Bi-zonal cartilaginous tissues engineered in a rotary cell culture system.

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.

Cartilage tissue engineering by expanded goat articular chondrocytes.

In this study we investigated whether expanded goat chondrocytes have the capacity to generate cartilaginous tissues with biochemical and biomechanical properties improving with time in culture. Goat chondrocytes were expanded in monolayer with or without combinations of FGF-2, TGF-beta1, and PDGFbb, and the postexpansion chondrogenic capacity assessed in pellet cultures. Expanded chondrocytes were also cultured for up to 6 weeks in HYAFF-M nonwoven meshes or Polyactive foams, and the resulting cartilaginous tissues were assessed histologically, biochemically, and biomechanically. Supplementation of the expansion medium with FGF-2 increased the proliferation rate of goat chondrocytes and enhanced their postexpansion chondrogenic capacity. FGF-2-expanded chondrocytes seeded in HYAFF-M or Polyactive scaffolds formed cartilaginous tissues with wet weight, glycosaminoglycan, and collagen content, increasing from 2 days to 6 weeks culture (up to respectively 2-, 8-, and 41-fold). Equilibrium and dynamic stiffness measured in HYAFF M-based constructs also increased with time, up to, respectively, 1.3- and 16-fold. This study demonstrates the feasibility to engineer goat cartilaginous tissues at different stages of development by varying culture time, and thus opens the possibility to test the effect of maturation stage of engineered cartilage on the outcome of cartilage repair in orthotopic goat models.

Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs.

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.

Properties of collagen in OIM mouse tissues.

The deletion of the alpha2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the alpha2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6 degrees and 1.9 degrees C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The alpha2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.

Ovine periodontitis as a potential model for periodontal studies. Cross-sectional analysis of clinical, microbiological, and serum immunological parameters.

OBJECTIVES: : To investigate infection and host immunity patterns in sheep with naturally occurring "broken-mouth" periodontitis. MATERIALS AND METHODS: : Eight periodontally healthy (HS) and eight periodontally diseased ewes (PDS) were selected. Subgingival plaque and sera were collected and examined for evidence of human periodontitis-associated pathogens. Serum IgG titers were measured by ELISA to multiple strains of Porphyromonas gingivalis, Bacteroides forsythus, Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum as well as several purified antigens (cysteine proteases, LPS, K, and fimbriae). RESULTS: : Neither the organism Aa nor antigens to Aa were found in any animal. Most animals were positive for Pg, Bf, and Pi, but DNA probes detected no difference between HS and PDS relative to amounts of pathogens in subgingival plaque. PDS had significantly higher serum IgG titers to all Pg strains, to 50% of Bf strains, to the Pi and Fn strains, and to fimbriae and the two cysteine proteases (p-values ranging from 0.05 to 0.001). Regression analysis demonstrated a significant association between number of teeth lost and serum IgG antibody titers to whole-cell sonicate antigens of P. gingivalis strains (p<0.01) and body weight (p<0.01). CONCLUSIONS: : The presence of pathogens associated with periodontitis was reflected in differences in serum IgG titers between healthy and diseased sheep. This may have influenced animal body weight and might have systemic health and economic consequences. The data suggest that susceptible and non-susceptible sheep can be identified for periodontal research.

Stretch receptor-associated expression of alpha 3 isoform of the Na+, K+-ATPase in rat peripheral nervous system.

Expression of the neuronal alpha(3) isoform of the Na(+),K(+)-ATPase (alpha(3) Na(+),K(+)-ATPase) was studied in the rat peripheral nervous system using histological and immunohistochemical techniques. Non-uniform expression of the alpha(3) Na(+),K(+)-ATPase was observed in L5 ventral and dorsal roots, dorsal root ganglion, sciatic nerve and its branches into skeletal muscle. The alpha(3) Na(+),K(+)-ATPase was not detected in nerve fibers in skin, saphenous and sural nerves. In dorsal root ganglion 12+/-2% of neurons were immunopositive for alpha(3) Na(+),K(+)-ATPase and all these neurons were large primary afferents that were not labeled by Griffonia simplicifolia isolectin B4 (marker of small primary sensory neurons). In dorsal and ventral roots 27+/-3% and 40+/-3%, respectively, of myelinated axons displayed immunoreactivity for alpha(3) Na(+),K(+)-ATPase. In contrast to the dorsal roots, strong immunoreactivity in ventral roots was observed only in myelinated axons of small caliber, presumably gamma-efferents. In the mixed sciatic nerve alpha(3) Na(+),K(+)-ATPase was detected in 26+/-5% of myelinated axons (both small and large caliber). In extensor hallicus proprius and lumbricales hind limb muscles alpha(3) Na(+),K(+)-ATPase was detected in some intramuscular axons and axonal terminals on intrafusal muscle fibers in the spindle equatorial and polar regions (regions of afferent and efferent innervation of the muscle stretch receptor, respectively). No alpha(3) Na(+),K(+)-ATPase was found in association with innervation of extrafusal muscle fibers or in tendon-muscle fusion regions. These data demonstrate non-uniform expression of the alpha(3) isoform of the Na(+),K(+)-ATPase in rat peripheral nervous system and suggest that alpha(3) Na(+),K(+)-ATPase is specifically expressed in afferent and efferent axons innervating skeletal muscle stretch receptors.

