by secondary competitive transplantations and cell cycle analysis. After transplantation, a donor chimerism of 97-98% was obtained, and by 15 weeks mild osteopetrosis had developed in recipients of cells from osteopetrotic mice. There were no alterations in the number of bone marrow cells. Colony formation was slightly reduced in Receptor Activator of Nuclear Factor Kappa B knockout recipients but unchanged in oc/oc recipients. Phenotypically, stem cells were marginally reduced in recipients of cells from osteopetrotic mice, but no significant difference was seen in cell cycle status and in competitive secondary transplantations all three groups performed equally well. Our results indicate that osteoclast function is not crucial for hematopoietic stem cell maintenance in adult mice.

Superoxide dismutase 3 is expressed in bone tissue and required for normal bone homeostasis and mineralization.

Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3-/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3-/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis.

Strontium and bone nanostructure in normal and ovariectomized rats investigated by scanning small-angle X-ray scattering.

The effect of SrCl(2) treatment on bone nanostructure in a rat ovariectomy model was studied using scanning small-angle X-ray scattering (sSAXS). Twelve 6-month-old female Wistar rats were used. Six animals were ovariectomized (+ovx) and six were left intact after sham surgery (-ovx). Six animals, three +ovx and three -ovx, were treated with 4 mmol SrCl(2) (aq)/kg/day (+Sr), whereas the remaining six received placebo (-Sr) for 140 days. Rats were labeled with flourochromes at days 7, 126, and 136. Femoral cross sections were studied using fluorescence microscopy, scanning electron microscopy including energy-dispersive X-ray analysis, and sSAXS. The SAXS data comprised about 5,500 measurements and provided information about mineral crystal thickness and orientation in new and old bone. The newly formed bone contained higher levels of Sr(2+) in +Sr than in -Sr animals, indicating that the Sr(2+) was incorporated into the new bone. Mineral plates were significantly thicker in old bone, 2.62 nm (95% CI 2.58-2.66), than in new bone, 2.41 nm (95% CI 2.36-2.46). Surprisingly, mineral plates in new bone were significantly thicker (2.52 [95% CI 2.47-2.57] nm vs. 2.41 [95% CI 2.36-2.46] nm, P = 0.017) in +ovx rats than in -ovx rats. However, no significant effect of SrCl(2) on mineral plate thicknesses in new bone was observed. The statistical model yielded estimates of the difference in bone mineral plate thickness induced by Sr. The estimated effect of Sr was -0.09 (95% CI -0.21 to 0.03) and 0.02 (95% CI -0.10 to 0.14) nm for new bone in -ovx and +ovx rats, respectively.

A novel acidification mechanism for greatly enhanced oxygen supply to the fish retina.

Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

Retinal oxygen supply shaped the functional evolution of the vertebrate eye.

The retina has a very high energy demand but lacks an internal blood supply in most vertebrates. Here we explore the hypothesis that oxygen diffusion limited the evolution of retinal morphology by reconstructing the evolution of retinal thickness and the various mechanisms for retinal oxygen supply, including capillarization and acid-induced haemoglobin oxygen unloading. We show that a common ancestor of bony fishes likely had a thin retina without additional retinal oxygen supply mechanisms and that three different types of retinal capillaries were gained and lost independently multiple times during the radiation of vertebrates, and that these were invariably associated with parallel changes in retinal thickness. Since retinal thickness confers multiple advantages to vision, we propose that insufficient retinal oxygen supply constrained the functional evolution of the eye in early vertebrates, and that recurrent origins of additional retinal oxygen supply mechanisms facilitated the phenotypic evolution of improved functional eye morphology.

Septins are critical regulators of osteoclastic bone resorption.

Septins are known to play key roles in supporting cytoskeletal stability, vesicular transport, endo-/exocytosis, stabilizing cellular membranes and forming diffusion barriers. Their function in mammalian cells is poorly investigated. The osteoclast offers an interesting tool to investigate septins because all cellular activities septins were reported to be involved in are critical for osteoclasts. However, the existence of septins in osteoclasts has not even been reported. Here we show that the SEPT9 gene and Septin 9 (SEPT9) protein are expressed and synthesized during differentiation of human osteoclasts. Pharmacological stabilization of septin filaments dose dependently inhibits bone resorption of human osteoclasts in vitro suggesting a role for septins in bone resorption. Attesting to this, conditional deletion of Sept9 in mice leads to elevated levels of trabecular bone and diminished femoral growth in vivo. Finally, systematic interrogation of the spatial organization of SEPT9 by confocal microscopy reveals that SEPT9 is closely associated to the structures known to be critical for osteoclast activity. We propose that septins in general and SEPT9 in particular play a previously unappreciated role in osteoclastic bone resorption.

