The proton-activated ovarian cancer G protein-coupled receptor 1 (OGR1) is responsible for renal calcium loss during acidosis.

Hypercalciuria is a common feature during metabolic acidosis and associates to nephrolithiasis and nephrocalcinosis. The mechanisms sensing acidosis and inducing increased urinary calcium excretion are still unknown. Here we tested whether mice deficient for proton-activated Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) have reduced urinary excretion of calcium during chronic metabolic acidosis. In the kidney, OGR1 mRNA was found in cells of the glomerulus, proximal tubule, and interstitium including endothelial cells. Wild type (OGR1+/+) and OGR1 knockout (OGR1-/-) mice were given standard chow without (control) or loaded with ammonium chloride for one or seven days to induce acute or chronic metabolic acidosis, respectively. No differences in responding to the acid load were observed in the knockout mice, except for higher plasma bicarbonate after one day. Bone mineral density, resorption activity of osteoclasts, and urinary deoxypyridinoline were similar between genotypes. During metabolic acidosis the expression levels of key proteins involved in calcium reabsorption, i.e. the sodium/proton exchanger (NHE3), the epithelial calcium-selective channel TRPV5, and the vitamin D-dependent calcium binding protein calbindin-D28k were all higher in the knockout mice compared to wild type mice. This is consistent with the previous demonstration that OGR1 reduces NHE3 activity in proximal tubules of mice. Wild-type mice displayed a non-linear positive association between urinary proton and calcium excretion which was lost in the knockout mice. Thus, OGR1 is a pH sensor involved in the hypercalciuria of metabolic acidosis by controlling NHE3 activity in the proximal tubule. Hence, novel drugs modulating OGR1 activity may improve renal calcium handling.

Effect of chronic metabolic acidosis on bone density and bone architecture in vivo in rats.

Chronic metabolic acidosis (CMA) might result in a decrease in vivo in bone mass based on its reported in vitro inhibition of bone mineralization, bone formation, or stimulation of bone resorption, but such data, in the absence of other disorders, have not been reported. CMA also results in negative nitrogen balance, which might decrease skeletal muscle mass. This study analyzed the net in vivo effects of CMA's cellular and physicochemical processes on bone turnover, trabecular and cortical bone density, and bone microarchitecture using both peripheral quantitative computed tomography and µCT. CMA induced by NH4Cl administration (15 mEq/kg body wt/day) in intact and ovariectomized (OVX) rats resulted in stable CMA (mean Δ[HCO3(-)]p = 10 mmol/l). CMA decreased plasma osteocalcin and increased TRAP5b in intact and OVX animals. CMA decreased total volumetric bone mineral density (vBMD) after 6 and 10 wk (week 10: intact normal +2.1 ± 0.9% vs. intact acidosis -3.6 ± 1.2%, P < 0.001), an effect attributable to a decrease in cortical thickness and, thus, cortical bone mass (no significant effect on cancellous vBMD, week 10) attributed to an increase in endosteal bone resorption (nominally increased endosteal circumference). Trabecular bone volume (BV/TV) decreased significantly in both CMA groups at 6 and 10 wk, associated with a decrease in trabecular number. CMA significantly decreased muscle cross-sectional area in the proximal hindlimb at 6 and 10 wk. In conclusion, chronic metabolic acidosis induces a large decrease in cortical bone mass (a prime determinant of bone fragility) in intact and OVX rats and impairs bone microarchitecture characterized by a decrease in trabecular number.

Proton-sensing G-protein-coupled receptors.

