Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles

Matrix vesicles are present in the calcifying front and in the site of callus formation of fracture heeling. In calcifying process, matrix vesicles have important roles. The metalloprotease was isolated from matrix vesicles and subsequently characterized. Matrix vesicles obtained from chicken epiphysial cartilage by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles and isolated by Sephadex G-150 gel filtration. Disc electrophoresis of the enzyme gave a single protein band. The matrix vesicle protease had a MW of 33,000 daltons, an optimal pH of 7.2, and was inhibited 100% by 0.1 mM EDTA and 0.2 mM o-Phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by o-phenanthroline was reversed by Co2+, Zn2+, Fe2+ and Cu2+. The protease released from the matrix vesicle at the calcifying front could degrade non-collagenous protein moieties which inhibit precipitation of minerals in the extra-vesicular matrix and thus facilitate mineralization.     

Human Osteoarthritic Cartilage Matrix Vesicles Generate Both Calcium Pyrophosphate Dihydrate and ApatiteIn Vitro

Calcium crystals in osteoarthritic (OA) joints promote enzymatic degradation of articular tissues. Matrix vesicles provide a nidus for calcium crystal formation in chick epiphyseal and mature porcine articular cartilage. In order to examine a potential role for matrix vesicles from OA cartilage in generating pathologic crystals, we sought to determine whether vesicles derived from human OA cartilage (OAMV) could mineralize; and we characterized the resultant mineral species. OAMV were isolated and examined for alkaline phosphatase (AP) and nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity. OAMV ATP-dependent and independent mineralization were measured in a radiometric biomineralization assay, and newly formed OAMV crystals were examined using Fourier transform infrared spectroscopy (FTIR) and compensated polarized light microscopy. The mean specific activity of OAMV AP was approximately 6 times higher and NTPPPH activity 11 times lower than that of previously characterized, mature, porcine, articular cartilage vesicles. OAMV progressively precipitated ⁴⁵Ca over time both in the presence and absence of ATP. The FTIR spectra of mineral formed in ATP-dependent assays most closely resembled the standard spectrum for calcium pyrophosphate dihydrate (CPPD). The FTIR spectra of OAMV mineral formed in the absence of ATP closely resembled apatite. These data support the hypothesis that OAMV may form mineral phases of two key crystals found in degenerating cartilage and provide further evidence for the role of matrix vesicles in pathologic articular cartilage biomineralization.     

FTIR microspectroscopic analysis of human osteonal bone

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of non-osteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 m steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate v ₁,v ₃ band at 900–1200 cm^-1 to the amide I band at 1585–1725 cm^-1, increased from the center to the periphery of the osteon. The total carbonate (based on the v ₂ band at 850–900 cm^-1) to phosphate v ₁,v ₃ ratio decreased as the mineral to matrix ratio increased. Analysis of the v ₂ CO₃ ^2- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in labile carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the v ₁,v ₃ phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at 1020 and 1030 cm^-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.     

Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles

A metalloprotease has been isolated from matrix vesicles of chicken epiphyseal cartilage and subsequently characterized. Matrix vesicles obtained by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles by treatment with deoxycholate and freeze-thawing, and then isolated by Sephadex G150 gel filtration. Disc electrophoresis of the enzyme, which displayed protease activity toward azocasein substrate, gave a single protein band. Based on molecular weight (MW) determination, lack of immunocross reactivity, and differences in electrophoretic migration, there is little possibility of any contamination with external protease from the commercial collagenase used for vesicle preparation. The matrix vesicle protease had a MW of 33,000 and a pH optimum of 7.2 and was completely inhibited by 0.1 mM EDTA and 0.2 mM o-phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by omicron-phenanthroline was reversed by Co2+, Zn2+, Fe2+, and Cu2+. Protease activity was most abundant in the heavy fraction of matrix vesicles fractionated by discontinuous sucrose density gradient centrifugation. Release of this protease at the calcifying front could degrade noncollagenous protein moieties that inhibit precipitation of minerals in the extravesicular matrix and thus facilitate mineralization.     

