Liquid-phase ASEM imaging of cellular and structural details in cartilage and bone formed during endochondral ossification: Keap1-deficient osteomalacia.

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1-/- femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Excessive salt consumption causes systemic calcium mishandling and worsens microarchitecture and strength of long bones in rats.

Excessive salt intake has been associated with the development of non-communicable diseases, including hypertension with several cardiovascular consequences. Although the detrimental effects of high salt on the skeleton have been reported, longitudinal assessment of calcium balance together with changes in bone microarchitecture and strength under salt loading has not been fully demonstrated. To address these unanswered issues, male Sprague-Dawley rats were fed normal salt diet (NSD; 0.8% NaCl) or high salt diet (HSD; 8% NaCl) for 5 months. Elevation of blood pressure, cardiac hypertrophy and glomerular deterioration were observed in HSD, thus validating the model. The balance studies were performed to monitor calcium input and output upon HSD challenge. The HSD-induced increase in calcium losses in urine and feces together with reduced fractional calcium absorption led to a decrease in calcium retention. With these calcium imbalances, we therefore examined microstructural changes of long bones of the hind limbs. Using the synchrotron radiation x-ray tomographic microscopy, we showed that trabecular structure of tibia and femur of HSD displayed a marked increase in porosity. Consistently, the volumetric micro-computed tomography also demonstrated a significant decrease in trabecular bone mineral density with expansion of endosteal perimeter in the tibia. Interestingly, bone histomorphometric analyses indicated that salt loading caused an increase in osteoclast number together with decreases in osteoblast number and osteoid volume. This uncoupling process of bone remodeling in HSD might underlie an accelerated bone loss and bone structural changes. In conclusion, long-term excessive salt consumption leads to impairment of skeletal mass and integrity possibly through negative calcium balance.

Effect of Drilling Techniques on Microcracks and Pull-Out Strength of Cortical Screw Fixed in Human Tibia: An In-Vitro Study.

The strength between the cortical screw and bone following an orthopaedic implant surgery is an important determinant for the success of osteosynthesis. An excessive axial cutting force during drilling produces microcracks in the bone surface, resulting in reduced strength between the screw and bone, resulting in loosening of implant. The present work, investigates the influence of drilling parameters on microcracks generated in the drilled surface and pull-out strength of screw fixed in cortical bone of human tibia. The holes were drilled by two different techniques: conventional surgical bone drilling (CSBD) and rotary ultrasonic bone drilling (RUBD), by a recently developed operation theatre (OT) compatible machine. Cutting force generated in drilling of human tibia using RUBD was 30-40% lesser than that of CSBD. Scanning electron microscopy (SEM) also revealed that RUBD produced significantly lesser and thinner microcracks than that of CSBD in human bones. Biomechanical pull-out test results showed that, the pull-out strength of screws inserted into drilled holes by RUBD was much higher (100-150%) than that of CSBD. A significant difference in pull-out strength (p < 0.05) between RUBD and CSBD was revealed by statistical analysis at 95% confidence interval.

Ultrastructural characterization of cells in the tibial stump of ruptured human anterior cruciate ligament, their changes and significance with duration of injury.

Fibroblasts and myofibroblasts have been known to be present in both ruptured and intact human anterior cruciate ligament (ACL), and although their relevant histology and immunochemistry have been studied in the past, ultrastructural features of these cells are largely lacking. Therefore, we aim to characterise the ultrastructural details of these cells with the help of transmission electron microscopy (TEM) and to study the changes and their significance with duration of injury. Samples from 60 ruptured human ACL undergoing surgery were obtained and categorised according to duration of injury and observed under TEM with main focus on the following ultrastructural features: cellular morphology, presence of rough endoplasmic reticulum, Golgi apparatus, lamina, myofilaments, and presence of myofibroblasts. These features were further correlated with the duration of injury and association, if any, determined using appropriate statistical analysis. A total of 54 male and 6 female patients with mean duration of the injury of 23.01 ± 26.09 weeks (2-108 weeks) were included in the study and categorised into five groups based on duration of injury as follows: I (< 6 weeks), II (7-12 weeks), III (13-20 weeks), IV (21-50 weeks) and V (> 50 weeks). There was a significant association between the above-mentioned ultrastructural features and the duration of injury (p < 0.05) except for the presence of ovoid fibroblast cells (p = 0.53). Furthermore, number of myofibroblasts and cells with Golgi apparatus and rough endoplasmic reticulum was seen to peak at 13-20 weeks following injury. We describe ultrastructural features of fibroblast of different morphology along with myofibroblasts in the ligaments following injury, the changes in which might have a potential bearing on ligament healing.

