Enhanced production of mineralized nodules and collagenous proteins in vitro by calcium ascorbate supplemented with vitamin C metabolites.

BACKGROUND: Vitamin C or ascorbate is important in wound healing due to its essential role in collagen synthesis. To study wound healing in the periodontium, cells adherent to expanded polytetrafluoroethylene (ePTFE) augmentation membranes, recovered from edentulous ridge augmentation procedures, have been established in culture in our laboratories. The objective of this study was to determine whether treatment of these cells with a calcium ascorbate, which contains vitamin C metabolites (metabolite-supplemented ascorbate), would increase the production of collagenous protein and mineralized tissue in vitro, as compared to unsupplemented calcium ascorbate (ascorbate). METHODS: Cells derived from ePTFE membranes were cultured with beta-glycerophosphate and the test agents for 2 to 5 weeks, and the surface areas of the cell cultures occupied by mineralized nodules were measured using computerized image analysis. One experiment tested the effects of calcium threonate, one of the vitamin C metabolites in metabolite-supplemented ascorbate. Incorporation of radioactive proline and glycine was used as a measure of total protein (radioactivity precipitated by trichloracetic acid) and collagenase-digestible protein (radioactivity released by collagenase digestion.) Co-localization of collagen and fibronectin was examined by immunofluorescence. RESULTS: In vitro treatment of these cells with metabolite-supplemented ascorbate increased the area of the cell cultures occupied by mineralized nodules after 5 weeks. Cell cultures treated with metabolite-supplemented ascorbate also exhibited significant increases in total protein. The increase in collagenous proteins in these cultures accounted for 85% of the increase in total protein. The greatest difference between treatment groups was observed in the cell-associated fraction containing the extracellular matrix. The additional collagen exhibited normal co-distribution with fibronectin. In cultures treated with ascorbate spiked with calcium threonate, the area of mineralized tissue was significantly greater than in ascorbate-treated cultures, but was less than that observed in cultures treated with metabolite-supplemented ascorbate. CONCLUSIONS: In vitro treatment with ascorbate containing vitamin C metabolites enhanced the formation of mineralized nodules and collagenous proteins. Calcium threonate may be one of the metabolites influencing the mineralization process. Identifying factors which facilitate the formation of mineralized tissue has significant clinical ramifications in terms of wound healing and bone regeneration.

Evidence that laminin-5 is a component of the tooth surface internal basal lamina, supporting epithelial cell adhesion.

Laminin-5 (Ln-5) is an extracellular matrix (ECM) glycoprotein found in epithelial basal laminae. We studied its expression on the surface of rat molars, in relationship to the location of the internal basal lamina (IBL) of the junctional epithelium (JE). In order to avoid disruption of the JE-tooth interface as much as possible, the surface of molars was prepared by mechanical removal of tissue debris and detergent/osmotic lysis of epithelial cell layers, and directly stained by immunohistochemistry, without sectioning. Antibodies to Ln-5 specifically stained a narrow band in the region of the cemento-enamel junction (CEJ), consistent with the expected location of the IBL. Western blotting of ECM material detergent--solubilized from the prepared tooth surfaces confirmed the molecular nature of Ln-5 identified by immunohistochemistry. By the use of a high-definition 3-D microscope, it appeared that Ln-5 coated the most apical part of the enamel and the most coronal portion of the cementum, on either side of the CEJ. In adhesion assays performed directly on tooth surfaces, epithelial cells adhered preferentially to the Ln-5 coated area of the tooth compared to the root surface, which is coated by other ECM components. Adhesion to the Ln-5 coated surface was specifically inhibited by a function-blocking monoclonal antibody to Ln-5. These results suggest that Ln-5 is a component of the IBL, and that it may be important in promoting adhesion of JE cells onto the tooth surface.

Production of matrix-degrading enzymes and inhibition of osteoclast-like cell differentiation by fibroblast-like cells from the periodontal ligament of human primary teeth.