Matrix Vesicles Mediate Mineralization of Human Thyroid Cartilage

Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca²⁺-P_i complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca²⁺ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca²⁺ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage. Received: 21 July 1999 / Accepted: 2 November 1999     

Influence of biochemical composition on endplate cartilage tensile properties in the human lumbar spine

Endplate cartilage integrity is critical to spine health and is presumably impaired by deterioration in biochemical composition. Yet, quantitative relationships between endplate biochemical composition and biomechanical properties are unavailable. Using endplate cartilage harvested from human lumbar spines (six donors, ages 51–67 years) we showed that endplate biochemical composition has a significant influence on its equilibrium tensile properties and that the presence of endplate damage associates with a diminished composition–function relationship. We found that the equilibrium tensile modulus (5.9 ± 5.7 MPa) correlated significantly with collagen content (559 ± 147 µg/mg dry weight, r² = 0.35) and with the collagen/GAG ratio (6.0 ± 2.1, r² = 0.58). Accounting for the damage status of the adjacent cartilage improved the latter correlation (r² = 0.77) and indicated that samples with adjacent damage such as fissures and avulsions had a diminished modulus–collagen/GAG relationship (p = 0.02). Quasi‐linear viscoelastic relaxation properties (C, τ₁, and τ₂) did not correlate with biochemical composition. We conclude that reduced matrix quantity decreases the equilibrium tensile modulus of human endplate cartilage and that characteristics of biochemical composition that are independent of matrix quantity, that is, characteristics related to matrix quality, may also be important. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:245–252, 2014.     

Isolation of matrix vesicles by isoelectric focusing in Pevikon-Sephadex

We have investigated the use of an isoelectric focusing (IEF) technique for isolating and characterizing matrix vesicles. Focusing was performed on crude preparations of matrix vesicles isolated from collagenase digests of chick epiphyseal cartilage and purified by discontinuous sucrose gradient centrifugation. Crude and partially purified vesicle preparations were subjected to flat bed IEF in a slurry of Pevikon-Sephadex. Partially purified matrix vesicles focused as a narrow band (pI congruent to to 6.5). Alkaline phosphatase, solubilized from matrix vesicles, focused with a pl of 4.0-4.5. The IEF profile of matrix vesicles also differed from that of chondrocyte membranes. Thus, the membrane pls were congruent to to 5.4 and 6.6-7.8, respectively. The latter peak probably corresponded to the pl of the matrix vesicle preparation. This observation lends support to the view that vesicles originate from distinct regions of the chondrocyte membrane.     

Dual-energy X-ray absorptiometry of the spine in anteroposterior and lateral projections

Dual-energy X-ray absorptiometry (DXA) of the lumbar spine provides an estimation of the bone mineral content (BMC) corrected by the projected area of the spine and expressed in g/cm². This two-dimensional estimate of the bone mineral density (BMD) is influenced by the skeletal size, assessed by the subject's height. In order to obtain an estimate of the volumetric BMD, we measured BMC with a new DXA device (Sophos L-XRA) equipped with 24 detectors and a rotating arm, thus allowing scanning of the lumbar spine in both an anteroposterior (AP) projection and a lateral (LAT) projection with the patient in a supine position. Comparison between the results obtained on the third (L3) and fourth (L4) lumbar vertebrae with automatic or manual analysis showed that the best precision was obtained with the lateral measurement of L3 alone with an automatic soft tissue baseline determination. Results were expressed in g/cm² and in g/cm³ (by dividing the g/cm² value by the width (AP area divided by the height of the vertebra) of L3), and were compared with those obtained by conventional AP scanning of L2–4 (g/cm²). The in vivo precision error evaluated by triplicate measurements on 10 controls was 17 mg/cm² (1.96%) and 5.2 mg/cm³ (2.31%) for LAT L3 as compared with 13 mg/cm² (1.15%) for AP L2–4. Volumetric BMD (g/cm³) measurement, assessed in vitro on a calibrated hydroxyapatite phantom, and the absolute values obtained in normal women were similar to those obtained by quantitative computed tomography (QCT). In 39 healthy adults (27±4 years) BMD expressed in g/cm² was correlated with height (r=0.36 for AP L2–4 andr=0.39 for LAT L3;p<0.05 for both) but not with LAT L3 BMD expressed in g/cm³ (r=0.02; NS). The age-related bone loss between 30 and 80 years of age, derived from the normal values for 101 healthy women (age range 19–73 years) was 36% for AP L2–4, 52% for LAT L3 (g/cm²) and 60% for LAT L3 (g/cm³). In a group of 22 women with untreated postmenopausal vertebral osteoporosis (one or more non-traumatic vertebral crush fractures) the mean decrease in BMD, expressed as a percentage of the age-adjusted normal value, was more pronounced (p<0.001) for LAT L3 BMD (–21% in g/cm²,Z-score –1.08; –22% in g/cm³,Z-score –0.94) than for AP L2–4 BMD (–9%,Z-score –0.66). We conclude that: 1) BMD measurement restricted to the vertebral body of L3 can be achieved with a low precision error with this new DXA device; 2) it allows an estimate of the volumetric density (g/cm³) which does not seem to be influenced by skeletal size; 3) lateral BMD appears to be more sensitive than conventional AP scanning for assessing age-related bone loss and should be useful in the investigation of trabecular osteoporosis.     