Increased glycolysis mediates Wnt7b-induced bone formation.

Wingless/integrated (Wnt) signaling has emerged as a major mechanism for promoting bone formation and a target pathway for developing bone anabolic agents against osteoporosis. However, the downstream events mediating the potential therapeutic effect of Wnt proteins are not fully understood. Previous studies have indicated that increased glycolysis is associated with osteoblast differentiation in response to Wnt signaling, but direct genetic evidence for the importance of glucose metabolism in Wnt-induced bone formation is lacking. Here, we have generated compound transgenic mice to overexpress Wnt family member 7B (Wnt7b) transiently in the osteoblast lineage of postnatal mice, with or without concurrent deletion of the glucose transporter 1 (Glut1), also known as solute carrier family 2, facilitated glucose transporter member 1. Overexpression of Wnt7b in 1-mo-old mice for 1 wk markedly stimulated bone formation, but the effect was essentially abolished without Glut1, even though transient deletion of Glut1 itself did not affect normal bone accrual. Consistent with the in vivo results, Wnt7b increased Glut1 expression and glucose consumption in the primary culture of osteoblast lineage cells, and deletion of Glut1 diminished osteoblast differentiation in vitro. Thus, Wnt7b promotes bone formation in part through stimulating glucose metabolism in osteoblast lineage cells.-Chen, H., Ji, X., Lee, W.-C., Shi, Y., Li, B., Abel, E. D., Jiang, D., Huang, W., Long, F. Increased glycolysis mediates Wnt7b-induced bone formation.

Effects of low-level laser therapy on the organization of articular cartilage in an experimental microcrystalline arthritis model.

The aim of this study was to evaluate the effects of low-level laser therapy using the gallium arsenide laser (λ = 830 nm) on the articular cartilage (AC) organization from knee joint in an experimental model of microcrystalline arthritis in adult male Wistar rats. Seventy-two animals were divided into three groups: A (control), B (induced arthritis), and C (induced arthritis + laser therapy). The arthritis was induced in the right knee using 2 mg of Na4P2O7 in 0.5 mL of saline solution. The treatments were daily applied in the patellar region of the right knee after 48 h of induction. On the 7th, 14th, and 21st days of treatment, the animals were euthanized and their right knees were removed and processed for structural and biochemical analysis of the AC. The chondrocytes positively labeled for the TUNEL reaction were lower in C than in B on the 14th and 21st days. The content of glycosaminoglycans and hydroxyproline in A and C was higher than B on the 21st day. The amount of tibial TNF-α in B and C was lower than in A. The amount of tibial BMP-7 in B and C was higher than in A. The femoral MMP-13 was lower in B and C than for A. The tibial TGF-ß for C was higher than the others. The femoral ADAMT-S4 content of A and C presented similar and inferior data to B on the 21st day. The AsGa-830 nm therapy preserved the content of glycosaminoglycans, reduced the cellular changes and the inflammatory process compared to the untreated group.

Degradation and interaction with bone of magnesium alloy WE43 implants: A long-term follow-up in vivo rat tibia study.

