Effects of low-intensity pulsed ultrasound on proliferation and chondroitin sulfate synthesis of cultured chondrocytes embedded in Atelocollagen gel.

The effects of low-intensity pulsed ultrasound (US) on the proliferation and chondroitin sulfate synthesis of cultured chondrocytes embedded in Atelocollagen gel in vitro were examined. Articular cartilage was harvested from the hip, knee, and shoulder joints of 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gel, Atelocollagen gel, and were cultured in Dulbecco's modified eagle's medium for 3 weeks. The US apparatus, SAFHS, was used to deliver an ultrasound signal with spatial and temporal average intensities of 30 mW/cm(2) (US group). The frequency was 1.5 MHz with a 200-microsecond tone burst repeated at 1.0 kHz. US treatments were administered for 20 min per day under culture dishes, with the medium replaced twice a week. Another group of cells was exposed to sham ultrasound as a control. Cell number, histological findings, synthesis of isomers of chondroitin sulfate, and stiffness of the chondrocyte-collagen gel composites were analyzed. US exposure promoted synthesis of chondroitin sulfate, especially chondroitin 6-sulfate, although it did not significantly enhance cell number and stiffness. In this three-dimensional culture model, these results suggest that US exposure may be clinically useful in improving the quality of chondrocyte-Atelocollagen implants for transplantation into articular cartilage defects.

Effects of hyaluronic acid and basic fibroblast growth factor on motility of chondrocytes and synovial cells in culture.

The capacity of chondrogenic precursor cells to migrate and proliferate in an injured area is considered to be essential for cartilage repair. We examined cell motility of chondrocytes and synovial cells in monolayer culture and the chemokinetic effects of hyaluronic acid (HA) and basic fibroblast growth factors (bFGF) on these cells. The velocity of chondrocyte migration was accelerated by giving bFGF and simultaneously administering of both HA and bFGF, but it was not affected by HA alone. The velocity of synovial cell migration was increased by HA, but not by bFGF. HA had a chemokinetic effect on synovial cells and bFGF had the same effect on chondrocytes. Treatment with exogenous HA and bFGF may be of value for repairing articular cartilage injury by recruiting chondrogenic cells and promoting migration of chondrocytes in the cartilage tissue.

Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin.

Anti-inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)-induced microvascular permeability change. Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400 microg site-1) from Salmonella typhimurium. Dye leakage in the skin was significantly increased 2 h after injection of LPS. This LPS-induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160 mg kg-1), milrinone (5 - 10 mg kg-1), rolipram (0.5 - 10 mg kg-1) and zaprinast (5 - 10 mg kg-1). The dye leakage was also inhibited by beta-adrenoceptor agonists, including isoproterenol (0.5 - 5 mg kg-1) and salbutamol (0.05 - 5 mg kg-1), an adenylate cyclase activator, forskolin (5 mg kg-1), and a cell permeable cyclic AMP analogue, 8-bromo-cyclic AMP (8-Br-cAMP, 10 mg kg-1). LPS caused a transient increase in serum TNF-alpha level peaking at 1 h after the injection. This increase in serum TNF-alpha was completely blocked by a pretreatment with pentoxifylline (160 mg kg-1), milrinone (5 mg kg-1), rolipram (1 mg kg-1), zaprinast (10 mg kg-1), salbutamol (0.5 mg kg-1), forskolin (1 mg kg-1) and 8-Br-cAMP (10 mg kg-1). LPS caused an increase in serum IL-1alpha level peaking at 3 h after injection. This increase in serum IL-1alpha was not significantly suppressed by the cyclic AMP elevating agents. Our study suggests that cyclic AMP elevating agents attenuate LPS-induced microvascular permeability change by suppressing TNF-alpha up regulation.

Alteration of collagen network and negative charge of articular cartilage surface in the early stage of experimental osteoarthritis.

Forty-eight mature male Japanese white rabbits were subjected to unilateral resection of a segment of the gluteal muscles at the sacral origin and a section of infrapatellar ligament. Animals were killed at 1, 2, 4, and 8 weeks postoperatively, and the articular cartilage of the femoral heads was evaluated. The collagen fibrillar network of the articular surface was observed by scanning electron microscopy (SEM) using microdissection by ultrasonication. Cationized ferritin (CF) was used for the labeling of negative charges on the articular surfaces and the thickness of CF layers was observed under the transmission electron microscope. Metachromasia of the matrix decreased remarkably at 4 weeks postoperatively, and fibrillation of the articular surface was evident at 8 weeks postoperatively. Derangement and rupture of the collagen network developed as early as 1 week after surgery. The thickness of the CF layer significantly decreased at 4 weeks postoperatively. This study confirms that alterations of the articular surface, such as derangement of the collagen network and loss of the negative charge, are some of the earliest changes in osteoarthritis. In addition, application of ultrasonication with proper frequencies to the articular cartilage effects an optimal removal of mucus, with the consequent exposure of a well-preserved articular surface for SEM study.

Intraosseous hypertension and venous congestion in osteonecrosis of the knee.