Blood pH is maintained in a narrow range around pH 7.4 mainly through regulation of respiration and renal acid extrusion. The molecular mechanisms involved in pH homeostasis are not completely understood. Here we show that ovarian cancer G-protein-coupled receptor 1 (OGR1), previously described as a receptor for sphingosylphosphorylcholine, acts as a proton-sensing receptor stimulating inositol phosphate formation. The receptor is inactive at pH 7.8, and fully activated at pH 6.8-site-directed mutagenesis shows that histidines at the extracellular surface are involved in pH sensing. We find that GPR4, a close relative of OGR1, also responds to pH changes, but elicits cyclic AMP formation. It is known that the skeleton participates in pH homeostasis as a buffering organ, and that osteoblasts respond to pH changes in the physiological range, but the pH-sensing mechanism operating in these cells was hitherto not known. We detect expression of OGR1 in osteosarcoma cells and primary human osteoblast precursors, and show that these cells exhibit strong pH-dependent inositol phosphate formation. Immunohistochemistry on rat tissue sections confirms the presence of OGR1 in osteoblasts and osteocytes. We propose that OGR1 and GPR4 are proton-sensing receptors involved in pH homeostasis.

TGFbeta3 and loading increases osteocyte survival in human cancellous bone cultured ex vivo.

The goal of this study was to assess the effect of the addition of TGFbeta(3), alone or in combination with loading, on the survival of osteocytes in 3D human explant cancellous bone during long-term culture in an ex vivo loading bioreactor. Human cancellous bone explants were cultured for up to 14 days with or without TGFbeta(3) (15 ng ml(-1)) and with or without loading (300 cycles, at 1 Hz, producing 4000 microstrain). Bone core response was visualized using undecalcified histology with morphological methods after embedding with Technovit 9100 New resin. Histological examination revealed normal gross level bone structure with or without the application of load or the addition of TGFbeta(3). The viability of the osteocytes within the bone was assessed by lactate dehydrogenase (LDH) activity. We demonstrate that this ex vivo loading bioreactor is able to maintain a high percentage (over 50%) of viable osteocytes throughout the bone explants after 14 days in ex vivo culture. Further to this, the combination of daily loading and TGFbeta(3) administration produced superior osteocyte survival at the core centres when compared to loading or TGFbeta alone.

A cathepsin K inhibitor reduces breast cancer induced osteolysis and skeletal tumor burden.

Osteoclasts mediate bone destruction in breast cancer skeletal metastases. Cathepsin K is a proteinase that is secreted by osteoclasts and degrades bone. Here, immunohistochemistry revealed that cathepsin K was expressed not only by osteoclasts but also by breast cancer cells that metastasize to bone. Following intratibial injection with cathepsin K-expressing human BT474 breast cancer cells, tumor-bearing mice treated with a clinical dosing regimen of cathepsin K inhibitor (CKI; 50 mg/kg, twice daily) had osteolytic lesions that were 79% smaller than those of tumor-bearing mice treated with the vehicle. The effect of CKI was also studied in a mouse model in which the i.v. inoculation of human B02 breast cancer cells expressing cathepsin K leads to bone metastasis formation. Drug administration was started before (preventive protocol) or after (treatment protocol) the occurrence of osteolytic lesions. In treatment protocols, CKI (50 mg/kg, twice daily) or a single clinical dose of 100 microg/kg zoledronic acid (osteoclast inhibitor) reduced the progression of osteolytic lesions by 59% to 66%. CKI therapy also reduced skeletal tumor burden by 62% compared with vehicle, whereas zoledronic acid did not decrease the tumor burden. The efficacy of CKI at inhibiting skeletal tumor burden was similar in the treatment and preventive protocols. By contrast, CKI did not block the growth of s.c. B02 tumor xenografts in animals. Thus, CKI may render the bone a less favorable microenvironment for tumor growth by inhibiting bone resorption. These findings raise the possibility that cathepsin K could be a therapeutic target for the treatment of bone metastases.

European Society of Biomechanics S.M. Perren Award 2008: using temporal trends of 3D bone micro-architecture to predict bone quality.