The aim of this in vivo study was to examine the degradation and biocompatibility of the WE43 magnesium alloy containing magnesium yttrium, rare earth elements, and zirconium over a one-year long-term follow-up period. Additionally, we compared anodized WE43 implants with monolithic ones and clarified the effect of the anodization. WE43 cylindrical implants with and without anodization (length, 10 mm; diameter, 0.3 mm) were transplanted into the rat tibia. In both groups, the development of corrosion and the change in implant volume were evaluated by in vivo micro-computed tomography until 12 months, and the bone tissue reaction was observed histologically. In the monolithic WE43 implants, hydrogen gas was evident until 14 days and the volume loss was 36.3% after 12 months. In the anodized WE43 implants, the development of hydrogen gas was inhibited and the volume loss was 27.7% after 12 months. The anodized WE43 implants showed a significantly slower corrosion process in the early phase. Therefore, these implants may require a prolonged period to degrade completely and may even resist complete degradation. At one year post surgery, bone maturation progressed and lamellar bone structure developed around the implant in both groups. In conclusion, the WE43 implants showed good long-term stability and biocompatibility in bone tissue.

Healing capacity of bone marrow mesenchymal stem cells versus platelet-rich fibrin in tibial bone defects of albino rats: an in vivo study.

Background: Various techniques for tissue engineering have been introduced to aid the regeneration of defective or lost bone tissue. The aim of this study was to compare the in vivo bone-forming potential of bone marrow mesenchymal stem cells (BM-MSCs) and platelet-rich fibrin (PRF) on induced bone defects in rats' tibiae. Methods: In total, one defect of 3-mm diameter was created in each tibia of 36 Wistar male rats. There were two groups: group A, left tibia bone defects that received PRF; and group B, right tibia bone defects of the same animal that received BM-MSCs loaded on a chitosan scaffold. Subsequently, Scanning electron microscope/energy-dispersive X-ray (SEM/EDX) analyses was performed at 3 and 10 days, and 3 weeks post­implantation and following euthanasia; (n=12). Results: The EDX analysis performed for each group and time point revealed a significant increase in the mean calcium and phosphorous weight percentage in the BM-MSC-treated group relative to the PRF-treated group at all-time intervals (P < 0.05). Moreover, the mean calcium and phosphorus weight percentage increased as time progressed since the surgical intervention in the PRF-treated and BM-MSCs groups (P < 0.05). Conclusions: In the present study, both BM-MSCs and PRF were capable of healing osseous defects induced in a rat tibial model. Yet, BM-MSCs promoted more adequate healing, with higher mean calcium and phosphorous weight percentages than PRF at all-time points, and showed greater integration into the surrounding tissues than PRF.

Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration.

Innate immune cells recruited to inflammatory sites have short life spans and originate from the marrow, which is distributed throughout the long and flat bones. While bone marrow production and release of leukocyte increases after stroke, it is currently unknown whether its activity rises homogeneously throughout the entire hematopoietic system. To address this question, we employed spectrally resolved in vivo cell labeling in the murine skull and tibia. We show that in murine models of stroke and aseptic meningitis, skull bone marrow-derived neutrophils are more likely to migrate to the adjacent brain tissue than cells that reside in the tibia. Confocal microscopy of the skull-dura interface revealed myeloid cell migration through microscopic vascular channels crossing the inner skull cortex. These observations point to a direct local interaction between the brain and the skull bone marrow through the meninges.

Lifelong intake of flaxseed or menhaden oil to provide varying n-6 to n-3 PUFA ratios modulate bone microarchitecture during growth, but not after OVX in Sprague-Dawley rats.

SCOPE: Skeletal health is a lifelong process impacted by environmental factors, including nutrient intake. The n-3 source and PUFA ratio affect bone health in growing rats, or following ovariectomy (OVX), but no study has investigated the longitudinal effect of PUFA-supplementation throughout these periods of bone development. METHODS AND RESULTS: One-month-old, Sprague-Dawley rats (n = 98) were randomized to receive one of four diets from 1 through 6 months of age. Diets were modified from AIN-93G to contain a varying amount and source of n-3 (flaxseed versus menhaden oil) to provide an n-6 to n-3 ratio of 10:1 or 5:1. At 3 (prior to SHAM or OVX) and 6 months of age, bone microarchitecture of the tibia was quantified using in vivo micro-computed tomography (SkyScan 1176, Bruker microCT). Providing 5:1 (flaxseed) resulted in lower trabecular thickness and medullary area and greater cortical area fraction during growth compared to diets with a 10:1 PUFA ratio, but many of these differences were not apparent following OVX. CONCLUSION: PUFA-supplementation at levels attainable in human diet modulates some bone structure outcomes during periods of growth, but is not an adequate strategy for the prevention of OVX-induced bone loss in rats.

