Apatite Mineralization in Teeth of the Chiton Acanthopleura echinata

Raman spectroscopy has been used to demonstrate, for the first time, that calcium mineralization in the core of the major lateral teeth of the chiton Acanthopleura echinata takes place as an ordered process, with crystalline carbonated apatite being the first mineral deposited. Deposition begins at the top of the tooth core, under the so-called tab region, progresses down the interior surface of the tab and lepidocrocite layer, and then extends outwards to the anterior surface. Mineralization is not initiated until the lepidocrocite layer has isolated the core of the tooth from the magnetite cap. The last region to be infiltrated is the anterior basal region of the tooth cusp, immediately above the junction zone. The junction zone is also a region of high ion density, as determined by energy dispersive spectroscopy (EDS) analysis, but we show here for the first time that it is free of mineral deposits, acting instead as a transfer and storage region. Received: 7 January 2000 / Accepted: 30 May 2000 / Online publication: 2 November 2000     

Effects of Magnesium Deficiency on Magnesium and Calcium Content in Bone and Cartilage in Developing Rats in Correlation to Chondrotoxicity

Quinolone-induced arthropathy has been described in juvenile rats between 3 and 6 weeks of age, but not in adult rats. The mechanism of this chondrotoxic effect is probably related to the Mg²⁺-chelating properties of the drugs, since identical cartilage lesions were observed in magnesium-deficient juvenile rats without quinolone treatment. However, the reasons for the phase-specificity of the effect are unknown. In the present study, we fed a magnesium-deficient diet to Wistar rats at different postnatal developmental stages. Cartilage lesions were only observed in magnesium-deficient rats between 3 and 5 weeks of age, but not in rats receiving the magnesium-deficient diet during weeks 5 to 8, weeks 8 to 11, or months 15 to 16. The formation of cartilage lesions was not related to the magnesium concentration in plasma, since magnesium concentrations in plasma were similarly reduced in rats with and without cartilage lesions. However, chondrotoxicity correlated with magnesium content in articular cartilage. In articular cartilage (articular and epiphyseal cartilage in immature rats) and bone, magnesium content was more reduced in rats receiving the magnesium-deficient diet between 3 and 5 weeks of age as compared with rats receiving the magnesium-deficient diet during weeks 8 to 11 postnatally. It was not possible to reduce the magnesium content in bone tissue of 15-month-old Wistar rats, which suggests a lower magnesium turnover in aged rats. Magnesium content in epiphyseal cartilage of 2-week-old rats (total femoral head) was 41.9 ± 16.9 mmol/kg dry weight. The magnesium content in joint hyaline cartilage was significantly lower in 4-week-old rats (19.5 ± 3.6 mmol/kg dry weight) and increased subsequently again to 48.5 ± 9.2 mmol/kg dry weight (mean ± SD; n= 8 to 16). Increase of the magnesium content in femoral bone between weeks 4 and 6 postnatally was less pronounced (139 ± 10 and 175 ± 15 mmol/kg dry weight, respectively). Taken together, these data show that in 4-week-old rats, magnesium concentration in joint hyaline cartilage is significantly lower than at other times during postnatal development. Only at this developmental stage can cartilage lesions be induced by feeding rats a magnesium-deficient diet. This period correlates well with the sensitive phase of immature rats toward the chondrotoxic action of quinolones. Received: 30 September 1996 / Accepted: 31 December 1996     

The effects of the aminobisphosphonate alendronate on thyroid hormone-induced osteopenia in rats

Summary We describe a detailed study of fluoride distribution with age in the human cortical rib bone. Human ribs were obtained from 110 subjects (M:68,F;42) aged 20–93 years. The fluoride distribution from the periosteal to endosteal surfaces of the ribs was determined by sampling each specimen using an abrasive micro-sampling technique, and the samples were analyzed using the fluoride electrode, as described by Weatherell et al. [1]. The concentration of fluoride was highest in the periosteal region, decreased gradually towards the interior of the tissue where the concentration of fluoride tended toward the plateau, and then rose again towards the endosteal surface. Patterns of fluoride distribution changed with age, and the difference between periosteal and endosteal fluoride levels increased with age. Although average fluoride concentrations increased with age in both sexes, there was a significant difference between males and females at the age of about 55 years (P<0.05).     

