PLIF surgery with titanium-coated PEEK or uncoated PEEK cages: a prospective randomised clinical and radiological study.

PURPOSE: This study aimed to compare clinical results and fusion rates of uncoated polyetheretherketone (PEEK) cages with titanium-coated PEEK cages in posterior lumbar interbody fusion (PLIF) surgery. METHODS: A prospective randomised study including 60 patients with one- or two-segment lumbar degenerative diseases. Patients received either titanium-coated PEEK cages (group A) or uncoated PEEK cages (group B). Fusion rates were evaluated on plain X-rays and CT scans after 6 and 12 months. Clinical follow-up (visual analogue scale, VAS; Oswestry Disability Index score, ODI; EQ-5D) was performed for 24 months. RESULTS: Fifty-five patients (92%) (36 female, 19 male) had a complete follow-up. There were no statistically significant differences in demographic, peri- or intraoperative data between groups A and B. ODI, VAS and EQ-5D improved significantly (p < 0.001) after surgery without statistically significant differences between the two groups. Overall, 65 operated segments could be evaluated for fusion (group A: 29 segments, group B: 36 segments, p = 0.6). Osseous integration of the cage surface improved significantly (p < 0.001) in both groups between 6 and 12 months after surgery. At 12-month follow-up, neither radiolucency nor signs of instability or dislocation were noted. Fusion was present in CT scans as follows: (a) bone growth through cage pores (A: 100%, B: 100%); (b) bone growth outside the cages (A: 48%, B: 61%; p = 0.3). CONCLUSIONS: PEEK and titanium-coated PEEK cages for PLIF produce equally favourable clinical and radiological results up to 24 months post-surgery. The fusion rate was not different.

Advances in the Preoperative Planning of Revision Trauma Surgery Using 3D Printing Technology.

The management of complex fractures at the time of revision surgery remains one of the most challenging tasks for orthopaedic trauma surgeons. As the major principle of treatment remains to achieve an anatomic reduction and a stable fixation, precise preoperative diagnostics and treatment planning are of utmost importance. Thus, knowledge of the 3-dimensional anatomy of the fracture site and its surrounding tissue is indispensable. However, radiographic tools have thus far mostly been unable to recapitulate the complexity of the fracture site in toto. In recent years, the development of 3-dimensional (3D) printers has led to novel opportunities in preoperative planning of complex operative procedures. Although the application of 3D printers has become increasingly popular in orthopaedic surgery, its implementation in trauma surgery is so far mostly limited to the preoperative planning of surgery in patients with pelvic and acetabular fractures/defects. Moreover, reports describing the advantages using this sophisticated methodology in revision trauma surgery are sparse. In this article, we report our experience using novel 3D printing technologies for the management of revision surgery in orthopaedic trauma. In particular, we describe the benefit of using 3D printing technologies in the preoperative planning of complex revision surgery of the proximal tibia, the elbow joint, the distal femur, the ankle joint, and several others. With the advantage to preoperatively plan the optimal surgical approach, implant placement, and contouring as well as the possibility to anticipate intraoperative difficulties, we believe that this emerging technology is of significant value for revision surgery in orthopaedic trauma.

Targeting of Extracellular RNA Reduces Edema Formation and Infarct Size and Improves Survival After Myocardial Infarction in Mice.