Mechanical properties of adult vertebral cancellous bone: correlation with collagen intermolecular cross-links.

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross-linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44-88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross-links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross-links, that is, hydroxylysyl-pyridinoline (HP), lysyl-pyridinoline (LP), and pyrrole cross-link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross-link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.

Cross-link profile of bone collagen correlates with structural organization of trabeculae.

Little is known regarding the mechanisms that govern the structural organization of cancellous bone. In this study, we compare the nature of the collagen in vertebral cancellous bone with the structural organization of its trabecular network. Cylindrical specimens of cancellous bone from vertebrae were obtained from nine autopsy subjects (ages 46-88). In each subject, eight pairs of corresponding samples were obtained from three levels in the spine and three areas within the vertebral body, leading to a total of 68 pairs of samples. The cylinders from one side were used for morphometry and the classical morphometrical parameters were obtained (BV/TV, bone volume fraction; Tb.Th, trabecular thickness; Tb.N, number; Tb.Sp, trabecular spacing) and strut analysis (TSL, total strut length; Nd, number of nodes; Fe, number of free-ends). The amount of osteoid bone was also quantified. The cylinders from the other side were powdered and used for collagen assessment, including the amount of collagen (% w/w), and its content in immature cross-links; such as hydroxylysinonorleucine (mol/mol of collagen) and dihydroxylysinornorleucine, as well as stable mature cross-links, such as hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), and the pyrrole cross-links. A random regression model was used to explore the correlations. None of the biochemical parameters correlated with the BV/TV except the ratio between immature and mature cross-links (eta(2) = 0.34, p < 0.05). There was no relationship between the amount of osteoid bone and the cross-link profile. However, the concentration of pyrrole and HP cross-links in the bone samples correlated with the structural organization of its trabeculae, but in an opposite direction. Hence, the pyrrole/HP ratio was a good predictor of Tb.Th, Tb.N, Tb.Sp, and TSL (eta(2) > 0.65 and p < 0.01) as well as Fe and star marrow space (eta(2) > 0.45 and p < 0.05). The cylinders from subjects with high pyrrole or low HP in their bone collagen had a relatively thick and simple structure. Those with low pyrrole and high HP had relatively thin trabeculae that were more numerous and spread over a complex network. The relative concentrations of the pyrrole and pyridinoline cross-links appear to reflect the structural organization of the trabeculae.

Differences in composition of avian bone collagen following genetic selection for resistance to osteoporosis.

1. Collagen characteristics were compared in the tibiotarsus and humerus from 103 females and 38 males aged 68 to 72 weeks from the G6 generation of lines of laying hen selected for resistance or susceptibility to osteoporosis (high and low bone index (BI) lines). 2. Selection over the latest generation resulted in further divergence in the breaking strengths of humerus (from 12.3 to 21.8%) and tibia (from 22.3 to 37.3%) in hens. Males also showed line differences in bone strengths. 3. Plasma pyridinoline concentration was higher in hens in the low BI line, suggesting a greater rate of bone resorption in this line. 4. There were few differences between the lines in collagen and calcium concentrations in humerus and tibiotarsus cortical bone. 5. There were no differences between the lines in either sex in reduced immature collagen cross-link content of humerus or tibiotarsus. 6. Mature collagen cross-link content was higher in the high BI line in the male humerus but this effect was not apparent in the male tibiotarsus nor in either bone in the females. 7. Pyrrolic cross-link contents were higher in the high BI line in the female humerus and tibiotarsus and in the male tibiotarsus. 8. Over both lines combined, there were positive correlations between humeral and tibiotarsal pyrrole contents and strengths in females and between tibiotarsal pyrrole content and strength in males. 9. It is concluded that an increase in cross-linking, particularly pyrrolic cross-linking, in the collagen matrix contributes in part to the improvement in bone strength in the high BI line.