The Effects of Magnesium on Hydroxyapatite Formation In Vitro from CaHPO4 and Ca4(PO4)2O at 37.4°C

The effects of magnesium ion on the formation of calcium-deficient hydroxyapatite [Ca₉HPO₄(PO₄)₅OH, CDHAp] from CaHPO₄ and Ca₄(PO₄)₂O dissolution were investigated using two magnesium sources: Mg₃(PO₄)₂ (chemical system 1) or MgCl₂· H₂O (chemical system 2) solutions. Because chloroapatite does not form from aqueous solutions, the use of two magnesium sources facilitated the determination of magnesium's role during synthetic hydroxyapatite formation in vitro and possible related effects during biomineralization. Isothermal calorimetry determined the progress of reactions. Two peaks are observed as heat is evolved during the formation of CDHAp in water at 37.4°C. The nucleation and growth of CDHAp are the corresponding mechanisms. Although the time for complete reaction and total heat-of-reaction ΔH_r remain constant, the height of the first peak is reduced as the concentration of magnesium ion approaches 4 mM in either chemical system. Magnesium does not substitute into CDHAp even though there are calcium vacancies available. Subsequent increases cause the remaining heat peak to broaden and the time required for complete reaction to approach 24 hours as the initial MgCl₂ concentration reaches 100 mM. Supersaturation limits chemical system 1 to Mg₃(PO₄)₂ concentrations below 10 mM. A MgCl₂ concentration of 3.16 M precludes CDHAp from forming for over 3 months; rather newberyite, MgHPO₄· 3H₂O, precipitates. The morphology and surface area of the CDHAp formed in 100 mM MgCl₂ solution are comparable to those of CDHAp formed in water. The surface areas are approximately 80 m²/g. Magnesium concentrations below 4 mM only inhibit nucleation whereas those above 4 mM inhibit growth as well. Magnesium phosphate complexes are more inhibitory than magnesium chloride complexes. Increasing the liquid-to-solids ratio or agitation significantly increases the induction period before reaction initiates. Increasing the liquid-to-solids ratio increases the time span for growth whereas increasing agitation decreases the time span for growth. The large inhibitory effect of agitation suggests quiescent systems are more suitable for determining the kinetics of HAp formation. A magnesium inorganic chemical activity (α_Mg=γ_Mg[Mg²⁺]) many times greater than that in biological fluids is required before inhibition of hydroxyapatite formation is realized. Received: 19 February 1996 / Accepted: 24 September 1996     

Calcification of the Lumbar Vertebrae During Human Fetal Development

The calcification rate of the human fetal vertebr? has not been previously documented. In bridging this gap in knowledge, this report will specifically provide reference values for mineral content in predominantly trabecular bone during its formation in the prenatal period. Such information would facilitate early detection of ossification defects which become apparent in the fetal spine as early as 20 weeks of gestation [1]. Lumbar vertebr? were obtained at autopsy from 33 human fetuses aged 17–41 weeks of gestation. Vertebral demineralization was effected using an acid-alcohol solution. The extracted concentrations of calcium and phosphorus were determined spectrometrically from the solutions. Atomic absorption spectroscopy was used to determine calcium concentration and phosphate concentration was assessed by ultraviolet colorimetry. Mineralization could be described by a linear relationship over the age range studied. Calcium was taken up by the fetal vertebr? at twice the rate of phosphorus (0.2% per week). When the fetuses were classed according to similar gestational ages, the Ca/P weight ratios increased from 1.7 in the youngest group to 2.2 in the oldest group. The time of onset of calcification, calculated by extrapolation of the calcium data, appeared to be earlier than ultrastructural studies by other investigators suggest, but was subject to many uncertainties. Received: 17 September 1996 / Accepted: 23 April 1997     