Characterization of nano-structural and nano-mechanical properties of osteoarthritic subchondral bone.

BACKGROUND: Although articular cartilage is the primary tissues affected by osteoarthritis (OA), the underlying subchondral bone also undergoes noticeable changes. Despite the growing body of research into the biophysical and mechanical properties of OA bone there are few studies that have analysed the structure of the subchondral sclerosis at the nanoscale. In this study, the composition and nano-structural changes of human osteoarthritis (OA) subchondral bone were investigated to better understand the site-specific changes. METHODS: OA bone samples were collected from patients undergoing total knee replacement surgery and graded according to disease severity (grade I: mild OA; grade IV: severe OA). Transmission electron microscopy (TEM), Electron Diffraction, and Elemental Analysis techniques were used to explore the cross-banding pattern, nature of mineral phase and orientation of the crystal lattice. Subchondral bone nano-hydroxyapatite powders were prepared and characterised using high resolution transmission electron microscopy (HR-TEM) and fourier transform infrared spectroscopy (FTIR). Subchondal bone mechanical properties were investigated using a nano-indentation method. RESULTS: In grade I subchondral bone samples, a regular periodic fibril banding pattern was observed and the c-axis orientation of the apatite crystals was parallel to the long axis of the fibrils. By contrast, in grade IV OA bone samples, the bulk of fibrils formed a random and undulated arrangement accompanied by a circular oriented pattern of apatite crystals. Fibrils in grade IV bone showed non-hierarchical intra-fibrillar mineralization and higher calcium (Ca) to phosphorous (P) (Ca/P) ratios. Grade IV OA bone showed higher crystallinity of the mineral content, increased modulus and hardness compared with grade I OA bone. CONCLUSIONS: The findings from this study suggest that OA subchondral sclerotic bone has an altered mineralization process which results in nano-structural changes of apatite crystals that is likely to account for the compromised mechanical properties of OA subchondral bones.

Combined high-fat-resveratrol diet and RIP140 knockout mice reveal a novel relationship between elevated bone mitochondrial content and compromised bone microarchitecture, bone mineral mass, and bone strength in the tibia.

SCOPE: While resveratrol (RSV) is associated with the prevention of high-fat (HF) diet-induced insulin resistance, the effects on bone health combined with an HF-diet is unknown. Therefore, we determined the effect of RSV on bone microarchitecture in the presence of an HF-diet, while also elucidating molecular adaptations within bone that could contribute to bone health status. METHODS AND RESULTS: Male C57BL6 mice were provided control (10% fat) or HF-diet (60% fat) in the presence or absence of RSV for 12 weeks. While RSV prevented HF diet-induced glucose intolerance, HF-RSV compromised tibial microarchitecture, mineral mass, and strength. The compromised outcomes following HF-RSV corresponded with higher markers of osteoclast-activation and bone-resorption (decreased OPG/RANKL ratio; increased cathepsin K), as well as higher markers of tibial mitochondrial content. A molecular model of elevated mitochondrial content (RIP140 knock out (KO) mice) was utilized to determine proof-of-principle that increasing mitochondrial content coincides with decrements in bone health. RIP140 KO mice displayed higher markers of mitochondrial content, and similar to HF-RSV, had compromised bone microarchitecture, lower BMD/strength, and higher markers of osteoclast-activation/bone-resorption. CONCLUSION: These data show that in the presence of an HF-diet, RSV negatively alters bone health, a process associated with increased mitochondrial content and markers of bone resorption.