Immunoglobulin G response of periodontitis patients to Porphyromonas gingivalis capsular carbohydrate and lipopolysaccharide antigens.

Porphyromonas gingivalis clonal types that participate in periodontal infections express serologically distinct surface antigens. This investigation sought to determine whether serum antibodies titers against the serotype-specific capsular carbohydrate K antigen and lipopolysaccharide antigens of P. gingivalis might reveal which serotypes are most likely to be responsible for subgingival infections in subjects with adult periodontitis. Immunoglobulin G (IgG) titers to purified K antigen and lipopolysaccharide from different P. gingivalis strains were measured by ELISA for 28 healthy controls and 51 patients with periodontal pockets known to be infected with genetically and serologically distinct P. gingivalis clonal types. Titers to purified K antigen from strains W50, HG184, A7A1-28, 49417, HG1690 and HG1691, representing serotypes K1-K6, respectively, and lipopolysaccharide from strains 381, HG1691 and W50, representing serotypes O1-O3, respectively, were measured for all subjects. Chi-square likelihood ratios, Mann-Whitney tests and receiver-operating characteristic sensitivity-specificity plots were used to compare the accuracy with which titer results for different target antigens classified subjects with or without disease. Results from assays targeting K2, K3, K4, K5, O1 and O2 generally gave poor diagnostic accuracy, whether evaluated separately or as summed titer pairs corresponding to the K/O combinations actually expressed by the target antigen parent strains. Exceptions were O3 (from W50) and K5+O2 (both from HG1690), which gave moderate accuracy in classifying subjects. In contrast, highly significant diagnostic accuracy was achieved using individual K1 (W50) and K6 (HG1691) titer data and K1+O3 (W50) and K6+O2 (HG1691) titer sum values. These observations suggest that P. gingivalis clonal types expressing K/O serotypes matching those of W50 (K1/O3) and HG1691 (K6/O2) are more likely than others to participate in periodontal infections in adult periodontitis patients and thus are more likely than others to express relevant virulence factors.

Anabolic effect of long-term estrogen replacement on bone collagen in elderly postmenopausal women with osteoporosis.

Estrogen has been shown to stimulate osteoblasts in cell culture and increase bone formation in animal models. Such an anabolic effect of estrogen replacement therapy (ERT) would be beneficial to postmenopausal women with osteoporosis. Hence, we assessed the total collagen content and collagen crosslink maturity in iliac crest bone biopsy from 18 such women before and after 6 years of higher-dose ERT. These results were compared with the serum estradiol level and bone mineral density (BMD). Total collagen content of both cortical and cancellous bone increased, showing a median (95% CI) percent change of 6.7 (0.3-14.2) and 25.6 (13.5-33.8), respectively. Increase in collagen synthesis was supported by a rise in intermediate crosslinks in both cortical and cancellous bone, and mature crosslinks in cortical bone only. At the same time, BMD showed a substantial rise both at the lumbar spine and proximal femur with a median (95% CI) percent change of 28.6 (19.8-37.3) and 14.5 (8.4-20.7), respectively. Serum estradiol and BMD results correlated with cortical bone collagen levels. Our results suggest that long-term higher-dose ERT has a therapeutic role due to its anabolic effect on bone in postmenopausal women with osteoporosis.

Treatment outcome for IDDM patients in relation to glutamic acid decarboxylase autoantibodies and serum IgG to periodontal pathogens.

BACKGROUND: Patients with insulin-dependent diabetes mellitus (IDDM) have elevated risk for periodontitis (PD) relative to subjects without diabetes. Whether refractory PD in IDDM patients is related to autoimmunity as indicated by serum glutamic acid decarboxylase autoantibody GAD Ab levels or to host bacterial immunity as reflected by serum antibody titers to periodontal pathogens is unknown. AIMS: To determine if non-surgical periodontal treatment outcome differs between GAD Ab-seropositive and -seronegative IDDM patients by assessing the following parameters: (1) pretreatment serum levels of GAD Ab, (2) pretreatment serum IgG titers to key periodontal pathogens, and (3) changes in periodontal pocket probing depth (PDC) after treatment. METHODS: Before and two months after periodontal treatment of 11 GAD Ab-seronegative and 7 -seropositive subjects, PDC was assessed and serum GAD Ab and IgG to Porphyromonas gingivalis (Pg), Bacteroides forsythus (BJ), and Actinobacillus actinomycetemcomitans (Aa) were studied using established radioligand precipitation and enzyme-linked immunosorbent assays, respectively. RESULTS: The PDC decrease was significantly better for GAD Ab-seronegative subjects than for seropositive subjects (median 1.4 mm+/-0.5 s.d. versus 0.5 mm+/-0.3 s.d., p<0.03, Mann-Whitney). GAD Ab levels and PDC were positively correlated (r=+0.71, p<0.05) for sero-positive subjects but were neutral (r=-0.07) for seronegative subjects. Serum IgG to Pg and GAD Ab levels were positively associated (r2=0.42) in seropositive subjects. Logistic regression analysis confirmed that GAD Ab status was the primary discriminator for PDC (p<0.04). CONCLUSION: Detection of elevated GAD Ab levels in combination with elevated IgG titers to Pg before treatment is indicative of IDDM patients with refractory PD.