BACKGROUND: Following myocardial infarction (MI), peri-infarct myocardial edema formation further impairs cardiac function. Extracellular RNA (eRNA) released from injured cells strongly increases vascular permeability. This study aimed to assess the role of eRNA in MI-induced cardiac edema formation, infarct size, cardiac function, and survival after acute MI and to evaluate the therapeutic potential of ribonuclease 1 (RNase-1) treatment as an eRNA-degrading intervention. METHODS AND RESULTS: C57BL/6J mice were subjected to MI by permanent ligation of the left anterior descending coronary artery. Plasma eRNA levels were significantly increased compared with those in controls starting from 30 minutes after ligation. Systemic application of RNase-1, but not DNase, significantly reduced myocardial edema formation 24 hours after ligation compared with controls. Consequently, eRNA degradation by RNase-1 significantly improved the perfusion of collateral arteries in the border zone of the infarcted myocardium 24 hours after ligation of the left anterior descending coronary artery, as detected by micro-computed tomography imaging. Although there was no significant difference in the area at risk, the area of vital myocardium was markedly larger in mice treated with RNase-1 compared with controls, as detected by Evans blue and 2,3,5-triphenyltetrazolium chloride staining. The increase in viable myocardium was associated with significantly preserved left ventricular function, as assessed by echocardiography. Moreover, RNase-1 significantly improved 8-week survival following MI. CONCLUSIONS: eRNA is an unrecognized permeability factor in vivo, associated with myocardial edema formation after acute MI. RNase-1 counteracts eRNA-induced edema formation and preserves perfusion of the infarction border zone, reducing infarct size and protecting cardiac function after MI.

Neovascularization of osteoporotic metaphyseal bone defects: A morphometric micro-CT study.

PURPOSE: Neovascularization is essential for bone regeneration in fractures. This study aimed to investigate the microvascular morphology and distribution in the non-injured femur and the neovascularization of the metaphyseal critical size defect in a small animal model of osteoporosis. MATERIALS AND METHODS: Female rats (n=7) were ovariectomized (OVX) and received a multideficiency diet. Three months after OVX, a 5mm wedge shaped critical size defect was cut at the distal femoral metaphysis and stabilized with a T-shaped mini-plate. After six weeks, the animals were euthanized, and femora were removed and decalcified for micro-CT measurement of fracture neovascularization. RESULTS: No fracture healing was observed along the critical size defects. In the non-injured bone, micro-vessel distribution showed a specific pattern, thereby enabling a differentiation between epi-, meta- and diaphysis. Micro-CT based morphometry revealed a significant reduction of the vascular volume fraction as well as the vascular thickness (p<0.001) in the critical size defect compared to the intact contralateral femur. Blood volume related vascular surface (vascular surface/volume) increased significantly (p<0.001). Connectivity density and tissue volume related vascular surface (vascular surface density) did not change significantly. CONCLUSIONS: Micro-CT based vascular morphometry demonstrated differences between epi-, meta- and diaphysis in the non-injured bone as well as differences between the critical size defect and the non-injured metaphysis. As angiogenesis is a crucial prerequisite that precedes osteogenesis, our results may influence further evaluation of osteoconductive or osteogenic biomaterials in this small animal model of osteoporosis.

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.

Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.

Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

BACKGROUND: Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT). METHODS: Vertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). RESULTS: Bone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N(+) (p < 0.05) and C4H8N(+) (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. CONCLUSIONS: Our results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7ß2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and ß-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

Systemic atherosclerosis causes detrusor overactivity: functional and morphological changes in hyperlipoproteinemic apoE-/-LDLR-/- mice.

PURPOSE: The prevalence of systemic atherosclerosis and overactive bladder/detrusor overactivity increases almost simultaneously with age but an association between these diseases has not yet been proved. We evaluated changes in bladder function and morphology, including vascularization, in apoE(-/-)LDLR(-/-) double knockout mice with systemic atherosclerosis but without central nervous system involvement. MATERIALS AND METHODS: Cystometry was performed in awake, freely moving 60-week-old apoE(-/-)LDLR(-/-) mice and C57BL/6N controls. The mice were sacrificed and perfused with Microfil® contrast medium. The bladder was excised, dissected and scanned by nano-computerized tomography, including 3-dimensional reconstruction. Samples then underwent histomorphological analysis. RESULTS: In apoE(-/-)LDLR(-/-) mice cystometry revealed a significant decrease in the peak-peak interval, micturition interval, functional bladder capacity and micturition volume. However, maximum bladder pressure increased. Nano-computerized tomography revealed a significant reduction in bladder wall thickness, segment volume, vascular volume and the vascular volume fraction. Histomorphologically bladder specimens showed a thickened media of intramural vessels, activated endothelial cells and intramural inflammatory cells. CONCLUSIONS: To our knowledge this study presents a new in vivo mouse model of nonneurogenic detrusor overactivity caused by systemic atherosclerosis. Decreased bladder wall vascularization seems to be a major factor for detrusor overactivity onset. Capillaries are rarified with reduced lumina due to thickened media. Activated endothelial cells and the infiltration of inflammatory cells in apoE(-/-)LDLR(-/-) mice underlines once more that atherosclerosis is an inflammatory process that may also be relevant to the onset of detrusor overactivity.