Dentin Sialoprotein (DSP) Has Limited Effects on In Vitro Apatite Formation and Growth

Sialoproteins such as bone sialoprotein (BSP) and dentin sialoprotein (DSP) accumulate at the mineralization fronts in bone and dentin, respectively, suggesting they have some function in the mineralization process. BSP, a highly phosphorylated protein rich in polyglutamate repeats, is an effective nucleator of hydroxyapatite (HA) formation in vitro. The present study examines the effect of DSP, a low phosphorylated but related sialoprotein, on the formation and growth of HA. In vitro, in a gelatin gel diffusion system, DSP at low concentrations (<25 μg/ml) slightly increased the yield of HA formed at 3.5 and 5 days, while at higher concentrations (50–100 μg/ml) it slightly inhibited accumulation. Fewer mineral crystals were formed in the presence of high concentrations of DSP but they tended to aggregate (making them appear larger by electron microscopic analysis) than those formed in DSP-free gels. X-ray diffraction line broadening analysis failed to show significant changes in c-axis crystal dimensions with increasing DSP concentration. When HA-seed crystals were coated with DSP before inclusion in the gelatin gel there was a reduction in mineral accumulation relative to HA-seeds which had not been coated with DSP, but the extent of inhibition was significantly less than that seen in this system with other mineralized tissue matrix sialoproteins, such as osteopontin or BSP. The low affinity of DSP for well-characterized seed crystals and the limited effect of this protein on HA formation and growth suggest that the role of DSP in dentin is not primarily that of a mineralization regulator. Received: 25 April 2000 / Accepted: 21 July 2000 / Online publication: 2 November 2000     

Treatment with Fluoride or Bisphosphonates Prevents Bone Loss Associated with Colitis in the Rat

Idiopathic inflammatory bowel disease (IBD) is associated with osteoporosis in over 30% of cases. We have previously shown that 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis in rats is associated with considerable bone loss. In the current study we tested the ability of sodium fluoride (NaF) or the bisphosphonate pamidronate to prevent the bone loss associated with TNBS-induced colitis in 22-week-old male Wistar rats. As previously found, there was a 43% decrease in cancellous bone volume in rats with TNBS-induced colitis after 4 weeks. This was associated with marked suppression of the bone formation rate to less than 25% of control animals. Treatment with NaF had no effect on the severity of colitis, but the bone volume and bone formation rate were increased to levels indistinguishable from those of control animals. In animals treated with pamidronate, cancellous bone volume was also restored to that of control animals despite persistence of the colitis. In these animals there was marked suppression of bone formation, associated with suppression of bone resorption. This data shows the bone loss associated with colitis may be prevented by treatment with NaF or bisphosphonates without requiring improvement in severity of the colitis. Received: 20 May 1999 / Accepted: 19 June 2000 / Online publication: 22 September 2000     

Fluoride Treatment Increased Serum IGF-1, Bone Turnover,and Bone Mass,but Not Bone Strength,in Rabbits