Alterations in the organisation, ultrastructure and biochemistry of the myocardial collagen matrix in doberman pinschers with dilated cardiomyopathy.

Remodelling of the collagen matrix of the myocardium has been implicated in the pathogenesis of dilated cardiomyopathy, a major cause of heart failure in Doberman pinschers. The aim of this study was to characterise the myocardial collagen matrix of Dobermans. In clinically normal Dobermans there was evidence of focal fibrosis. Collagen cross-links were altered in both diseased and clinically normal Doberman myocardium as compared with myocardium from control dogs. Extensive remodelling, in the form of a loss of collagen tethers, increased collagen synthesis and alterations in the collagen cross-links, occurs in diseased Doberman myocardium. Changes in the collagenous matrix are also present in apparently normal Dobermans. These changes are likely to be involved in the progression of the disease and may explain the predisposition of this breed to dilated cardiomyopathy.

Schwann cell-induced loss of synapses in the central nervous system.

Synaptophysin immunostaining of areas of spinal gray matter occupied by radiation-induced intraspinal Schwann cells revealed a loss of immunoreactivity from the neuropil. In contrast, synaptophysin immunoreactivity was preserved on the somata and proximal dendrites of motor neurons. The present study extended these observations to the ultrastructural level and confirmed the absence not only of synapses but also of astrocytes and small- and medium-sized dendrites. These neural elements were abundant and appropriately organized in contiguous areas of irradiated neuropil not occupied by Schwann cells.

Identification of an intermediate state in the helix-coil degradation of collagen by ultraviolet light.

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.

Synaptophysin immunoreactivity in spinal white matter of young adult rats.

Patterns of synaptophysin immunoreactivity were examined in the ventral and lateral funiculi of rat lumbosacral spinal cords. In normal young adults, dendrites from neurons in the spinal gray matter extended into the ventral and lateral white matter as finger-like projections, immunopositive for synaptophysin. These projections appeared to diminish in size as they extended peripherally and, in general, did not reach the surface of the spinal cord, so that the outer one-third to one-fourth of the funiculi contained little or no immunoreactivity. The spinal cords of some of the animals studied were X-irradiated on the third postnatal day. When examined 6 weeks to 5 months later, the pattern of synaptophysin immunoreactivity was found to be markedly altered in these animals. In general, the synaptophysin immunoreactivity in the white matter was less organized than in the non-irradiated rat. As a result, the finger-like projections, particularly into the lateral funiculi, were not as distinct, and the immunoreactivity appeared to be more diffusely distributed in the white matter. Further, the immunoreactivity was present throughout the thickness of the white matter in the irradiated animals and subpial concentrations were evident, especially along the lateral aspect of the spinal cord. Ultrastructural evaluation of the synaptic profiles revealed no differences between irradiated and non-irradiated animals. The synapses occurred on both the shafts of the dendrites and on the spines. In general, both dendrites and axon terminals were covered by astrocyte processes except at synaptic sites, and the synaptic complexes were surrounded by astrocyte processes. Although the mechanisms underlying the altered pattern of synaptophysin immunoreactivity are not yet understood, they may be related to radiation-induced effects on the glial populations previously reported by the investigators and/or to radiation-induced alterations in reorganization or maturation of dendritic trees.

Isinglass/collagen: denaturation and functionality.