In longitudinal studies, three-dimensional (3D) bone images are acquired at sequential time points essentially resulting in four-dimensional (4D) data for an individual. Based on the 4D data, we propose to calculate temporal trends and project these trends to estimate future bone architecture. Multiple consecutive deformation fields, calculated with Demons deformable image registration algorithm, were extrapolated on a voxel-by-voxel basis. Test data were from in vivo micro-computed tomography (microCT) scans of the proximal tibia of Wistar rats that were either ovariectomized (OVX; N=5) or sham operated (SHAM; N=6). Measurements performed at baseline, 4 and 8 weeks were the basis to predict the 12 week data. Predicted and actual 12 week data were compared using qualitative (3D rendering) and quantitative (geometry, morphology and micro-finite element, microFE) methods. The results indicated a voxel-based linear extrapolation scheme yielded mean geometric errors that were smaller than the voxel size of 15 microm. Key morphological parameters that were estimated included bone volume ratio (BV/TV; mean error 0.4%, maximum error 9%), trabecular thickness (Tb.Th; -1.1%, 11%), connectivity density (Conn.D; 9.0%, 18.5%) and the apparent Young's modulus (E(1); 6.0%, 32%). These data demonstrated a promising and novel approach for quantitatively capturing in vivo bone dynamics at the local trabecular level. The method does not require an a priori understanding of the diseases state, and can provide information about the trends of the bone remodeling process that may be used for better monitoring and treatment of diseases such as osteoporosis.

Dlk1/FA1 is a novel endocrine regulator of bone and fat mass and its serum level is modulated by growth hormone.

Fat and bone metabolism are two linked processes regulated by several hormonal factors. Fetal antigen 1 (FA1) is the soluble form of dlk1 (delta-like 1), which is a member of the Notch-Delta family. We previously identified FA1 as a negative regulator of bone marrow mesenchymal stem cell differentiation. Here, we studied the effects of circulating FA1 on fat and bone mass in vivo by generating mice expressing high serum levels of FA1 (FA1 mice) using the hydrodynamic-based gene transfer procedure. We found that increased serum FA1 levels led to a significant reduction in total body weight, fat mass, and bone mass in a dose-dependent manner. Reduced bone mass in FA1 mice was associated with the inhibition of mineral apposition rate and bone formation rates by 58 and 72%, respectively. Because FA1 is colocalized with GH in the pituitary gland, we explored the possible modulation of serum FA1 by GH. Serum levels of IGF-I and IGF binding proteins did not change in FA1 mice, whereas increasing serum GH in normal mice using hydrodynamic-based gene transfer procedure dramatically reduced serum FA1 levels by 60%. Conversely, serum FA1 was increased 450% in hypophysectomized mice, and this high level was reduced by 40% during GH treatment. In conclusion, our data identify the FA1 as a novel endocrine factor regulating bone mass and fat mass in vivo, and its serum levels are regulated by GH. FA1 thus provides a novel class of developmental molecules that regulate physiological functions of the postnatal organisms.

The relative merits of anabolics versus anti-resorptive compounds: where our targets should be, and whether we are addressing them.

Currently available results from fracture trials provide evidence that the most potent anti-resorptive agents reduce vertebral and non-vertebral fractures maximally by 61% and 51%, respectively. Results from the Phase III trial with denosumab, the human monoclonal antibody, are eagerly awaited. Denosumab leads to sustained 80-90% reduction of bone resorption markers, which is below the level commonly achieved with bisphosphonates, and it will be interesting to see whether this leads to an improvement in its anti-fracture efficacy over bisphosphonates. If the majority of the anti-fracture efficacy of anti-resorptive agents results from the reduction of the remodelling space (removal of stress raisers) and the conservation of structural integrity of cancellous bone, a further decrease in bone resorption might not be desirable, especially as suppression of the residual remodelling capacity could lead to an increased risk for accumulation of microdamage. In contrast to anti-resorptive agents, the bone anabolic parathyroid hormone activates modelling drifts, which act to increase trabecular thickness and add bone predominantly on the endocortical, and to a lesser degree the periosteal, surface. Despite its anabolic nature, reduction of vertebral and non-vertebral fractures is only marginally better than those achieved with anti-resorptive agents. Ageing compromises locomotor capacity and is associated with an increased risk of falls. Perhaps it is time to shift our attendance to the age-related deterioration of muscle or neuromuscular function as a target and add this 'adjuvant therapy' to the potent anti-remodelling and bone anabolic agents available for the treatment of osteoporosis if we truly want to reduce fracture incidence beyond what is possible today.

Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography.

The ovariectomized (OVX) rat is a well established model for osteoporosis research. The recent development of in vivo micro-computed tomography (micro-CT) provides new possibilities to monitor individual bone changes over time. The purpose of this study was to establish the normal time course of bone loss in the OVX rat model, and to determine the ability to detect morphological changes in vivo compared to cross-sectional study designs where animals are sacrificed at each time point. Eight-month-old female Wistar rats were randomly assigned to one of two groups: OVX (N = 10) or sham-operated (N = 10). In vivo micro-CT scanning of the right proximal tibial metaphyses occurred at 1-month intervals for 6 months. Morphological analyses were performed at each time step for every animal, and a two-way ANOVA with repeated measures was used to analyze the data. A second statistical analysis was performed without repeated measures for analysis as a cross-sectional study design. The repeated measures analysis was more sensitive to early changes than the cross-sectional study analysis. Changes were detected by longitudinal analysis in the sham-operated and OVX animals over time (P < 0.001) with the exception of trabecular separation in the sham animals. The OVX animals had decreases of bone volume ratio of 33% after 1 month, and 72% after 3 months relative to baseline measurements. Significant changes in bone volume ratio, trabecular number and separation were detected early using a longitudinal analysis, thus in vivo assessment is well poised to enable the study of early treatment protocols on the effects of bone architecture. The in vivo analysis found significant changes in the sham animals which were not detected by the cross-sectional analysis, and the changes to the OVX animal morphology was detected sooner. A substantial variation of baseline morphometry within the homogenous group of rats and response to OVX was observed, thus emphasizing the advantage of performing in vivo analysis where each animal acts as its own control. These data provide new insight into individual bone changes following OVX, and can be used as baseline information upon which future in vivo studies can be designed.

A single intravenous administration of zoledronic acid prevents the bone loss and mechanical compromise induced by aromatase inhibition in rats.

Recent evidence has demonstrated that long-term estrogen deprivation using aromatase inhibitor therapy in postmenopausal women with breast cancer results in bone loss and increased fracture risk. Bisphosphonates are potent inhibitors of bone resorption and have demonstrated efficacy in preventing bone loss in postmenopausal women with low bone mineral density (BMD) and in patients with breast cancer receiving estrogen deprivation therapy. Therefore, this study investigated the effects of the bisphosphonate zoledronic acid on BMD and bone strength in rats treated with the aromatase inhibitor, letrozole. Peripheral quantitative computed tomography demonstrated that treatment of rats with daily oral letrozole (1 mg/kg) induced significant bone loss and cortical thinning compared with control animals (P < 0.01). A single prior intravenous dose of zoledronic acid dose dependently protected against letrozole-induced bone loss and cortical thinning, with the highest evaluated dose (20 microg/kg) resulting in BMD values that were not significantly different from controls over the 24 weeks of letrozole treatment. Furthermore, biomechanical testing of the distal femoral metaphysis demonstrated that zoledronic acid (20 microg/kg) significantly prevented the decrease in stiffness and elastic modulus induced by letrozole treatment. Taken together, these data support the use of zoledronic acid for the prevention of bone loss in women with breast cancer receiving aromatase inhibitor therapy.

Annual intravenous zoledronic acid for three years increased cancellous bone matrix mineralization beyond normal values in the HORIZON biopsy cohort.