Cancellous bone structure of iliac crest biopsies following 370 days of head-down bed rest.

INTRODUCTION: Static bone histomorphometry was applied to existing iliac bone sections originating from a 370-d 5 degrees head-down bed rest experiment. This bed rest experiment is the longest ever to have been conducted. We hypothesized that bed rest would decrease cancellous bone volume fraction and that this effect would be reversed by countermeasures. METHODS: Eight healthy male subjects underwent 370 d of 5 degrees head-down bed rest. Three subjects were treated with bisphosphonate (Xidifon, potassium salt of ethane-1-hydroxy-1-disphosphonate, EHDP) combined with an exercise regimen (1-2 h x d(-1)) for the entire study period. Five subjects underwent 120 d of bed rest without countermeasures followed by 250 d of bed rest with the exercise regimen. Transiliac bone biopsies were obtained either at baseline and day 366, or at baseline, day 116, and day 366 at alternating sides of the ileum. Static histomorphometry was performed using a computerized method. RESULTS: The 120 d of head-down bed rest without countermeasures resulted in decreased bone volume fraction BV/TV (-6.3%, p = 0.046) and trabecular number (Tb.N; -10.2%, p = 0.080) and increased trabecular separation (Tb.Sp; 14.7%, p = 0.020), whereas the 250 d of subsequent head-down bed rest with exercise treatment prevented further significant deterioration of the histomorphometric measures. DISCUSSION: The 120 d of 5 degrees head-down bed rest without countermeasures induced significant deterioration of iliac crest cancellous bone histomorphometric properties. On average, the countermeasures consisting of either bisphosphonate and exercise, or exercise alone were able to either prevent or stop immobilization-induced changes of the iliac cancellous bone structure.

The influence of hemostatic agents on bone healing after sternotomy in a porcine model.

BACKGROUND: Sternotomy is the preferred access to the mediastinum. During sternotomy, trabecular bone is exposed, often resulting in bleeding, which can be treated with mechanical hemostatic agents; however, their influence on the healing process is relatively unexplored. The aim of this study was to investigate the influence of two hemostatic agents: bone wax (BW) and a water-soluble polymer wax, Ostene (WSW), on the mechanical and histologic characteristics of healing sternal bone. METHODS: Twenty-four pigs underwent sternotomy and were randomized into three groups: WSW, BW, or no hemostatic treatment (control). Bone samples were obtained 6 weeks postoperatively. RESULTS: Fracture strength (Fmax) and maximum stiffness (dF/dx) was lower in the BW group than in controls (Fmax: 175.2 vs. 255.8 N, dF/dx: 165.2 vs. 375.4 N/mm,) (p < 0.05). The stiffness did not differ statistically between the WSW and BW groups (298.4 vs 165.2 N/mm) nor did the fracture strength (211.4 vs 175.2 N). The fraction of granulomatous tissue was higher in the BW group compared with both the WSW group (79.1 vs. 16.52%) (p < 0.001) and controls (79.1 vs. 11.2%) (p < 0.001). There was more calcified tissue in controls than in the BW group (23.4 vs. 10.8%) (p < 0.05). CONCLUSIONS: In a porcine model, BW significantly inhibited sternal healing and was associated with chronic inflammation and reduced mechanical integrity. The WSW did not, to the same degree as BW, inhibit bone healing and thus presents an alternative treatment option for sternal bleeding.

Towards Automated Three-Dimensional Tracking of Nephrons through Stacked Histological Image Sets.

An automated approach for tracking individual nephrons through three-dimensional histological image sets of mouse and rat kidneys is presented. In a previous study, the available images were tracked manually through the image sets in order to explore renal microarchitecture. The purpose of the current research is to reduce the time and effort required to manually trace nephrons by creating an automated, intelligent system as a standard tool for such datasets. The algorithm is robust enough to isolate closely packed nephrons and track their convoluted paths despite a number of nonideal, interfering conditions such as local image distortions, artefacts, and interstitial tissue interference. The system comprises image preprocessing, feature extraction, and a custom graph-based tracking algorithm, which is validated by a rule base and a machine learning algorithm. A study of a selection of automatically tracked nephrons, when compared with manual tracking, yields a 95% tracking accuracy for structures in the cortex, while those in the medulla have lower accuracy due to narrower diameter and higher density. Limited manual intervention is introduced to improve tracking, enabling full nephron paths to be obtained with an average of 17 manual corrections per mouse nephron and 58 manual corrections per rat nephron.