Isinglass is widely used commercially to clarify alcoholic beverages by aggregation of the yeast and other insoluble particles. It is derived from swim bladders of tropical fish by solubilisation in organic acids and consists predominantly of the protein collagen. The low content of intermolecular cross-links allows ready dissolution of swim bladder compared to bovine hide which is cross-linked by a high proportion of stable bonds and requires enzymic digestion to solubilise. Isinglass is no longer effective as a clarifying agent if thermally denatured hence the collagenous triple helical structure must be maintained. Thermal denaturation of isinglass occurs at 29 degrees C, compared to 40-41 degrees C for mammalian collagens, primarily due to the lower hydroxyproline content. The hydroxyproline is essential for the formation of H-bonded water-bridges through the hydroxyl group and the peptide chain thereby stabilising the triple helix. Based on the lower enthalpy determined by differential scanning calorimetry we have calculated that the thermally labile domain of the isinglass molecule was 41 residues compared to 66 for mammalian collagen. The fining efficiency was unaffected by pH, chelating agents, detergents and removal of surface proteins from yeast cells. Studies on the mechanism of action of isinglass have shown that higher molecular weight aggregates that increase the length of the collagen molecules (trimers, tetramers, etc.) increase efficiency and that their surface charge are important in the clarification process. By chemical modification, we have shown that blocking positively charged groups had no effect on the fining process, whilst negative charges are clearly essential and that increasing the negative charge by succinylation increases its efficacy. Solutions of bovine hide collagen were shown to be equally effective in refining beers and standard yeast preparations. The higher thermal denaturation temperature, ready availability and reproducibility of bovine collagen preparations gives it considerable advantages over isinglass.

Age related changes in the non-collagenous components of the extracellular matrix of the human lamina cribrosa.

AIMS: To investigate age related alterations in the non-collagenous components of the human lamina cribrosa. METHODS: Fibronectin, elastin, and glial fibrillary acidic protein (GFAP) staining were assessed in young and old laminae cribrosae. An age range (7 days to 96 years) of human laminae cribrosae were analysed for lipid content (n=9), cellularity (n=28), total sulphated glycosaminoglycans (n=28), elastin content (n=9), and water content (n=56), using chloroform-methanol extraction, fluorimetry, the dimethylmethylene blue assay, and ion exchange chromatography, respectively. RESULTS: Qualitatively, an increase in elastin and a decrease in fibronectin and GFAP were demonstrated when young tissue was compared with the elderly. Biochemical analysis of the ageing human lamina cribrosa demonstrated that elastin content increased from 8% to 28% dry tissue weight, total sulphated glycosaminoglycans decreased, and lipid content decreased from 45% to 25%. There were no significant changes in total cellularity or water content. CONCLUSION: These alterations in composition may be indicative of the metabolic state of the lamina cribrosa as it ages, and may contribute to changes in mechanical integrity. Such changes may be implicated in the susceptibility of the elderly lamina cribrosa and also its response to glaucomatous optic neuropathy.

Antigenic cross-reactivity among Porphyromonas gingivalis serotypes.

The goal of our research program is to develop a Porphyromonas gingivalis vaccine. Vaccine development requires identification of antigenic components shared by the many clonal types of P. gingivalis. The purpose of the present study was to evaluate the extent and nature of antigenic cross-reactivity among serotypes of P. gingivalis and to identify shared antigenic components. Strains selected to represent serotypes A-D were 33277, A7A1-28 W50 and 381, respectively. Using intact cells, antibodies were raised in rabbits. Titers were assessed by enzyme-linked immunosorbent assay (ELISA) using intact cells as antigen, Western blots were prepared and biologic activity was measured as opsonization (chemiluminescence expressed as mV) and enhancement of phagocytosis and killing by polymorphonuclear leukocytes. Extensive cross-reactivity that varied greatly among serotypes was observed by ELISA. The Western blots showed an even greater extent of cross-reactivity, with shared protein components at approximately 140, 130, 37, 32 and 28 kDa and a shared variable molecular mass smear considered to be lipopolysaccharide and other carbohydrate. Additional protein components at 110, 85, 35 and 20 kDa appeared to be shared by some but not all serotypes. In the functional assays, strains 33277 and 381 were equally well opsonized by anti-33277 and anti-381 (500-650 mV) but opsonized to a much lesser extent by anti-A7A1-28 and anti-W50 (roughly 125 mV and 350 mV respectively). A7A1-28 and W50 were opsonized by all four immune sera almost equally but to a much lower extent (roughly 400 mV and 250 mV respectively). Enhancement of phagocytosis and killing in the presence of active complement mirrored opsonization with the exception that 381 was reasonably well opsonized by anti-A7A1-28 (400 mV) and anti-W50 (350 mV), but poorly killed. The protein components at 140, 130, 37 and 28 kDa shared by all of the four serotypes appear to have potential as vaccine candidate antigens.