We hypothesized that fluoride partly acts by changing the levels of circulating calcium-regulating hormones and skeletal growth factors. The effects of oral fluoride on 24 female, Dutch-Belted, young adult rabbits were studied. The rabbits were divided into two study groups, one control and the other receiving about 16 mg fluoride/rabbit/day in their drinking water. After 6 months of fluoride dosing, all rabbits were euthanized and bone and blood samples were taken for analyses. Fluoride treatment increased serum and bone fluoride levels by over an order of magnitude (P < 0.001), but did not affect body weight or the following serum biochemical variables: urea, creatinine, phosphorus, total protein, albumin, bilirubin, SGOT, or total alkaline phosphatase. No skeletal fluorosis or osteomalacia was observed histologically, nor did fluoride affect serum PTH or Vitamin D metabolites (P > 0.4). BAP was increased 37% (P < 0.05) by fluoride; serum TRAP was increased 42% (P < 0.05); serum IGF-1 was increased 40% (P < 0.05). Fluoride increased the vertebral BV/TV by 35% (P < 0.05) and tibial ash weight by 10% (P < 0.05). However, the increases in bone mass and bone formation were not reflected in improved bone strength. Fluoride decreased bone strength by about 19% in the L5 vertebra (P < 0.01) and 25% in the femoral neck (P < 0.05). X-ray diffraction showed altered mineral crystal thickness in fluoride-treated bones (P < 0.001), and there was a negative association between crystal width and fracture stress of the femur (P < 0.02). In conclusion, fluoride's effects on bone mass and bone turnover were not mediated by PTH. IGF-1 was increased by fluoride and was associated with increased bone turnover, but was not correlated with bone formation markers. High-dose fluoride treatment did not improve, but decreased, bone strength in rabbits, even in the absence of impaired mineralization. Received: 5 November 1996 / Accepted: 3 January 1997     

A Comparative Infrared Spectroscopic Study of Hydroxide and Carbonate Absorption Bands in Spectra of Shark Enameloid, Shark Dentin, and a Geological Apatite

The purpose of the present work was to investigate the infrared (IR) spectrum of shark enameloid, especially with regard to hydroxide and carbonate bands. With thin sections placed directly in the IR beam it was possible to get high concentrations of ions without interfering effects from a dispersion medium (e.g., alkali halides). For comparison, spectra of shark dentin and a geo-apatite were also recorded. In spectra of shark enameloid and geo-apatite medium strong hydroxide absorption bands were found around 3535 cm⁻¹, and in shark dentin and geo-apatite spectra weak shoulders were observed at about 3570 cm⁻¹. Hydroxide libration bands at about 740 cm⁻¹ were found in shark enameloid and geo-apatite spectra; in the latter, also a band at 680 cm⁻¹. Carbonate bands were found in shark enameloid spectra at 1480 (weak shoulder), 1453, 1423, and 868 cm⁻¹. In shark dentin spectra there were carbonate bands at 1452, 1417, and 875 cm⁻¹, and probably also a carbonate band at about 1530 cm⁻¹ overlapped by an amide II band. Weak carbonate bands were also found in the spectra of the geo-apatite at 1452 cm⁻¹, and at about 1425 and 880 cm⁻¹. The relative intensities of the bands at 1453 cm⁻¹ (contributed from A and B sites) and around 1420 cm⁻¹ (B sites) changed from shark enameloid to shark dentin, and also from shark enameloid to the geo-apatite. More A sites seem to be occupied by carbonate in shark dentin than in shark enameloid, supposedly owing to fluoride occupation of A sites in shark enameloid. In geo-apatite and shark enameloid there are hydroxide ions hydrogen bonded to fluoride. Both shark enameloid and the geo-apatite are fluoride rich, and geo-apatite seems to have the highest fluoride concentration. There are, however, indications that the hydroxide concentration is also higher in the geo-apatite than in shark enameloid. This can be explained by the much higher carbonate content, and partly also by the higher water content in shark enameloid. There are A sites in geo-apatite and probably also in shark enameloid which are occupied by carbonate, but the proportion of occupied A sites relative to occupied B sites is greater in geo-apatite than in shark enameloid. This difference can be explained by the preference of A sites when the carbonate concentration is very low. On the other hand, for greater amounts of carbonate such as we have in shark enameloid, B sites are preferred. Received: 1 June 1998 / Accepted: 12 March 1999     

Fluoride Analysis of Apatite Crystals with a Central Planar OCP Inclusion: Concerning the Role of F− Ions on Apatite/OCP/Apatite Structure Formation