Western diet in ApoE-LDLR double-deficient mouse model of atherosclerosis leads to hepatic steatosis, fibrosis, and tumorigenesis.

Nonalcoholic fatty liver disease has been linked to cardiovascular diseases and atherosclerosis. The aim of the current study was to characterize the hepatic pathology leading to fibrosis and tumors in a murine model of atherosclerosis. Male apolipoprotein E/low-density lipoprotein receptor double-knockout mice (AL) mice were fed with a high fat and high cholesterol western diet for 35 weeks (AL mice on WD). Protein and mRNA analysis as well as micro-computed tomography (micro-CT) were performed to assess oxidative stress, liver damage, inflammation, fibrosis, signaling pathways, vascularization, and tumorigenesis. Controls were chosen to distinguish between genetically and dietary effects in steatohepatitis and associated tumorigenesis. Hepatic inflammation and dyslipidemia were increased in AL mice on WD compared with wild-type mice on WD. Uniquely, AL mice on WD showed a spontaneous development of tumors (30% of cases) and thickening of intrahepatic vessel walls. Functionally relevant underlying signaling pathways such as NF-κB, Stat3, JNK, and AKT were differentially regulated between AL and wild-type mice on WD. Micro-CT was capable of visualizing and quantitatively distinguishing tumor neovascularization from vascularization in non-neoplastic liver tissue. AL mice on WD diet represent a novel model combining atherosclerosis and nonalcoholic fatty liver disease. Signaling pathways of liver cell damage and compensatory liver regeneration in combination with enhanced inflammation appear to be crucial for the spontaneous development of tumors in AL mice on WD. Micro-CT represents a new and powerful technique for the ultrastructural and three-dimensional assessment of the vascular architecture of liver tumors.

Change of mechanical vertebrae properties due to progressive osteoporosis: combined biomechanical and finite-element analysis within a rat model.

For assessing mechanical properties of osteoporotic bone, biomechanical testing combined with in silico modeling plays a key role. The present study focuses on microscopic mechanical bone properties in a rat model of postmenopausal osteoporosis. Female Sprague-Dawley rats were (1) euthanized without prior interventions, (2) sham-operated, and (3) subjected to ovariectomy combined with a multi-deficiencies diet. Rat vertebrae (corpora vertebrae) were imaged by micro-CT, their stiffness was determined by compression tests, and load-induced stress states as well as property changes due to the treatment were analyzed by finite-element modeling. By comparing vertebra stiffness measurements with finite-element calculations of stiffness, an overall microscopic Young's modulus of the bone was determined. Macroscopic vertebra stiffness as well as the microscopic modulus diminish with progression of osteoporosis by about 70 %. After strong initial changes of bone morphology, further decrease in macroscopic stiffness is largely due to decreasing microscopic Young's modulus. The micromechanical stress calculations reveal particularly loaded vertebra regions prone to failure. Osteoporosis-induced changes of the microscopic Young's modulus alter the fracture behavior of bone, may influence bone remodeling, and should be considered in the design of implant materials.

Thalidomide influences atherogenesis in aortas of ApoE(-/-)/LDLR (-/-) double knockout mice: a nano-CT study.