Body fat accumulation and postoperative morbidity in colorectal-cancer surgery

Summary BACKGROUND: Obesity is reported to increase the risk of colorectal cancer. We investigated whether it was associated with mortality or morbidity after colorectal-cancer surgery. METHODS: 70 colorectal-cancer patients who underwent elective surgery were investigated in this study. The intra-abdominal fat (IAF) and subcutaneous fat (SCF) areas were quantified by CT scan preoperatively. We investigated whether body mass index (BMI), IAF, or SCF were associated with postoperative mortality or morbidity. RESULTS: The mortality rate was 0%. 11 (16%) of 70 patients had medical complications (pneumonia, 9; arrhythmia, 2) and 16 (23%) had surgery-related complications (wound infection, 12; anastomotic leakage, 5) postoperatively. There was no significant difference in the postoperative medical or surgery-related morbidity rate between the IAF⁺ (males, 160 cm²; females, 120 cm²) and IAF^– groups. The surgery-related morbidity rate was significantly higher in the SCF⁺ group (male, 180 cm²; female, 250 cm²) than in the SCF^– group and multilogistic regression analysis confirmed that diabetes mellitus and SCF were significant for surgery-related morbidity. CONCLUSIONS: Subcutaneous fat accumulation is significantly associated with postoperative surgery-related morbidity after colorectal-cancer surgery.     

Exposure to low-intensity ultrasound increases aggrecan gene expression in a rat femur fracture model

The effects of ultrasound stimulation on various parameters of bone repair after diaphyseal injury were assessed in a standard rat femur fracture model. Bilateral closed femoral fractures were made in 79 skeletally mature male Long-Evans rats. An ultrasound signal consisting of a 200 microsecond burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 50 or 100 mW/cm² spatial and temporal average, was applied to one fracture in each animal. The contralateral fracture was not exposed to ultrasound and served as a control. Mechanical testing of the healing fracture was performed 3 weeks after injury. In fractures treated with a 50 mW/cm² ultrasound signal, the average maximum torque (223.5 ± 50.5 Nmm compared with 172.6 ± 54.9 Nmm, p = 0.022, paired t test) and average torsional stiffness (13.0 ± 3.4 Nmm/° compared with 9.5 ± 2.9 Nmm/°, p = 0.017) were significantly greater in treated than in control fractures. In animals treated with a 100 mW/cm² ultrasound signal, the average maximum torque and torsional stiffness were greater in treated than in control fractures, but this trend did not reach statistical significance. Biochemical analysis of callus in ultrasound-treated and control fractures failed to demonstrate significant differences in cell number, collagen content, or calcium content. Evaluation of gene expression in fractures treated with 50 mW/cm² ultrasound demonstrated a shift in the expression of genes associated with cartilage formation; aggrecan gene expression was significantly higher on day 7 after fracture and significantly lower on day 21 (p = 0.033 and 0.035, respectively). αl (11) procollagen gene expression was similarly modified, but this trend did not reach statistical significance. Expression of genes coding for bone-related proteins, including α1(I) procollagen, bone γ-carboxyglutamic acid protein, alkaline phosphatase, and transforming growth factor-β1, did not differ between ultrasound-treated and control fractures. These data suggest that ultrasound stimulation increased the mechanical properties of the healing fracture callus by stimulating earlier synthesis of extracellular matrix proteins in cartilage, possibly altering chondrocyte maturation and endochondral bone formation.     