To study the roles of F⁻ ions in the formation of apatite crystals embedding octacalcium phosphate (OCP) lamella in the center of apatite (Ap), a range of the Ap/OCP/Ap lamellar-mixed crystals were synthesized under various concentrations of fluoride ion (F⁻) from 0.1–1.0 ppm at pH 6.5 and 37°C. The products were analyzed for the F⁻ incorporation, F⁻ distribution, and the amount of OCP and Ap by chemical analysis, X-ray diffraction (XRD), electron probe microanalysis (EPMA), and nuclear magnetic resonance (NMR) techniques. The F⁻ content and the amount of apatite in the crystalline product increased with an increase in the F⁻ concentration in solution, whereas the amount of OCP and the yield of total product decreased. EPMA indicated that F⁻ ions are distributed in the crystals almost homogeneously. The combined analysis suggested that a low-substituted fluoridated hydroxyapatite (FHAp) grew on a small amount of F⁻-containing OCP or on a surface-reaction layer of OCP, which has accumulated a small amount of F⁻. The roles of F⁻ ions were hypothesized as the reduction of the growth rate and/or the critical thickness in the a*-axis direction of OCP, the enhancement of hydrolysis of OCP, and the activation of the growth of FHAp, resulting in thinner OCP lamella and thicker apatite lamella in the a*-axis direction with an increase in F⁻ concentration. Received: 11 November 1995 / Accepted: 26 February 1996     

Multiple Cellular Phenotypes in an Insertional Mutation in Mouse Affecting Bone Development

The 6093 line of transgenic mice exhibits altered bone development as a result of an insertional mutation by the transgene. Female transgenic mice show a marked kyphosis as early as 2 weeks of age. Vertebrae from female mice have lower total bone area and mineral content than age-matched, gender-matched controls, although the bone mineral density is not changed. The femur and tibia exhibit the opposite effect—increased bone area and mineral content. Fluorescent bone label experiments indicated an increased rate of bone mineral deposition in the femur during the early postnatal growth period, and bone marrow from femurs of 6093 females had increased numbers of fibroblast colony-forming units. Transgenic females also are obese and have altered thymocyte development, suggesting that the insertional mutation affects multiple cell populations. We hypothesize that these phenotypes arise as a result of an alteration in the function or developmental potential of a stromal cell or mesenchymal stem cell. Received: 17 April 2000 / Accepted: 24 August 2000 / Online publication: 22 December 2000     

Morphological and Functional Features of Clasts in Low Phosphate, Vitamin D-Deficiency Rickets

Focusing on resorption processes, we have extended our previous studies on chondroclasts and osteoclasts in normally developing tissues, using a model of nutritionally induced vitamin D-deficiency rickets. To analyze the resorption process, we investigated the matrix-resorbing cells in this modified and poorly mineralized tissue regarding morphological features and expression of tartrate-resistant acid phosphatase (TRAP) at the subcellular level. Our goal was to test the hypotheses that initiation of resorption is impaired with unmineralized matrix, and that such alterations involve changes in the subcellullar distribution of TRAP, implicating a role for this enzyme in the resorption process. Our results reveal distinctly different morphological appearances of clast-like cells in rickets compared with normal osteoclasts and chondroclasts. Ordinary resorption structures of osteoclasts and chondroclasts at the cell-matrix border, i.e., ruffled borders and clear zones, are profoundly altered in favor of a less well-defined intermediate zone. TRAP distribution at the subcellullar level is also clearly different from that in osteoclasts and chondroclasts from normal rodents, with impaired secretion; consequently, the enzyme is unable to function in the matrix outside the ruffled border. Our ultrastructural observations demonstrate that in rickets, the clasts are incapable of degrading the poorly mineralized cartilage and bone efficiently. Rachitic clasts seem to be recruited to the matrix surface and interaction between cell and matrix is also initiated, but definitive resorption structures at the cell-matrix border are not normally developed. Whether resorption is inhibited by the mere lack of mineral or mineral-associated proteins, or by other mechanisms remains to be settled. Received: 5 January 2000 / Accepted: 12 May 2000 / Online publication: 2 November 2000     

