Dragica Bobinac

Changes in articular cartilage and subchondral bone histomorphometry in osteoarthritic knee joints in humans.

In this study, we have examined the correlation between the histological and histochemical changes of articular cartilage and bone parameters of the underlying subchondral bone. The aim was to elucidate patterns of bone parameter changes within different depths of subchondral bone in the joints with macroscopically normal cartilage and in joints with osteoarthritis (OA). Ten tibial plateaus were taken from patients during total knee replacement surgery due to severe OA. They were compared with 10 sets of tibial condyles obtained from autopsy subjects with no history of bone or joint disease. The cylindrical cartilage-bone samples were taken out from the anterior, posterior, external, and internal areas of the condyles for cartilage assessment (Mankin score) and subchondral bone histomorphometry. Four histomorphometric parameters were measured: bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.S). Our study showed that subchondral bone from the OA group had significantly higher bone volume (54.1 +/- 10.6%) than control group (37.8 +/- 8.1%) (P < 0.01). In addition, trabecular parameters from the OA subchondral bone showed a smaller number of sparsely distributed and thicker trabecules than in control group (P < 0.05). Medial and lateral condyle from the control group did not differ significantly, while medial condyle from OA group showed a high increase of bone volume (62.8 +/- 13.3) and consecutively different trabecular parameters when compared with the lateral condyle from the same group. Also, it was shown that there are regional differences (anterior, posterior, internal, and external) in bone parameters between both condyles within both, control and OA groups. Comparison of bone parameters from three different stage of articular cartilage degeneration (Mankin score) showed that higher degree of cartilage degeneration is followed by significant changes in subchondral bone architecture. Furthermore, we have found that progression of cartilage degeneration leads to changes in bone parameters which affected deeper levels of subchondral bone. According to these results, it can be suggested that changes in histomorphometric parameters of subchondral bone are secondary to cartilage damage and proceed deeper into subchondral bone with increasing cartilage degeneration.

Bone Morphogenetic Protein–7 (Osteogenic Protein–1) Promotes Tendon Graft Integration in Anterior Cruciate Ligament Reconstruction in Sheep

Background Bone morphogenetic proteins induce new bone both in patients with bone defects and at extraskeletal sites in animals. After anterior cruciate ligament rupture, tendon graft fixation into a bone tunnel is a widely used method for anterior cruciate ligament reconstruction. Hypothesis Bone morphogenetic protein–7 applied to the bone-tendon interface enables better integration of a free tendon graft into the surrounding bone. Study Design Controlled laboratory study. Methods The anterior cruciate ligament was reconstructed using a free tendon graft in the right rear knees of 30 one-year-old male sheep. Recombinant human bone morphogenetic protein–7 (25 μg) was applied randomly to the bone-tendon interface in 15 animals, and a vehicle was applied in 15 control animals. At 3 weeks, 10 animals from each group were sacrificed, and the remaining sheep were sacrificed at 6 weeks after surgery. Subsequently, histologic analysis and mechanical testing were performed. In another group of 20 sheep, the same procedure was used and mechanical testing was performed after 3 weeks. Results More new bone was formed at the bone-tendon interface in the knees treated with bone morphogenetic protein–7 as compared histologically with similar areas in control animals, creating areas of dense trabecular network with significantly greater invasion of the tendon fibrous tissue into the bone marrow space. Mechanical testing showed greater strain resistance to force (368 N) in the knees treated with bone morphogenetic protein–7 than in control specimens (214 N). There was no difference between mechanical testing of samples from 3 and 6 weeks after surgery. Conclusion Bone morphogenetic protein–7 promotes complete tendon graft integration into the newly formed surrounding trabecular bone in the reconstruction of the anterior cruciate ligament. Clinical Relevance Bone morphogenetic protein–7 in tendon graft integration might be successfully used in reconstructive surgery of ligaments.

Expression of bone morphogenetic proteins and cartilage-derived morphogenetic proteins during osteophyte formation in humans

Bone‐ and cartilage‐derived morphogenetic proteins (BMPs and CDMPs), which are TGFβ superfamily members, are growth and differentiation factors that have been recently isolated, cloned and biologically characterized. They are important regulators of key events in the processes of bone formation during embryogenesis, postnatal growth, remodelling and regeneration of the skeleton. In the present study, we used immunohistochemical methods to investigate the distribution of BMP‐2, ‐3, ‐5, ‐6, ‐7 and CDMP‐1, ‐2, ‐3 in human osteophytes (abnormal bony outgrowths) isolated from osteoarthritic hip and knee joints from patients undergoing total joint replacement surgery. All osteophytes consisted of three different areas of active bone formation: (1) endochondral bone formation within cartilage residues; (2) intramembranous bone formation within the fibrous tissue cover and (3) bone formation within bone marrow spaces. The immunohistochemistry of certain BMPs and CDMPs in each of these three different bone formation sites was determined. The results indicate that each BMP has a distinct pattern of distribution. Immunoreactivity for BMP‐2 was observed in fibrous tissue matrix as well as in osteoblasts; BMP‐3 was mainly present in osteoblasts; BMP‐6 was restricted to young osteocytes and bone matrix; BMP‐7 was observed in hypertrophic chondrocytes, osteoblasts and young osteocytes of both endochondral and intramembranous bone formation sites. CDMP‐1, ‐2 and ‐3 were strongly expressed in all cartilage cells. Surprisingly, BMP‐3 and ‐6 were found in osteoclasts at the sites of bone resorption. Since a similar distribution pattern of bone morphogenetic proteins was observed during embryonal bone development, it is suggested that osteophyte formation is regulated by the same molecular mechanism as normal bone during embryogenesis.