Antigen-free bovine cancellous bone loaded with recombinant human bone morphogenetic protein-2 for the repair of tibial bone defects in goat model.

Antigen-free bovine cancellous bone has good performances of porous network structures and mechanics with antigen extracted. To develop a bioactive scaffold for enhancing bone repair and evaluate its biological property, rhBMP-2 loaded with antigen-free bovine cancellous bone was used to treat tibial bone defect. Twenty-four healthy adult goats were chosen to establish goat defects model and randomly divided into four groups. The goats were treated with rhBMP-2/antigen-free bovine cancellous bone scaffolds (group A), autogenous cancellous bone graft (group B), porous tricalciumphosphate scaffolds (group C) and nothing (group D). Animals were evaluated with radiological and histological methods at 4, 8 and 12 weeks after surgery. The gray value of radiographs was used to evaluate the healing of the defects, which revealed that the group A had a better outcome of defect healing compared with group C at 4, 8 and 12 weeks, respectively (p < 0.05), while the difference between groups A and B was without significance at each time (p > 0.05). The newly formed bone area was calculated from histological sections, and the results indicated that the amount of new bone in group A increased significantly compared with that in group C (p < 0.05) but was similar to that in group B (p > 0.05) at 4, 8 and 12 weeks, respectively. In addition, the expression of collagen I and vascular endothelial growth factor by real-time polymerase chain reaction at 12 weeks in group A was significantly higher than that in group C (p = 0.034, p = 0.032, respectively), but no significant differences were found when compared with that in group B (p = 0.36, p = 0.54, respectively). At the same time, group C presented better results than group D on bone defects healing. Therefore, the composites of antigen-free bovine cancellous bone loaded with rhBMP-2 have a good osteoinductive activity and capacity to promote the repair of bone defects.

Reacción del tejido óseo de la rata a un sellador endodóntico a base de óxido de cinc y eugenol con acetato de hidrocortisona / Response of the bone tissue of the rat to a zin-oxide and eugenol/hydrocortisone acetate-based endodontic sealer

Objetivos: analizar comparativamente la respuesta del tejido óseo de la rat a la implantación de Kleppmethasona (KMS), un sellador endodóntico a base de óxido de cinc y eugenol que contiene un corticoide en su composición y Pulp Canal Sealer EWT (PCS), un sellador a base de óxido de cinc y eugenol convencional. Materiales y métodos: se implantaron en ambas tibias de 12 ratas, tubos de silicona obturados a ras en los dos extremos con una preparación fresca de Kleppmethasona (KMS; Klepp/Raysan SA) o Pulp Canal Sealer (PCS; Sybron-Endo) (control positivo). Como grupo negativo se utilizó la pared lateral de los tuobs de silicona (SCN). Luego de los 14 y 94 días, los implantes fueron removidos, fijados en solución de formol al 10 por ciento y procesados para su estudio histológico. Resultados: a los 14 días posimplantación, el análisis histológico reveló una reacción inflamatoria severa compuesta por polimorfonucleares neutrófilos, linfocitos, plasmocitos, macrófagos, fibroblastos y vasos de neoformación en los especímenes que se hallaban en contgacto directo con KMS y PCS. A los 94 días, en los casos en contacto con KMS, la reacción inflamatoria se redujo sustancialmente y se observó un proceso de reparación con presencia de una cápsula fibrosa sin céluals inflamatorias y el desarrollo de nuevas trabéculas óseas. En los especímenes en contacto con PCS la reacción inflamatoria inicial se redujo, pero se observó la persistencia de algunas células inflamatorias. Sin embargo, esta situación no pareció impedir el desarrollo incipiente de nuevas trabéculas óseas. En contacto con el SCN la reacción de los tejidos a los 14 días fue considerada mínima en un solo caso. Al finalizar la experiencia, los tejidos circundantes se encontraban normales en todos los casos. A los 14 días se observaron diferencias significativas (p<0,05) entre KMS/PCS y los SCN, mientras que no las hubo (p>0,05) entre ambos selladores. A los 94 día...