In vitro precocious accumulation of calcium and matrix vesicles formation in young cartilage cells: Specific effects of corticosteroids

Summary The present study examined the effects of various corticosteroid and noncorticosteroid hormones upon the ultrastructure of chondroprogenitor cells and chondroblasts in an organ-culture system of late fetal condylar cartilage. Corticosteroids, (triamcinolone acetonide, dexamethasone, corticosterone) at concentrations of 10⁻⁶–10⁻⁸M stimulated markedly a precocious formation of matrix vesicles by chondroblasts. This stimulation was accompanied by a significant accretion of calcium complexes intra- and extracellularly in both the chondroprogenitor cell population and chondroblastsin vitro, as well as in the newly induced matrix vesicles. Nonglucocorticoid steroids (progesterone, estradiol, testosterone, cortexolone) did not evoke similar effects. Progesterone and testosterone, however, seemed to adversely affect the ultrastructure of the cartilage cells, whereas estradiol appeared to have a favorable effect on the morphology of cultured condylar cartilage.     

The occurrence of chloride ions in the apatite lattice of Holly Springs hydroxyapatite and dental enamel

The hydroxyl stretching mode in hydroxyapatite occurs as a single absorption band in the infra-red spectrum at 3570 cm^–1. When there is approximately 10% replacement of hydroxyl ions by chloride ions, an extra band appears at 3498 cm^–1 which is assigned to hydroxyl ions forming weak hydrogen bonds with adjacent chloride ions. A similar extra band appears at 3545 cm^–1 in the infra-red spectrum of a synthetic fluor-hydroxyapatite. Mineral Holly Springs hydroxyapatite contains fluorine and was also found to contain chlorine. The infra-red spectrum showed, in addition to the unperturbed hydroxyl stretching mode at 3570 cm^–1, two sharp bands at 3543 cm^–1 and 3498 cm^–1 which are assigned to hydroxyl ions hydrogen-bonded with fluorine and chlorine respectively. The infra-red spectrum of human enamel, which is known to contain about 0.3% by weight of chlorine shows a weak band at 3500 cm^–1 and it is proposed that this is due to chloride ions present in the apatite lattice.

---

Zusammenfassung Die Schwingung (stretching) der Hydroxylgruppe in Hydroxyapatit findet sich als einzelne Absorptionsbande im Infrarot-Spektrum bei 3570 cm^–1. Werden etwa 10% der Hydroxylionen durch Chloridionen ersetzt, so erscheint bei 3498 cm^–1 eine besondere Bande, die den Hydroxylionen zugeschrieben wird, welche schwache Wasserstoffbindungen mit benachbarten Chloridionen eingehen. Eine ähnliche zusätzliche Bande wird bei 3545 cm^–1 im Infrarot-Spektrum des synthetischen Fluor-Hydroxyapatites sichtbar. Anorganisches Holly Springs Hydroxyapatit enthält Fluor und es konnte auch ein Gehalt an Chlor nachgewiesen werden. Das Infrarot-Spektrum zeigte, zusätzlich zum unveränderten Bild der Hydroxylgruppen-Schwingung bei 3570 cm^–1, zwei scharf abgegrenzte Banden bei 3543 cm^–1 und 3498 cm^–1, welche den mit Fluor- bzw. Chlorwasserstoff-gebundenen Hydroxylionen zugeschrieben werden. Das Infrarot-Spektrum von menschlichem Zahnschmalz, der bekanntlich etwa 0,3 Gewichtsprozente Chlor enthält, zeigt eine schwache Bande bei 3500 cm^–1, die möglicherweise durch die im Apatitgitter vorliegenden Chloridionen verursacht wird.

---

Résumé L'élongation de l'hydroxyle au niveau de l'hydroxyapatite se traduit par une bande d'absorption unique dans le spectre infra-rouge à 3570 cm^–1. Lorsqu'il y a environ 10% de remplacement des ions hydroxyles par des ions de chlore, une bande supplémentaire apparait à 3498 cm^–1, qui semble dûe aux ions hydroxyles formant des liaisons d'hydrogène faibles avec les ions de chlore adjacents. Une bande supplémentaire similaire apparait à 3545 cm^–1 dans le spectre infra-rouge réalisé à partir d'un fluoro-hydroxyapatite synthétique. L'hydroxyleapatite minéral de type Holly Springs contient du fluor et du chlore. Le spectre infra-rouge présente, en plus de l'élongation non perturbée de l'hydroxyapatite à 3570 cm^–1, deux bandes nettes à 3543 cm^–1 et 3498 cm^–1, qui semblent être en rapport avec les ions hydroxyles liés par l'hydrogène respectivement avec le fluor et le chlore. Le spectre infra-rouge de l'émail humain, qui contient environ 0,3% en poids de chlore, présente une bande faible à 3500 cm^–1. Il semble que ce fait soit lié à la présence d'ions de chlore dans la maille de l'apatite.