Plaque progression in atherosclerosis is closely connected to angiogenesis due to vasa vasorum (VV) growth. Objective of this study was to determine the unknown long-term effect of thalidomide on adventitial VV neovascularization and plaque progression using nano-focussed computed tomography (nano-CT). Proliferation and migration assays in human coronary artery endothelial cells (HCAEC) measured number of viable cells after incubation with thalidomide. Male ApoE(-/-)/LDLR(-/-) (AL) mice (n = 5) received a thalidomide containing western diet (WD) over 29 weeks. Another five male AL mice (WD without thalidomide) served as control group. Descending aortas were scanned with nano-CT at (1.5 µm)(3) isotropic voxel size. Number and area of adventitial VV as well as plaque cross sectional area were measured. Results were complemented by histology. Thalidomide inhibited proliferation and migration of HCAEC dose-dependently. VV neovascularization decreased in number per cross section (7.66 ± 0.301 vs. 8.62 ± 0.164, p < 0.001) and in cross sectional area (0.0183 ± 0.0011 vs. 0.0238 ± 0.0008 mm(2), p < 0.001). Cross sectional area of plaque decreased significantly when treated with thalidomide (0.57 ± 0.0187 vs. 0.803 ± 0.0148 mm(2), p < 0.001). Nano-CT imaging revealed a reduced plaque growth and VV neovascularization after long-term application of thalidomide. Therefore, nano-CT can be considered as a new method to detect therapeutic effects in experimental models of atherosclerosis.

Bone matrix, cellularity, and structural changes in a rat model with high-turnover osteoporosis induced by combined ovariectomy and a multiple-deficient diet.

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.

Differences of bone healing in metaphyseal defect fractures between osteoporotic and physiological bone in rats.

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n=14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n=14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p=0.048) but no differences at 10 mm lever span. Microcomputer tomography (µCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3mm lever span biomechanical testing and histology.

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures.

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.

Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat.

Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.

Implications of combined ovariectomy/multi-deficiency diet on rat bone with age-related variation in bone parameters and bone loss at multiple skeletal sites by DEXA.

BACKGROUND: Osteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period. MATERIAL/METHODS: Sprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14. RESULTS: BMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below -5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression. CONCLUSIONS: Combined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures.

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.

Micro-computed tomography imaging of composite nanoparticle distribution in the lung.

Nanomedicine comprises a significant potential to approach the therapy of severe diseases. Knowledge of nanoparticle behavior at the target site would contribute to the development of specialized tools for respiratory medicine. Here, we were interested in the potential of micro-computed tomography (µCT) imaging to monitor the pulmonary distribution of polymeric nanoparticles. Composite nanoparticles were analyzed for physicochemical properties, morphology and composition. µCT was employed to visualize the pulmonary distribution of composite nanoparticles in an ex vivo lung model. Employed composite nanoparticles were composed of poly(styrene) cores coated by a thin shell of colloidal iron oxide. Particles were mainly located in the interstitial space and associated with pulmonary cells, as observed by light microscopy. µCT detected enhanced X-ray opacities in the conducting (linear pattern) and respiratory airways (aciniform X-ray attenuations). In conclusion, multifunctional nanoparticles will prompt the development of novel therapeutic and diagnostic tools in respiratory medicine.

Quantitative analyses of bone composition in acetylcholine receptor M3R and alpha7 knockout mice.

AIMS: Increasing collagen synthesis was observed in lung after stimulation of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) on fibroblasts. Since collagen synthesis is an important process during fracture healing and bone remodelling, we asked whether cholinergic receptors are involved in bone collagen production. MAIN METHODS: In the present study we analysed 16 week old male knockout mice for nAChRα7 (α7-KO) and mAChR M3R (M3R-KO) in correlation to their corresponding wild types (WT). Microarchitecture of right femora, vertebrae Th13 and L1 were analysed by 3D Micro-CT, left femora by a three-point bending test and humeri by real-time RT-PCR. KEY FINDINGS: A significant decrease in relative bone volume, trabecular thickness, trabecular number, bone surface density, and a significant increase in trabecular separation and structure model index were measured for the M3R-KO using Micro-CT analysis. Bending stiffness of M3R-KO was significantly reduced in comparison to WT as well as the collagen 1α1 and 1α2 mRNA expression was down-regulated. No changes were detected for α7-KO using Micro-CT, biomechanical testing, and collagen mRNA expression. SIGNIFICANCE: Our results indicate that nAChRα7 are not involved in the regulation of bone collagen synthesis whereas M3R exert stimulatory effects on cancellous bone microarchitecture, flexural rigidity, and bone matrix synthesis. Since the M3R-KO exhibit bone structures similar to systemically diseased bone it might be valuable to establish new therapeutic strategies using administration of agonists for the M3R to improve bone qualities.