Structural and Qualitative Bone Remodeling Around Repetitive Loaded Implants in Rabbits.

BACKGROUND: Bone mechanical function is regulated by bone quality and bone mineral density (BMD) that reflect bone strength. The preferential alignment of biological apatite (BAp) c-axis/collagen fibers and osteocytes is a determinant factor of bone quality. However, the effect of mechanical loading on bone quality around dental implants is unclear. PURPOSE: The aim of this study was to clarify the effects of mechanical loading on osseointegration, bone volume BMD, and bone quality around dental implants. MATERIALS AND METHODS: Twenty anodized Ti-6Al-4V alloy implants (KYOCERA Co., Kyoto, Japan) were placed in the proximal tibial metaphysis of 10 rabbits. Twelve weeks after surgery, mechanical loading was applied along the long axis of the implant (50 N, 3 Hz, 1,800 cycles, 2 days/week) for 8 weeks. Osseointegration, bone volume, BMD, and bone quality were evaluated using light microscopy, microcomputed tomography, polarized light microscopy, and microbeam X-ray diffractometer. RESULTS: Mechanical loading increased osseointegration, bone volume, and BMD. Bone quality around dental implant was altered with increased osteocyte numbers and the preferential alignment direction and degree of BAp c-axis/collagen fibers. CONCLUSIONS: These findings suggest that mechanical loading effectively induces bone anabolic responses around dental implants. Altered bone quality may upregulate bone strength, contributing to long-term implant stability.

Effects of manganese deficiency on the microstructure of proximal tibia and OPG/RANKL gene expression in chicks.

Manganese (Mn) deficiency can result in perosis in chicks, but the mechanism of Mn deficiency on tibia development remains poorly understood. Ninety one-day-old Arbor Acres male broiler chickens administered with control diet (60 mg Mn/kg) and Mn-deficient diets (40 mg Mn/kg, 8.7 mg Mn/kg) to investigate the effects of Mn deficiency on morphology of tibia and related signal transduction pathways in broiler chickens. At the age of 42 days, the bone trabecula, damaged osteoblasts and OPG/RANKL mRNA expression levels were investigated by histological assessment, electron microscopic examination and real-time quantitative PCR analysis, respectively. Results of histological observations showed that decreased trabecular thickness, trabecular number and trabecular bone area (%) together with increased trabecular bone separation were involved in perosis induced by Mn deficiency. The most striking ultrastructural modifications involved disruption of nuclear membrane and mitochondria outer membrane, loss of mitochondrion cristae and alteration in endoplasmic reticulum in osteoblasts of the Mn-deficient groups. Likewise, Mn deficiency results in a significant (P < 0.05) decrease in the relative mRNA expression levels of OPG and RANKL with a significantly higher RANKL/OPG ratio (P < 0.05). In conclusion, Mn deficiency can affect the development of tibia in broiler chickens, leading to metaphyseal osteoporosis which may be due to decreased OPG/RANKL mRNA expression.

Reacción del tejido óseo de la rata a un material de obturación de conductos radiculares de tercera generación: un ensayo piloto / Reaction of the rat osseous tissue to a third-generation root canal filling material: a pilot assay