Bone morphogenetic protein-7 reduces the severity of colon tissue damage and accelerates the healing of inflammatory bowel disease in rats

Bone morphogenetic protein‐7 (BMP‐7) is a growth and differentiation factor and belongs to the TGF‐β superfamily of proteins. Previous studies have shown an abundant expression of BMP‐7 in the developing intestine and an association with a perturbed BMP/SMAD downstream signaling leading to a malignant phenotype and inflammation in the gut. In the present study, we have evaluated the effect of systemically administered recombinant human BMP‐7 against trinitrobenzenesulfonic (TNBS) acid induced inflammatory bowel disease (IBD) in rats. The TNBS administered rats treated with BMP‐7 have developed much less severe form of colitis based on macroscopic and histological scoring when administered 1.5 h before or 24 h after colitis induction. Bioavailability studies in healthy rats have revealed that significant portion (3.6%) of i.v. administered BMP‐7 is targeted for BMP‐7 receptors in the stomach and ileum, respectively, suggesting its availability to target tissue upon administration. Immunohistochemical and RT‐PCR analyses have shown elevated expression of pro‐inflammatory (IL‐6, TNF‐β, ICAM‐1) and pro‐fibrogenic (TGF‐β) cytokines, and BMP‐7 treatment significantly reduced their expression in the intestine; among which the suppression of IL‐6 appeared to be the most important. Taken together, the results of this study suggest that BMP‐7 plays an important role in the regulation of anti‐inflammatory response in the adult gut tissue. J. Cell. Physiol. 196: 258–264, 2003.

Variations of the great arteries in the carotid triangle

The variations of the common carotid artery, as well as of the external and internal carotid arteries, are described. During anatomic dissection on adult cadavers, we investigated the variability of appearance of 40 carotid arterial systems. Special consideration was given to the topographic relations such as the level of the bifurcation of the common carotid artery, the relationship between the external and internal carotid arteries, and the origin of the great collateral branches. Special attention was paid to the origin of the superior thyroid artery. In this article the practical importance of these variations is stressed.

Tributaries of the Human and Canine Coronary Sinus

The coronary sinus is an anatomic landmark and conduit for many diagnostic and therapeutic procedures. We studied 40 human and 40 canine coronary sinuses of the heart in order to ascertain various functional anatomic features of the coronary sinus and its tributaries. We performed a comparative analysis of human and canine cardiac veins. We also studied the formation, tributaries, length, diameter and the ostium of the coronary sinus. The tributaries of the coronary sinus were highly variable in human and dog. The main and constantly present tributaries of both the human and canine coronary sinus were the great and the middle cardiac veins. We also found a rare case of a small cardiac vein connecting two cardiac venous systems--the coronary sinus and its tributaries and the anterior cardiac veins. In two cases we found a common trunk of the small cardiac vein and middle cardiac vein which opened into the coronary sinus. An intramyocardial course of the anterior interventricular vein was found in 2 cases and in 1 case the vein curved twice around the anterior interventricular branch of the left coronary artery. The Thebesian and Vieussens valves and ostial valves of other cardiac veins were also found.

Age- and region-dependent changes in human lumbar vertebral bone: a histomorphometric study.