The efficacy of 3 years of annual intravenous administration of zoledronic acid (ZOL) in reducing vertebral and nonvertebral fractures in postmenopausal osteoporosis has been shown by the HORIZON pivotal fracture trial. Histomorphometric analysis of transiliac bone biopsies from the HORIZON participants revealed significantly improved trabecular architecture and reduced bone remodeling for the ZOL-treated versus placebo-treated patients. The aim of our study was to evaluate the cancellous and cortical bone mineralization density distribution (BMDD) in these biopsies by quantitative backscattered electron imaging (qBEI). The study cohort comprised 82 patients on active treatment (ZOL, yearly doses of 5 mg) and 70 treated with placebo, and all received adequate Ca and VitD supplementation. Comparison of ZOL-treated versus placebo-treated cancellous (Cn.) and cortical (Ct.) BMDD-derived variables resulted in significantly higher average (Cn.CaMean + 3.2%, Ct.CaMean + 2.7%) and mode calcium concentrations (Cn.CaPeak + 2.1%, Ct.CaPeak + 1.5%), increased percentages of highly mineralized bone areas (Cn.CaHigh + 64%, Ct.CaHigh + 31%), lower heterogeneity of mineralization (Cn.CaWidth -14%, Ct.CaWidth -13%), and decreased percentages of low mineralized bone areas (Cn.CaLow -22%, Ct.CaLow -26%) versus placebo (all p < 0.001). Cn. BMDD from the patients on active treatment also revealed a statistically significant shift to higher Ca concentrations when compared to a historical normal reference BMDD. These differences in BMDD from ZOL patients compared to the other groups were in line with the correlation of BMDD variables with previously determined cancellous mineralizing surface per bone surface (Cn. MS/BS, a primary histomorphometric index for bone turnover), showing that those with lower Cn. MS/BS had a higher degree of bone matrix mineralization. However, the differences in BMDD variables between the study groups remained when adjusted for Cn. MS/BS, suggesting that other factors in addition to reduced bone turnover might contribute to the higher bone matrix mineralization after ZOL treatment.

Bone measurements by peripheral quantitative computed tomography in rodents.

This chapter provides information for the use of peripheral quantitative computed tomography in small animals, including suggestions for study design, instrument setting, and data interpretation.

Effects of intravenous zoledronic acid plus subcutaneous teriparatide [rhPTH(1-34)] in postmenopausal osteoporosis.

Clinical data suggest concomitant therapy with bisphosphonates and parathyroid hormone (PTH) may blunt the anabolic effect of PTH; rodent models suggest that infrequently administered bisphosphonates may interact differently. To evaluate the effects of combination therapy with an intravenous infusion of zoledronic acid 5 mg and daily subcutaneous recombinant human (rh)PTH(1-34) (teriparatide) 20 µg versus either agent alone on bone mineral density (BMD) and bone turnover markers, we conducted a 1-year multicenter, multinational, randomized, partial double-blinded, controlled trial. 412 postmenopausal women with osteoporosis (mean age 65 ± 9 years) were randomized to a single infusion of zoledronic acid 5 mg plus daily subcutaneous teriparatide 20 µg (n = 137), zoledronic acid alone (n = 137), or teriparatide alone (n = 138). The primary endpoint was percentage increase in lumbar spine BMD (assessed by dual-energy X-ray absorptiometry [DXA]) at 52 weeks versus baseline. Secondary endpoints included change in BMD at the spine at earlier time points and at the total hip, trochanter, and femoral neck at all time points. At week 52, lumbar spine BMD had increased 7.5%, 7.0%, and 4.4% in the combination, teriparatide, and zoledronic acid groups, respectively (p < .001 for combination and teriparatide versus zoledronic acid). In the combination group, spine BMD increased more rapidly than with either agent alone (p < .001 versus both teriparatide and zoledronic acid at 13 and 26 weeks). Combination therapy increased total-hip BMD more than teriparatide alone at all times (all p < .01) and more than zoledronic acid at 13 weeks (p < .05), with final 52-week increments of 2.3%, 1.1%, and 2.2% in the combination, teriparatide, and zoledronic acid groups, respectively. With combination therapy, bone formation (assessed by serum N-terminal propeptide of type I collagen [PINP]) increased from 0 to 4 weeks, declined minimally from 4 to 8 weeks, and then rose throughout the trial, with levels above baseline from 6 to 12 months. Bone resorption (assessed by serum ß-C-telopeptide of type I collagen [ß-CTX]) was markedly reduced with combination therapy from 0 to 8 weeks (a reduction of similar magnitude to that seen with zoledronic acid alone), followed by a gradual increase after week 8, with levels remaining above baseline for the latter half of the year. Levels for both markers were significantly lower with combination therapy versus teriparatide alone (p < .002). Limitations of the study included its short duration, lack of endpoints beyond DXA-based BMD (e.g., quantitative computed tomography and finite-element modeling for bone strength), lack of teriparatide placebo, and insufficient power for fracture outcomes. We conclude that while teriparatide increases spine BMD more than zoledronic acid and zoledronic acid increases hip BMD more than teriparatide, combination therapy provides the largest, most rapid increments when both spine and hip sites are considered.