Induction of osteoporosis with its influence on osteoporotic determinants and their interrelationships in rats by DEXA.

BACKGROUND: As women are the population most affected by multifactorial osteoporosis, research is focused on unraveling the underlying mechanism of osteoporosis induction in rats by combining ovariectomy (OVX) either with calcium, phosphorus, vitamin C and vitamin D2/D3 deficiency, or by administration of glucocorticoid (dexamethasone). MATERIAL/METHODS: Different skeletal sites of sham, OVX-Diet and OVX-Steroid rats were analyzed by Dual Energy X-ray Absorptiometry (DEXA) at varied time points of 0, 4 and 12 weeks to determine and compare the osteoporotic factors such as bone mineral density (BMD), bone mineral content (BMC), area, body weight and percent fat among different groups and time points. Comparative analysis and interrelationships among osteoporotic determinants by regression analysis were also determined. RESULTS: T scores were below-2.5 in OVX-Diet rats at 4 and 12 weeks post-OVX. OVX-diet rats revealed pronounced osteoporotic status with reduced BMD and BMC than the steroid counterparts, with the spine and pelvis as the most affected skeletal sites. Increase in percent fat was observed irrespective of the osteoporosis inducers applied. Comparative analysis and interrelationships between osteoporotic determinants that are rarely studied in animals indicate the necessity to analyze BMC and area along with BMD in obtaining meaningful information leading to proper prediction of probability of osteoporotic fractures. CONCLUSIONS: Enhanced osteoporotic effect observed in OVX-Diet rats indicates that estrogen dysregulation combined with diet treatment induces and enhances osteoporosis with time when compared to the steroid group. Comparative and regression analysis indicates the need to determine BMC along with BMD and area in osteoporotic determination.

Assessment of angiogenesis in osseointegration of a silica-collagen biomaterial using 3D-nano-CT.

Bony integration of biomaterials is a complex process in which angiogenesis plays a crucial role. We evaluated micro- and nano-CT imaging to demonstrate and quantify neovascularization in bony integration of a biomaterial and to give an image based estimation for the needed resolution for imaging angiogenesis in an animal model of femora defect healing. In 8 rats 5mm full-size defects were created at the left femur that was filled with silica-collagen bone substitute material and internally fixed with plate osteosynthesis. After 6 weeks the femora were infused in situ with Microfil, harvested and scanned for micro-CT (9 µm)(3) and nano-CT (3 µm)(3) imaging. Using those 3D images, the newly formed blood vessels in the area of the biomaterial were assessed and the total vascular volume fraction, the volume of the bone substitute material and the volume of the bone defect were quantitatively characterized. Results were complemented by histology. Differences were statistically assessed using (ANOVA). High-resolution nano-CT demonstrated new blood vessel formation surrounding the biomaterial in all animals at capillary level. Immunohistochemistry confirmed the newly formed blood vessels surrounding the bone substitute material. The mean vascular volume fraction (VVF) around the implant was calculated to be 3.01 ± 0.4%. The VVF was inversely correlated with the volume of the bone substitute material (r=0.8) but not with the dimension of the fracture zone (r=0.3). Nano-CT imaging is feasible for quantitative analysis of angiogenesis during bony integration of biomaterials and a promising tool in this context for the future.