A simple laparoscopic method to provide access to the gastroesophageal junction in obese patients

By now, the feasibility of laparoscopic surgery in obese patients is well established; a conversion rate of 1.4–4.3% has been reported [1, 2]. The main reason for conversion in these cases is the difficulty encountered in exposing the gastroesophageal junction due to a huge fatty liver that covers the entire upper abdomen (``the invisible stomach' [1]). We report here a simple method that allows easy access to the upper stomach in such cases. This technique involves the exposure of the gastroesophageal junction using a laparoscopic suprahepatic route. Received: 30 April 1999/Accepted: 5 October 1999/Online publication: 24 March 2000     

Laparoscopic pancreaticoduodenectomy in the porcine model

Background: Minimally invasive techniques offer theoretical advantages for treating resectable periampullary neoplasms. Laparoscopic pancreaticoduodenectomy (LPD) was first reported in 1992 and has been performed clinically despite lack of animal data to support the operation. The purpose of this study was to develop LPD in an acute porcine model and to assess safety and efficacy before considering clinical trials. Methods: LPD was initiated in six domestic pigs under general anesthesia. Once pneumoperitoneum was created, five 10-mm access ports were placed (one central and two in each flank). After cholecystectomy, the duodenum was mobilized and the proximal jejunum was divided distal to the ligament of Treitz. The neck of the pancreas was separated from the superior mesenteric vein, and the midstomach was divided by a stapler. Pancreaticojejunostomy (PJ), choledochojejunostomy (CDJ), and gastrojejunostomy (GJ) were performed using interrupted sutures. The animals were immediately sacrificed and the operative site was examined. Results: LPD was aborted in three animals due to complications: intestinal perforation with fecal contamination (one) and prolonged resection time ≥ 2.5 h (two). LPD was completed in three animals (operative time ranged from 5.0 to 7.5 h, blood loss < 200 cc); however, at sacrifice one PJ and two CDJs had small posterior leaks. The efferent loop of the GJ was narrowed by the staple line in one pig. All animals had extensive ecchymosis of the jejunal serosa due to excessive manipulation. Conclusion: Despite a significant number of anastomotic leaks in the immediate postoperative period, laparoscopic pancreaticoduodenectomy is feasible in a porcine model. Further studies and technical development are necessary before laparoscopic pancreatic resection can be performed on a more widespread basis. Received: 22 April 1996/Accepted: 10 July 1996     

Prednisolone Induces an Increase in Serum Calcium Concentration: Possible Involvement of the Kidney, the Bone, and the Intestine

To evaluate the early effect of glucocorticoids on calcium metabolism, 15 subjects aged 22–58 years (5 males, 10 females) with chronic glomerulonephritis were orally treated with 40 mg daily of prednisolone. Five of these subjects were diagnosed with nephrotic syndrome and none had a serum creatinine concentration of more than 1.4 mg/dl. Serum specimens and 24-hour urine specimens were obtained just before and 24 hours after a single oral dose of prednisolone. Serum calcium, ionized calcium, phosphate, intact parathyroid hormone (PTH), intact osteocalcin and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and urinary excretion of calcium, phosphate, and deoxypyridinoline were measured. Both serum calcium and ionized calcium concentrations were significantly increased from 4.39 ± 0.10 to 4.47 ± 0.09 mEq/liter (P= 0.037) and from 2.48 ± 0.04 to 2.55 ± 0.04 mEq/liter (P= 0.002), respectively, 24 hours following a single oral dose of prednisolone. Serum intact PTH concentration slightly decreased, but the difference was not significant by statistical analysis. Serum intact osteocalcin concentration was markedly suppressed. In contrast, no significant changes were observed in urinary excretion of deoxypyridinoline. Serum 1,25(OH)₂D₃ concentration measured in five patients was significantly increased. No significant changes in urinary excretion of calcium was observed in the face of these findings. It thus follows that a single oral dose of prednisolone administration increases serum calcium and ionized calcium concentrations, possibly mediated by suppressed bone formation, increased intestinal absorption of calcium, and impaired urinary excretion of calcium. Received: 19 February 1998 / Accepted: 12 March 1999     