The effect of haemostatic devices on bone healing 6 months postoperatively in sternotomized pigs.

OBJECTIVES: Bone wax is frequently used to diminish bleeding after sternotomy. Water-soluble polymer wax has been shown to diminish postoperative bleeding and, unlike traditional bone wax, to be absorbed and removed by the organism in an unchanged state. We have previously shown that bone wax impairs early bone healing after sternotomy, whereas polymer wax does not. This difference was observed 6 weeks postoperatively and questions arose as to whether these effects were long term. Therefore, we hypothesized that bone wax impairs bone healing in sternotomized pigs 6 months postoperatively, whereas polymer wax does not. METHODS: Fourteen Landrace/Yorkshire pigs were sternotomized and then randomly assigned to haemostasis by either bone wax (WAX-group) or water-soluble polymer wax (POL-group). After 6 months, the pigs were euthanized and the sternum was removed and prepared for further assessment. Bone fracture strength and bone stiffness were determined using a modified three-point bending test, whereas bone healing was examined by means of quantitative histology. Six pigs died before the end of the study due to failure to thrive, valve prosthesis endocarditis and coronary artery occlusion. RESULTS: The mechanical testing showed no difference between groups with regard to fracture strength [WAX-group versus POL-group; 214.8 (85.5-478.5) vs 203.8 (90.4-478.5) N, P = 0.986] or maximum stiffness [213.0 (81.5-409.5) vs 348.5 (23.3-689.5) N/mm, P = 0.128]. Histology showed predominance of fibroblast-covered surfaces [10.6% (1.8-23.3%) vs 4.1% (0.0-13.0%), P < 0.001] and fibrous tissue volume [45.4% (6.9-82.0%) vs 17.4% (2.9-55.0%), P < 0.001] in animals treated with bone wax. The volume fraction of calcified bone tended to be higher in the POL-group [26.8% (4.3-35.8%) vs 16.7% (1.5-35.8%), P = 0.065]. Granulomas comprised 12.5% (0.0-78.9%) of the volume fraction in the WAX-group compared with 0.0% (0.0-0.0%) in the POL-group (P < 0.001). CONCLUSION: Bone wax and water-soluble polymer wax had similar long-term effects on bone mechanical properties. Histology confirmed our hypothesis and showed a more extensive foreign body reaction in animals treated with bone wax than in those treated with water-soluble polymer wax.

Prevention of bone loss in ovariectomized rats by combined treatment with risedronate and 1alpha,25-dihydroxyvitamin D3.

Bisphosphonates inhibit bone loss through inhibition of osteoclast-mediated bone resorption. At low doses, vitamin D metabolites can prevent bone loss in models of osteopenia in rats by an antiresorptive effect, while at high doses they also stimulate osteoblast activity and show an anabolic effect. Therefore, combined therapy with bisphosphonates and vitamin D analogs might be expected to be more effective than either treatment alone. It was the aim of this study to compare the efficacy of risedronate and of the naturally occurring vitamin D hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol), alone and in combination, for the prevention of ovariectomy-induced bone loss in rats. One hundred ten female 4-month-old Sprague-Dawley rats were used for this experiment. Ninety rats were bilaterally ovariectomized (OVX), 10 rats were sham-operated (SHAM), and 10 rats were killed at the time of surgery as a baseline control. Groups of rats (10 rats/group) received vehicle or daily doses of 0.1 mg or 0.5 mg of risedronate or 0.05 microg or 0.1 microg of calcitriol/kg body weight, alone and in combination. Both compounds were administered orally via gavage, commencing on the day after surgery. Although estrogen deficiency-induced bone loss was prevented by individual prophylactic administration of risedronate or calcitriol, OVX rats treated with a combination of risedronate and calcitriol had higher bone mineral density (BMD), cancellous bone area (B.Ar), and bone strength in long bones and vertebrae compared with rats receiving risedronate alone. Furthermore, calcitriol enhanced the suppressive effects of risedronate on osteoclast number and partially counteracted the suppressive effects of risedronate on bone formation and histomorphometric indices of osteoblast team performance. Risedronate did not reduce the anabolic effect of calcitriol, and at the high dose it normalized hypercalcemia in calcitriol-treated OVX rats. Therefore, this study in OVX rats suggests that combined therapy with bisphosphonates and vitamin D analogs may offer advantages over the treatment with bisphosphonates or vitamin D analogs alone.