     

Effect of glycosaminoglycans on calcium pyrophosphate crystal formation in collagen gels

Summary Formation of calcium pyrophosphate dihydrate (CPPD) crystals in native collagen gels represent anin vitro model system for the study of pathological cartilage calcification. The conditions under which CPPD forms in collagen gels have been determined. At low Ca x pyrophosphate product, CPPD forms directly. At high Ca x pyrophosphate product, CPPD forms via the amorphous intermediate calcium magnesium pyrophosphate (CMPP). Chondroitin sulfate (CS) inhibits formation of CPPD by both pathways, but apparently by different mechanisms. Direct CPPD formation is inhibited with low potency by CS, apparently by binding of Ca²⁺ ions. Indirect formation of CPPD is inhibited with high potency by CS, apparently by stabilization of the CMPP intermediate. Comparison of the inhibition of direct CPPD formation by the two glycosaminoglycan species occurring in cartilage proteoglycan showed that CS is a more potent inhibitor than keratan sulfate (KS), in agreement with the greater Ca²⁺-binding affinity of CS. The increase in KS/CS ratio which occurs in human hyaline cartilage with aging may facilitate deposition of CPPD crystals by decreasing the exclusion of pyrophosphate anions.     

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

Summary Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy [1], attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or [γ-P]ATP was labeled with³²P.³²P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1×10 anion exchange chromatography. The results of the analysis indicate that the (³²P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with³²P. In contrast,³²P was found to be associated with inorganic pyrophosphate (PPi) when [γ-³²P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form. The moles of Ca deposited were approximately equal to the sum of moles Pi and PPi deposited. Levamisole at 10 mM inhibited 70% of ATPase (specific release of³²Pi from [γ-³²P]ATP at pH 7.6) activity, 18% of Ca deposition, and 34% of Pi deposition during 3 hours of incubation. Adenosine monophosphate (AMP) or adenosine diphosphate (ADP) at 1 mM exerted earlier and greater inhibition on Ca and P (Pi and PPi) deposition than did 10 mM levamisole. Adenosine (1 mM) had little effect on Ca P deposition. Prolonged incubations which allowed enzymatic degradation of ADP and AMP substantially reduced the ADP and AMP inhibition. Levamisole was able to potentiate the AMP and ADP inhibition since it prevents further breakdown of AMP or ADP by alkaline phosphatase. Thus, we concluded that alkaline phosphate is not the major factor in thein vitro Ca P deposition by fetal bovine matrix vesicles. Instead, it appears that nucleoside trisphosphate pyrophosphohydrolase is directly involved in the early stages of deposition since both the enzyme and Ca P deposition were inhibited by AMP or ADP. The possible involvement of ATP phyophosphohydrolase in chondrocalcinosis is suggested.     

Fourier transform infrared spectroscopic study of the carbonate ions in bone mineral during aging

Summary The environment of CO₃ ²⁻ ions in the bone mineral of chickens of different ages and in bone fractions of different density have been investigated by resolution-enhanced Fourier Transform Infrared (FTIR) Spectroscopy. Three carbonate bands appear in thev ₂ CO₃ domain at 878, 871, and 866 cm⁻¹, which may be assigned to three different locations of the ion in the mineral: in monovalent anionic sites of the apatitic structure (878 cm⁻¹), in trivalent anionic sites (871 cm⁻¹), and in unstable location (866 cm⁻¹) probably in perturbed regions of the crystals. The distribution of the carbonate ions among these locations was estimated by comparing the intensities of the corresponding FTIR spectral bands. The intensity ratio of the 878 and 871 cm⁻¹ bands remains remarkably constant in whole bone as well as in the fractions obtained by density centrifugation. On the contrary, the intensity ratio of the 866 cm⁻¹ to the 871 cm⁻¹ band was found to vary directly and decreased with the age of the animal. In bone of the same age, the relative content of the unstable carbonate ion was found to be highest in the most abundant density centrifugation fraction. A resolution factor of the CO₃ ²⁻ band (CO₃ RF) was calculated from the FTIR spectra which was shown to be very sensitive to the degree of crystallinity of the mineral. The crystallinity was found to improve rapidly with the age of the animal. The CO₃ RF in the bone samples obtained by density centrifugation from bone of the same animal was found to be essentially constant. This indicates a fairly homogeneous, crystalline state of the mineral phase. A comparison of the maturation characteristics of synthetic carbonated apatites with bone mineral indicates that a simple, passive, physicochemical maturation process cannot explain the changes observed in the mineral phase of whole bone tissue or in the density centrifugation fractions of bone during aging and maturation.     