To determine whether an angiogenic factor affects the pathogenesis of the idiopathic osteonecrosis of the medial femoral condyle, intraosseous pressure and venogram in 11 knees with osteonecrosis were compared with intraosseous pressure and venogram in 11 knees with the medial type of osteoarthritis. Patients were matched by age, gender, obesity index, blood pressure, tibiofemoral angle, and clinical evaluation. The intraosseous pressure of the medial condyle of the knees with osteonecrosis (62.8 +/- 27.3 mm Hg) was significantly higher than that in the lateral condyle of the knees with osteonecrosis (25.4 +/- 18.9 mm Hg) and those of both condyles of the knees with osteoarthritis (medial, 31.6 +/- 17.4 mm Hg; lateral, 29.5 +/- 11.0 mm Hg). In contrast, there was no significant difference in the pressure between the medial and lateral condyles of the knees with osteoarthritis. Venography showed a marked disturbance of venous drainage in all patients with osteonecrosis. In addition, the average clearance time of the medium in the medial femoral condyle was significantly more prolonged in patients with osteonecrosis (17.7 +/- 6.1 minutes) than in patients with osteoarthritis (5.5 +/- 1.6 minutes). These data support the hypothesis that venous stasis within the medullar canal in the condyle increases intraosseous pressure and decreases arteriovenous pressure difference, leading to osteonecrosis.

A Ca(2+) channel blocker-like effect of dehydrocurdione on rodent intestinal and vascular smooth muscle.

Effects of dehydrocurdione, a zedoary-derived sesquiterpene, on smooth muscle were investigated by recording the mechanical activity of intestines and aorta from guinea pigs and rats. Dehydrocurdione (0.1-3 mM) induced a sustained relaxation of rat duodenum and inhibited spontaneous motility. Dehydrocurdione (0.1-1 mM) inhibited the contractile response of guinea pig ileum induced by acetylcholine (0.01-10 microM), histamine (0.03-10 microM) and substance P (0.1-30 nM) in a non-competitive manner. Acetylcholine (0.5 microM) elicited a transient contraction followed by a sustained contraction of guinea pig ileum, and dehydrocurdione pretreatment inhibited the sustained component, which depends on Ca(2+) entry from the extracellular space. The high K(+)-induced contraction of rat aortic ring is reported to be blocked by Ca(2+) channel blockers, while the norepinephrine-induced contraction includes a Ca(2+) channel blocker-resistant component. Dehydrocurdione (1 mM) blocked the high K(+) (60 mM)-induced contraction of rat aortic ring by 81%, while it inhibited the norepinephrine (1 microM)-induced contraction by only 28%. Dehydrocurdione (1 mM) significantly reduced the high K(+)-stimulated increase in cytosolic Ca(2+) level of Fura-2-loaded mesenteric artery from rats. These results suggest that the inhibitory effects of dehydrocurdione on intestinal and vascular smooth muscle are mediated by blockade of Ca(2+) entry from the extracellular space.

Evaluation of core decompression for early osteonecrosis of the femoral head.

Twenty-six hips (19 patients) with osteonecrosis of the femoral head with stage I or II of the disease, according to the Ficat and Arlet classification, underwent core decompression. Osteonecrosis was confirmed histologically in all 26 hips. Of 19 patients, 7 had prognostic factors traditionally associated with poor outcome including collagen vascular disease and continued use of steroids. The follow-up period averaged 7 years 10 months (range: 2 years 5 months-13 years 8 months) for 17 patients with 24 hips. Two patients died secondary to systemic illness. Seventeen hips (65.4%) had very good or good results using the Ficat criteria. Eight hips (30.8%) needed further operation [total hip arthroplasty (THA) for 7 hips, osteotomy for 1 hip]. Of the 12 hips in patients who had used steroids, 6 hips (50%) were converted to THA. Four hips in patients with systemic lupus erythematosus (SLE) needed THA (100%). We conclude that core decompression provides an effective treatment for steroid-associated osteonecrosis other than in cases with SLE, as well as providing effective treatment for non-steroid-associated osteonecrosis in the early stages of the disease.

Apoptosis during inner ear development in human and mouse embryos: an analysis by computer-assisted three-dimensional reconstruction.

Apoptosis in the developing inner ear tissue of human (Carnegie stage 14 to 21, approximately 5 to 8 weeks of gestation) and mouse (10.5 to 14 days of gestation) embryos was systematically analyzed by a computer-assisted three-dimensional reconstruction of the serial histological sections and by the TUNEL method. Morphogenetic events such as folding between the utricular portion and endolymphatic duct, constriction of the junction of the saccule with the cochlea and folding of the vestibular portion to form the semicircular ducts were accompanied by a localized distribution of apoptosis. The apoptosis was also related to the innervation of the cochlear and vestibular epithelia from the sensory ganglion of the eighth cranial nerve and the differentiation of the otic epithelia into the sensory epithelia. These results suggest that apoptosis plays an important role in the development of the inner ear.