Long-term protective effects of zoledronic acid on cancellous and cortical bone in the ovariectomized rat.

UNLABELLED: Current bisphosphonate therapies effectively prevent bone loss in postmenopausal women. We studied the effect of a single intravenous dose of ZOL in ovariectomized rats. Protection from bone loss was dose dependent, lasting for up to 32 weeks, supporting the rationale for an annual intravenous dosing regimen of ZOL for treatment of postmenopausal osteoporosis. INTRODUCTION: Once-yearly dosing with zoledronic acid (ZOL) 5 mg can increase BMD and reduce fracture rate in postmenopausal women with low BMD. The primary objective of this study was to determine the duration of bone protective effects of a single dose of ZOL in ovariectomized rats, an animal model of postmenopausal osteopenia. Secondary objectives were to determine the effects on bone turnover and mechanical properties. MATERIALS AND METHODS: Female Wistar rats (10 per group) received single intravenous doses of ZOL 0.8, 4, 20, 100, or 500 microg/kg, alendronate 200 microg/kg, or isotonic saline 4 days before bilateral ovariectomy. Sham-operated controls were pretreated with saline. Mass and density of cancellous and cortical bone (pQCT) were measured at 4-wk intervals for 32 wk. Bone architecture (microCT), bone formation dynamics (fluorochrome label-based histomorphometry), and biomechanical strength in compression testing were also assessed at 32 wk. RESULTS: Ovariectomy-associated BMD loss was significantly attenuated for 32 wk by ZOL >or=4 microg/kg for total BMD, ZOL >or=20 microg/kg for cortical BMD, and ZOL >or=4 microg/kg for cancellous BMD (p < 0.01 versus ovariectomized controls). Alendronate 200 microg/kg was of equivalent potency to ZOL 20 microg/kg. Ovariectomy-associated decreases in trabecular architectural parameters were dose-dependently attenuated by ZOL. Alendronate 200 microg/kg was equivalent to ZOL 20 microg/kg. The bone resorption marker TRACP5b indicated transient suppression of elevated osteoclast activity by ZOL relative to OVX-rats even at the lowest dose of 0.8 microg/kg, whereas at 100-500 microg/kg, the effect was significant relative to the OVX control for the entire duration of the study of 32 wk. Bone formation parameters were not significantly affected by ZOL 20 microg/kg but were significantly reduced by ZOL 100-500 microg/kg. Alendronate 200 microg/kg was equivalent to ZOL 100 microg/kg. ZOL produced dose-related improvements in bone strength parameters after ovariectomy. Alendronate 200 microg/kg was of similar potency to ZOL 20 microg/kg. CONCLUSIONS: The duration and magnitude of the bone-protecting effect of a single intravenous dose of ZOL in ovariectomized rats is dose dependent and lasts for up to 32 wk. Compared with alendronate, ZOL shows 10-fold higher potency in preventing bone loss. These data support the use of an annual intravenous ZOL dosing regimen for the treatment of osteoporosis.