Measurement of radiometric surface temperature and integrated backscattered light intensity during feedback-controlled laser-assisted cartilage reshaping

Cartilage undergoes characteristic mechanical stress relaxation following laser irradiation below the ablation threshold. Porcine auricular cartilage (1–2 mm thickness) was irradiated with a Nd:YAG laser (=1.32 m) at two power levels (W/cm²). Surface temperature (S _c (t) (°C)) (monitored using a single element HgCdTe infrared detector, 10-14 m spectral range), and integrated back scattered light intensityI(t) were measured during laser irradiation. A HeNe laser beam (=632.8 nm) was incident on the back surface of the cartilage specimen and fractional integrated backscattered light intensity was measured using an integrating sphere anda silicon photodiode. Laser irradiation (5.83 W/cm², 50 Hz pulse repetition rate (PRR)) continued until surface temperature reached approximately 70°C, during which cartilage mechanical stress relaxation was observed. Integrated back scattered light intensity reached a plateau at about 70°C). At higher laser power (39.45 W/cm², 50 Hz PRR), a feedback-controlled cryogen spray was used to maintain surface temperature below 50°C. A similar plateau response was noted in integrated backscattered light intensity. This signal may be used to optimise the process of stress relaxation in laser cartilage reshaping. Several clinical applications involving reconstructive surgery are proposed.     

Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in "tiptoe walking" (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12-14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-beta and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited approximately 80-90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent 45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.     

Measurement of articular cartilage volumes in the normal knee by magnetic resonance imaging: can cartilage volumes be estimated from physical characteristics?

In recent times several studies have been performed on magnetic resonance imaging (MRI) sequences for imaging cartilage. A fat-suppressed three-dimensional sequence is one such noteworthy example. More recent studies have reported that the total volume of cartilage in a knee joint can be elucidated using this sequence. Based on these studies, we hypothesized that the total volume of cartilage in the knee joint may reflect certain other physical characteristics. The purpose of the current study was to clarify the articular cartilage volumes of the patella and femur in the human knee joints of healthy adults using MRI and to analyze the correlation of these volumes with other physical characteristics. The material comprised 68 knees of 68 Japanese healthy volunteers, aged from their twenties to their forties (37 men and 31 women) who had no past history of joint disease or trauma in the legs. The knees were imaged by MRI with a fat-suppressed three-dimensional sequence, and the cartilage volumes were calculated by computer processing. The factors analyzed were age, body weight, height, leg length, foot size, circumferences of the thigh and lower leg, the distance between medial and lateral femoral condyles, the diameter of the tibial head, body mass index, general joint laxity, quadriceps angle, and leg-heel alignment. The mean cartilage volume was 7.6 ± 1.6cm³ (8.3 ± 1.6cm³ in men, 6.7 ± 0.9cm³ in women). It was significantly larger in men than in women. However, the volume positively correlated with body weight, height, leg length, and foot size, without distinction of gender or age. Based on these data, a multiple regression analysis was developed: cartilage volume = 0.113 × height – 11.053. We concluded that the cartilage volume depends on physical size regardless of gender, and it can be estimated from factors of physical size.     

Pyrophosphatase and ATPase of isolated cartilage matrix vesicles

Some of the characteristics of the pyrophosphatase and ATPase activities studied in isolated cartilage matrix vesicles were found to be similar to those already reported for the solubilized and purified bone alkaline phosphatase. Thus, the pH optimum of the pyrophosphatase activity responded similarly to changes in the concentration of Mg²⁺, Ca²⁺, and PP_i. Further, the ATPase activity was not activated by Ca²⁺ in the presence of an optimal Mg²⁺ concentration. It is proposed that a function of the alkaline phosphatase of matrix vesicles in vivo is to hydrolyze the substrates PP_i, ADP, and ATP, which are known inhibitors of calcium phosphate precipitation.