Increased Serum Levels of N-Telopeptides (NTx) of Bone Collagen in Postmenopausal Nigerian Women

Serum levels of cross-linked N-telopeptides (NTx) of bone collagen, alkaline phosphatase (ALP), and intact parathyroid hormone (PTH) were determined in 64 premenopausal (PRM) and 86 postmenopausal (PSM) women living in northern Nigeria. Serum NTx values were correlated with ALP activity (r = 0.31–0.58, P < 0.01) and PTH (0.32–0.35, P < 0.01)) in all of the subjects studied, and were also related to age (−0.47, P < 0.001) and body mass index (−0.45, P < 0.001) in PRM women. Menopause had the effect of increasing the circulating concentrations of NTx and ALP activity by 15% (P= 0.001) and 11% (P= 0.02), respectively; however, serum levels of PTH were not different between these two groups of women. Compared with Caucasian counterparts matched for age and body mass index, PSM Nigerian women had significantly increased circulating concentrations of NTx (21.7 versus 16.2 nmol BCE/liter, P= 0.01) and demonstrated a trend towards higher ALP activities and PTH levels. These results indicate that (1) discrete reference intervals should be defined for biochemical markers of bone metabolism in African populations, (2) Nigerian women have relatively higher rates of bone turnover, and (3) further investigation of the implications of increased serum NTx should be undertaken using physical methods such as dual X-ray absorptiometry (DXA) and bone ultrasound attenuation. Received: 16 September 1998 / Accepted: 10 January 1999     

Effects of Calcium on Gap Junctions Between Osteoblast-Like Cells in Culture

The present study investigated the effects of elevated cytoplasmic free calcium concentrations ([Ca²⁺]_i) on the permeability of gap junctions between cultured osteoblast-like (OB) cells derived from calvarial and periosteal fragments of newborn rats. This was studied using the double whole cell patch clamp technique and intracellular dye injections. To increase [Ca²⁺]_i, patch pipette solutions contained 100 μmol/liter Ca²⁺. About 1–2 minutes after whole cell modes had been attained, the total number of gap junction channels was reduced from an average of 400 in normal Ca²⁺ to 20 in high Ca²⁺. Thereafter, remaining gap junction channels were active for up to 8 minutes. In normal rat kidney (NRK) cells, gap junction channels were closed by high Ca²⁺ within 1 minute, pointing to a similar sensitivity of Connexin43 gap junction channels in OB and NRK cells. To study the effects of elevated [Ca²⁺]_i on the dye permeability of gap junctions between extended OB cells, the spread of Lucifer Yellow to neighboring cells was evaluated. [Ca²⁺]_i was gradually increased from 1.5- to 14-fold the normal value by application of either ouabain, Na⁺-free/ouabain, or A23187. Reduced dye spread correlated with the increase of [Ca²⁺]_i measured by analyzing the fluorescence of fura-2. These data show that gap junctions in OB cells are sensitive to Ca²⁺. Received: 15 August 1995 / Accepted: 12 February 1996     