Models of tibial fracture healing in normal and Nf1-deficient mice.

Delayed union and nonunion are common complications associated with tibial fractures, particularly in the distal tibia. Existing mouse tibial fracture models are typically closed and middiaphyseal, and thus poorly recapitulate the prevailing conditions following surgery on a human open distal tibial fracture. This report describes our development of two open tibial fracture models in the mouse, where the bone is broken either in the tibial midshaft (mid-diaphysis) or in the distal tibia. Fractures in the distal tibial model showed delayed repair compared to fractures in the tibial midshaft. These tibial fracture models were applied to both wild-type and Nf1-deficient (Nf1+/-) mice. Bone repair has been reported to be exceptionally problematic in human NF1 patients, and these patients can also spontaneously develop tibial nonunions (known as congenital pseudarthrosis of the tibia), which are recalcitrant to even vigorous intervention. pQCT analysis confirmed no fundamental differences in cortical or cancellous bone in Nf1-deficient mouse tibiae compared to wild-type mice. Although no difference in bone healing was seen in the tibial midshaft fracture model, the healing of distal tibial fractures was found to be impaired in Nf1+/- mice. The histological features associated with nonunited Nf1+/- fractures were variable, but included delayed cartilage removal, disproportionate fibrous invasion, insufficient new bone anabolism, and excessive catabolism. These findings imply that the pathology of tibial pseudarthrosis in human NF1 is complex and likely to be multifactorial.

Spatial and temporal patterns of bone formation in ectopically pre-fabricated, autologous cell-based engineered bone flaps in rabbits.

Biological substitutes for autologous bone flaps could be generated by combining flap pre-fabrication and bone tissue engineering concepts. Here, we investigated the pattern of neotissue formation within large pre-fabricated engineered bone flaps in rabbits. Bone marrow stromal cells from 12 New Zealand White rabbits were expanded and uniformly seeded in porous hydroxyapatite scaffolds (tapered cylinders, 10-20 mm diameter, 30 mm height) using a perfusion bioreactor. Autologous cell-scaffold constructs were wrapped in a panniculus carnosus flap, covered by a semipermeable membrane and ectopically implanted. Histological analysis, substantiated by magnetic resonance imaging (MRI) and micro-computerized tomography scans, indicated three distinct zones: an outer one, including bone tissue; a middle zone, formed by fibrous connective tissue; and a central zone, essentially necrotic. The depths of connective tissue and of bone ingrowth were consistent at different construct diameters and significantly increased from respectively 3.1+/-0.7 mm and 1.0+/-0.4 mm at 8 weeks to 3.7+/-0.6 mm and 1.4+/-0.6 mm at 12 weeks. Bone formation was found at a maximum depth of 1.8 mm after 12 weeks. Our findings indicate the feasibility of ectopic pre-fabrication of large cell-based engineered bone flaps and prompt for the implementation of strategies to improve construct vascularization, in order to possibly accelerate bone formation towards the core of the grafts.

Conditional transgene expression mediated by the mouse beta-actin locus.

Transgenic mice are an effective model to study gene function in vivo; however, position effects can complicate tissue-specific transgene analysis. To facilitate precise targeting of a transgenic construct into the mouse genome, we combined the Cre/lox and Flp/FRT recombination systems to allow for rapid transgene replacement and conditional transgene expression from the endogenous beta-actin locus. Flp/FRT recombination was used to rapidly exchange FRT-flanked transgene cassettes by recombinase-mediated cassette exchange in embryonic stem cells, while transgene expression can be activated in mice after Cre-mediated excision of a floxed STOP cassette. To validate our system, we analyzed the expression profile of an EGFP reporter gene after integration into the beta-actin locus and Cre-mediated excision of the floxed STOP cassette. Breeding of EGFP reporter mice with various Cre mouse lines resulted in the expected expression profiles, demonstrating the feasibility of the model to facilitate predictable and strong transgene expression in a spatially and temporally controlled manner.