Objetivos: analizar la respuesta del tejido óseo de la rata a la implantación de Licon-D (LND). Material y métodos: se implantaron en tibias de 18 ratas, tubos de silicona obturados con LND o pasta lentamente reabsorbible (PLR) utilizada como control positivo. La pared lateral de los tubos (SCN) fue utilizada como control negativo. A los 7, 30 y 90 días post implantación, los implantes y tejidos circundantes fueron fijados en formol al 10 por ciento y procesados para su estudio histológico. Resultados: 7 días: se observó una reacción inflamatoria severa de los tejidos en contacto con LND y PLR, mientras que en contacto con SCN se observó un tejido conectivo sano, con excepción de dos casos. 30 días: la reacción inflamatoria en contacto con LDN se redujo, observándose un proceso de reparación en progreso con trabéculas óseas incipientes. 90 días: en contacto con LDN se observó una cápsula fibrosa y reparación ósea. Para PLR se observó a los 30 días un tejido fibroso que rodeaba un foco de tejido granulomatoso, mientras que los SCN se encontraban rodeados por una cápsula constituida por tejido fibroso sano. 90 días: en contacto con PLR, se observó una cápsula fibrosa y trabéculas óseas incipientes. En contacto con SCN, los tejidos circudantes se encontraban normales en todos los casos. Conclusiones: a pesar que LND y PLR produjeron una reacción inflamatoria inicial severa, luego de 90 días, ambos materiales fueron bien tolerados por el tejido óseo de la rata. Se observó una reparación completa en contacto con LND y un proceso de reparación en progreso en contacto con PLR.

The cn/cn dwarf mouse. Histomorphometric, ultrastructural, and radiographic study in mutants corresponding to human acromesomelic dysplasia Maroteaux type (AMDM).

BACKGROUND: The cn/cn dwarf mouse is caused by a loss-of-function mutation in the natriuretic peptide receptor 2 (NPR-2) gene which helps positively regulate endochondral longitudinal bone growth. The gene mutation corresponds to that in the human skeletal dysplasia Acromesomelic Dysplasia Maroteaux type (AMDM). This study assesses histomorphometric, ultrastructural and radiographic correlates of the growth abnormality. METHODS: Ten litters of cn/cn and cn/+littermates at ages ranging from 2.5 to 6.5 weeks were studied by skeletal radiographs, histomorphometry and physeal ultrastructure. Skeletal radiographs were done on 2 cn/cn and 2 cn/+littermates at 5 weeks of age. Humeral, femoral, and tibial lengths were measured from 34 intact bones (17 cn/cn, 17 cn/+) at 2.5 to 6.5 weeks. Growth plate histomorphometry in 50 bones (26 cn/cn and 24 cn/+) determined the hypertrophic zone/entire physeal cartilage ratios in 204 sections (87 cn/+, 117 cn/cn) at 3 time periods (2.5-3, 4-4.5, and 6-6.5 weeks). Electron microscopy assessed 6 cn/cn and 6 cn/+age and site-matched physeal cartilage. RESULTS: Cn/cn mice were two thirds the size of the cn/+. Cn/cn bones were normal in shape or only minimally deformed except for the radius with mid-diaphyseal bowing. Length ratios of cn/cn humeri, femurs, and tibias were a mean of 0.65 (± 0.03, n = 34, 17 ratios) compared to cn/+bones. The main physeal abnormality was a markedly shortened hypertrophic zone with the ratio of hypertrophic zone to entire physis 0.17 (± 0.063) in the cn/cn and 0.30 (± 0.052) in the cn/+mice. Ratio assessments were similar comparing humeral, femoral, and tibial growth plates as were ratios from each of the 3 time periods. Ultrastructural assessments from the resting zone to the lower hypertrophic zone-metaphyseal junction showed no specific individual cell abnormalities in cn/cn compared to cn/+physes. CONCLUSIONS: The disorder causes a shortened physeal hypertrophic zone but normal ultrastructure of cn/cn chondrocytes points to abnormality primarily affecting the hypertrophic zone rather than a structural cell or matrix synthesis problem.

Contemporary management of critical lower limb ischemia in TASC D lesions with subintimal angioplasty in femoro-popliteal lesions, tibial angioplasty and sequential compression biomechanical device for infra-inguinal arterial occlusion. Experience and quality of life outcome learned over 25 years.