Study Design. Histomorphometric evaluation of autopsy material. Objectives. To explore region-dependent changes that occur with aging in trabecular and cortical bone of the human vertebral body. Summary of Background Data. Human vertebral bone is continuously subjected to external forces (loads) that promote changes in inner architecture. This functional adaptability is limited, however, and when lost, vertebral bone progressively deteriorates and becomes subject to injury with increases in mechanical loading. Methods. Bone cylinders were drilled with a trephine from three regions (central anterior, central posterior, and lateral) of the third lumbar vertebral bodies of 48 autopsy cases 31 to 76 years old. Two consecutive 5-&mgr;m sections obtained 150 &mgr;m apart were stained with toluidine blue and Masson trichrome and photographed at ×40. Differences in numerous morphometric factors were evaluated by age and region of the vertebra using repeated-measures analysis of variance and Tukey’s Honestly Significant Difference test. Results. Starting at about 50 years of age, significant, linearly progressive decreases occurred in trabecular and cortical bone volume (P < 0.005), trabecular surface area (P < 0.001), number of trabeculae (P < 0.001), and thickness of trabeculae (P < 0.001). Space between trabeculae increased from ages 31 to 70 years and then decreased (P < 0.001). Trabecular deterioration was significantly more pronounced in central versus lateral regions (P < 0.001). Cortical bone thickness decreased significantly with aging in central regions but increased in lateral regions between ages 61 and 70 years (P < 0.001). Conclusions. The balance between cortical and trabecular bone maintains the strength of the vertebral body until about the age of 50 years, when irreversible deterioration begins in central regions and subsequently involves lateral regions.

Mn porphyrin-based SOD mimic, MnTnHex-2-PyP5+, and non-SOD mimic, MnTBAP3−, suppressed rat spinal cord ischemia/reperfusion injury via NF-κB pathways

Abstract Herein we have demonstrated that both superoxide dismutase (SOD) mimic, cationic Mn(III) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP5+), and non-SOD mimic, anionic Mn(III) meso-tetrakis(4-carboxylatophenyl)porphyrin (MnTBAP3−), protect against oxidative stress caused by spinal cord ischemia/reperfusion via suppression of nuclear factor kappa B (NF-κB) pro-inflammatory pathways. Earlier reports showed that Mn(III) N-alkylpyridylporphyrins were able to prevent the DNA binding of NF-κB in an aqueous system, whereas MnTBAP3− was not. Here, for the first time, in a complex in vivo system—animal model of spinal cord injury—a similar impact of MnTBAP3−, at a dose identical to that of MnTnHex-2-PyP5+, was demonstrated in NF-κB downregulation. Rats were treated subcutaneously at 1.5 mg/kg starting at 30 min before ischemia/reperfusion, and then every 12 h afterward for either 48 h or 7 days. The anti-inflammatory effects of both Mn porphyrins (MnPs) were demonstrated in the spinal cord tissue at both 48 h and 7 days. The downregulation of NF-κB, a major pro-inflammatory signaling protein regulating astrocyte activation, was detected and found to correlate well with the suppression of astrogliosis (as glial fibrillary acidic protein) by both MnPs. The markers of oxidative stress, lipid peroxidation and protein carbonyl formation, were significantly reduced by MnPs. The favorable impact of both MnPs on motor neurons (Tarlov score and inclined plane test) was assessed. No major changes in glutathione peroxidase- and SOD-like activities were demonstrated, which implies that none of the MnPs acted as SOD mimic. Increasing amount of data on the reactivity of MnTBAP3− with reactive nitrogen species (RNS) (.NO/HNO/ONOO−) suggests that RNS/MnTBAP3−-driven modification of NF-κB protein cysteines may be involved in its therapeutic effects. This differs from the therapeutic efficacy of MnTnHex-2-PyP5+ which presumably occurs via reactive oxygen species and relates to NF-κB thiol oxidation; the role of RNS cannot be excluded.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

Effect of salbutamol on innervated and denervated rat soleus muscle

The objective of the present investigation was to perform a 14-day time-course study of treatment with salbutamol, a beta2 adrenoceptor agonist, on rat soleus muscle in order to assess fiber type selectivity in the hypertrophic response and fiber type composition. Male Wistar rats were divided into four groups: control (N = 10), treated with salbutamol (N = 30), denervated (N = 30), and treated with salbutamol after denervation (N = 30). Salbutamol was injected intraperitoneally in the rats of the 2nd and 4th groups at a concentration of 0.3 mg/kg twice a day for 2 weeks. The muscles were denervated using the crush method with pean. The animals were sacrificed 3, 6, 9, 12, and 14 days after treatment. Frozen cross-sections of soleus muscle were stained for myosin ATPase, pH 9.4. Cross-sectional area and percent of muscle fibers were analyzed morphometrically by computerized image analysis. Treatment with salbutamol induced hypertrophy of all fiber types and a higher percentage of type II fibers (21%) in the healthy rat soleus muscle. Denervation caused marked atrophy of all fibers and conversion from type I to type II muscle fibers. Denervated muscles treated with salbutamol showed a significantly larger cross-sectional area of type I muscle fibers, 28.2% compared to the denervated untreated muscle. Moreover, the number of type I fibers was increased. These results indicate that administration of salbutamol is able to induce changes in cross-sectional area and fiber type distribution in the early phase of treatment. Since denervation-induced atrophy and conversion from type I to type II fibers were improved by salbutamol treatment we propose that salbutamol, like other beta2 adrenoceptor agonists, may have a therapeutic potential in improving the condition of skeletal muscle after denervation.