Calcium Mobilization is Required for Spreading in Human Osteoblasts

Adhesion-induced changes in intracellular calcium concentration ([Ca²⁺]_i) were measured in populations of human osteoblasts spreading on bone matrix proteins. In cells spreading on collagen type I, fibronectin, or laminin, average values for [Ca²⁺]_i were found to increase approximately 2× over baseline and then decline. The speed with which [Ca²⁺]_i increased and declined was dependent upon the matrix protein on which the cells were plated but was generally complete within 1 hour from the time of plating. Calcium mobilization was found to be due to influx of calcium across the osteoblast plasma membrane and was integrin dependent. Carboxyamido triazole (CAI), a specific inhibitor of nonvoltage-dependent calcium channels, or BAPTA-AM, a chelator of intracellular calcium, inhibited osteoblast adhesion and spreading on collagen type I, fibronectin and laminin in a dose-dependent manner. In conclusion, these results demonstrate that calcium mobilization is induced upon integrin-ligand contact and that calcium influx is required for cell adhesion and spreading. Received: 10 September 1999 / Accepted: 18 January 2000     

Chondrocyte Differentiation in Human Osteoarthritis: Expression of Osteocalcin in Normal and Osteoarthritic Cartilage and Bone

Osteocalcin (OC), which is a marker of the mature osteoblasts, can also be found in posthypertrophic chondrocytes of the epiphyseal growth plate, but not in chondrocytes of the resting zone or in adult cartilage. In human osteoarthritis (OA), chondrocytes can differentiate to a hypertrophic phenotype characterized by type X collagen. The protein- and mRNA-expression pattern of OC was systematically analyzed in decalcified cartilage and bone sections and nondecalcified cartilage sections of human osteoarthritic knee joints with different stages of OA to investigate the differentiation of chondrocytes in OA. In severe OA, we found an enhanced expression of the OC mRNA in the subchondral bone plate, demonstrating an increased osteoblast activity. Interestingly, the OC protein and OC mRNA were also detected in osteoarthritic chondrocytes, whereas in chondrocytes of normal adult cartilage, both the protein staining and the specific mRNA signal were negative. The OC mRNA signal increased with the severity of OA and chondrocytes from the deep cartilage layer, and proliferating chondrocytes from clusters showed the strongest signal for OC mRNA. In this late stage of OA, chondrocytes also stained for alkaline phosphatase and type X collagen. Our results clearly show that the expression of OC in chondrocytes correlates with chondrocyte hypertrophy in OA. Although the factors including this phenotypic shift in OA are still unknown, it can be assumed that the altered microenvironment around osteoarthritic chondrocytes and systemic mediators could be potential inducers of this differentiation. Received: 20 May 1999 / Accepted: 10 February 2000     

Time Pattern of Exercise-Induced Changes in Type I Collagen Turnover after Prolonged Endurance Exercise in Humans

Type I collagen is known to adapt to physical activity, and biomarkers of collagen turnover indicate that synthesis can be influenced by a single intense exercise bout, but the exact time pattern of these latter changes are largely undescribed. In the present study, 17 healthy young males had their plasma concentrations of the carboxyterminal propeptide of type I procollagen (PICP), a marker of collagen formation, and the immunoactive carboboxyterminal cross-linked telopeptide (ICTP), a marker of collagen resorption, measured before and immediately postexercise, as well as 1, 2, 3, 4, 5, and 6 days after completion of a marathon run (42 km). Serum concentrations of creatine kinase (S-CK) were measured as an indicator of muscular breakdown in response to the exercise bout. After a transient decrease in collagen formation immediately after exercise (plasma PICP concentration: 176 ± 17 μg/liter to 156 ± 9 μg/liter)(P < 0.05), concentrations rose in the days following the marathon, peaked 72 hours after exercise (197 ± 8 μg/liter)(P < 0.05 versus basal), and returned to basal values similar to those 5 days postexercise (170 ± 10 μg/liter). Apart from a short increase immediately after exercise, collagen resorption did not change from basal levels throughout the remaining period (P > 0.05). Muscle breakdown was elevated during the days following the exercise and peaked 24 hours after the exercise (S-CK concentration: 3133 ± 579 U/liter). The findings in the present study indicate that type I collagen synthesis is accelerated in response to prolonged strenuous exercise, reaching a peak after 3 days and returning to preexercising levels 5 days after the completion of a marathon run. Received: 21 July 1999 / Accepted: 10 February 2000