AIM: Patients with end-stage critical limb ischemia (CLI) survive on borrowed time and amputation is inevitable if an aggressive management stratagem is not instigated. Our primary aim was to equate effectiveness of subintimal angioplasty (SIA) and tibial balloon angioplasty (TBA) in sustaining clinical improvement and amputation free survival (AFS) in patients with CLI TASD II D. Moreover, patients with severe CLI, who were not suitable for revascularization and who were offered therapy with a sequential compression biomechanical device (SCBD) were scrutinised as part of a comprehensive lower limb salvage program. METHODS: From 2002-2012, 5876 patients were referred with peripheral vascular disease (PVD); 987 presented with CLI and 798 had intervention; 189 patients presenting with CLI were not candidates for revascularisation, out of which 171 were offered SCBD. We formed a prospective observational group study of 441 patient who had TASC D disease. All of these patients presented as emergencies and were allocated to the next available treatment list. Duplex ultrasound arterial mapping (DUAM) was the sole preoperative investigation tool in 92% of all cases. Of the 441 patients studied, 190 patients (206 procedures) has SIA for TASC D femero-popliteal occlusions, 80 patients (89 procedures) had TBA and cool eximer laser angioplasty (CELA) for tibial artery occlusions and 171 patients with severe CLI were not suitable for revascularization and joined the SCBD program. Mean age (SIA 73±13 years vs. TBA/CELA 74±8 years vs. SCBD 75±13 years), and comorbidity severity scores (P>0.05) were similar between groups. RESULTS: Perioperative mortality within the SIA group was 1.6% vs. 0% within the TBA group and 0.6% in SCBD. Length of hospital stay within the TBA group was 3.8±2 days vs. SIA 14±16 days, P<0.0001. The 5-year freedom from major adverse events (MAE) for the SIA group was 68% that was comparable to the results obtained for both the TBA group; 59%, and SCBD group: 62.5% (P=0.1935). Five-year freedom from target lesion revascularization was 85.9% within the SIA group and 79% within the TBA group. A sustained clinical improvement was seen in 82.8% of primary SIA and 68% of TBA, which mimics the outcome of SCBD at 68% at one year. A total of 83% SCBD patients had no rest pain within one week of starting the program and gangrene remained dry and non-progressive. Ulceration healed in all but 12 patients. There were no device-related complications. Limb salvage was 94% at 5 years. All-cause survival was 69%. Quality time spent without symptoms of disease or toxicity of treatment (Q-TWiST) was 24.7 months for SIA and 8.5 months for TBA and was 38.13 for SCBD for a total of 708 months of usage. Cost per quality adjusted-life years (QALY) for SIA was € 5662.79, € 12,935.18 for TBA and € 2943.56 for SCBD. CONCLUSION: All treatment pathways augmented patient-specific Q-TWiST with substantial cost reduction. SIA, TBA and SCBD expand AFS and symptom-free survival. All treatment modalities are minimally invasive and allow for a high patient turnover without compromising limb salvage, once they are performed by experienced vascular surgeons in high deliberate practice volume centers.

Anatomic changes in the macroscopic morphology and microarchitecture of denervated long bone tissue after spinal cord injury in rats.

To study the effects of mechanical loading on bones after SCI, we assessed macro- and microscopic anatomy in rats submitted to passive standing (PS) and electrical stimulation (ES). The study design was based on two main groups of juvenile male Wistar rats with SCI: one was followed for 33 days with therapies starting at day 3 and the other was followed for 63 days with therapies starting at day 33. Both groups were composed of four subgroups (n = 10/group): (1) Sham, (2) SCI, (3) SCI + PS, and (4) SCI + ES. Rehabilitation protocol consisted of a 20-minute session, 3x/wk for 30 days. The animals were sequentially weighed and euthanized. The femur and tibia were assessed macroscopically and microscopically by scanning electronic microscopy (SEM). The SCI rats gained less weight than Sham-operated animals. Significant reduction of bone mass and periosteal radii was observed in the SCI rats, whereas PS and ES efficiently improved the macroscopic parameters. The SEM images showed less and thin trabecular bone in SCI rats. PS and ES efficiently ameliorated the bone microarchitecture deterioration by thickening and increasing the trabeculae. Based on the detrimental changes in bone tissue following SCI, the mechanical loading through weight bearing and muscle contraction may decrease the bone loss and restore the macro- and microanatomy.