Clinically, the most apparent difference between the primary and permanent dentitions is the physiologic loss of the primary tooth by root resorption. Root resorption is associated with loss of integrity of the periodontal ligament (PDL), followed by recruitment of resorptive cells that remove root structure. We therefore cultured primary dentition PDL fibroblasts (PPDL cells) to investigate in vitro their production of matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs), and the effects of soluble factors produced by these cells on osteoclast-like cell differentiation. These studies demonstrate that PPDL cells in vitro have a heterogeneous morphology, and they constitutively synthesize 92-kDa gelatinase, 72-kDa gelatinase, and 53/57-kDa procollagenase as well as TIMP-1, -2, and a third inhibitor of matrix metalloproteinase, as determined by substrate gel zymography and immunoblot analysis. Compared with PDL cells from the permanent dentition, PPDL cells generally produced a greater amount of collagenase but similar amounts of the gelatinases and inhibitors. PPDL cells were treated with pro-inflammatory cytokines to determine their effect on the expression of matrix-degrading enzymes and inhibitors. Interleukin-1alpha and tumor necrosis factor-alpha enhanced the constitutive expression of proteinases but not that of inhibitors in PPDL cells. Conditioned media from PPDL cell lines inhibited the differentiation of osteoclast-like cells in mouse bone marrow cultures. These findings indicate that PPDL cells may modulate the cascade of root resorption both by their regulated production of proteinases and inhibitors and by synthesis of unknown soluble factor(s) that may regulate osteoclast development.

A clinical study of the Ahmed glaucoma valve implant in advanced glaucoma.

OBJECTIVE: To assess clinical outcomes and establish the incidence and management of a hypertensive phase (HP) (defined as intraocular pressure [IOP] > 21 mmHg in the first 6 postoperative months) in patients with Ahmed glaucoma valve implant. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: A total of 85 patients were included in the current study, including consecutive patients who had intractable glaucoma and underwent Ahmed valve implant insertion at the University Glaucoma Center, Tampa, Florida (DWR and WEL) and the Massachusetts Eye and Ear Infirmary (PAN) between January 1993 and June 1997. Only patients with a minimum of 6 months' follow-up were included. INTERVENTION: Ahmed glaucoma valve implant insertion to control intractable glaucoma was performed. MAIN OUTCOME MEASURES: Success was defined as IOP less than 22 mmHg and greater than 4 mmHg on the last two visits, a decrease of no more than two lines in the visual acuity and no additional surgical interventions to control IOP. RESULTS: The cumulative probability of success at 12 months was 77%. A total of 26 patients (30.6%) failed during the study period, and 70 patients (82%) exhibited HP. Hypertensive phase peaked at 1 month after the operation and stabilized at 6 months. There were 34 patients (48%) with HP who were controlled with additional medications: 14 (20%) with needling and 5-fluorouracil injections and 20 (28%) who needed secondary surgical intervention. There were 8 patients (9.4%) who exhibited hypotony (< 5 mmHg) on postoperative day 1 and 3 (3.5%) at 3 months. Visual acuity returned to baseline between 3 and 6 months after the operation. The major complications associated with the valve were hyphema in 14 cases (16.5%), suprachoroidal hemorrhage in 4 cases (4.7%), end-plate exposure in 10 cases (11.7%), tube exposure in 6 cases (7%), tube block in 4 cases (4.7%), loss of vision in 5 cases (5.8%), and corneal graft failure in 4 (30%) of 13 cases with clear grafts. CONCLUSIONS: The overall success rate is comparable to that of prior studies using different implants. The majority of the patients exhibit an HP that peaks at 1 month, with gradual stabilization over 6 months. One third of the patients needed secondary surgical intervention to control the HP. The incidence of postoperative hypotony and flat or shallow anterior chambers is very low after Ahmed glaucoma valve insertion.

Cystoid macular edema associated with latanoprost in aphakic and pseudophakic eyes.

PURPOSE: To describe four patients who developed cystoid macular edema shortly after onset of treatment with latanoprost. METHOD: Retrospective review of medical records of patients with open-angle glaucoma who developed cystoid macular edema shortly after starting latanoprost. RESULTS: The use of topical latanoprost was temporally related to the development of cystoid macular edema in four patients (six eyes; two aphakic eyes and four pseudophakic eyes). Cystoid macular edema resolved in all patients after latanoprost was discontinued. CONCLUSIONS: Cystoid macular edema is a potential complication of latanoprost therapy. Further observations are needed to determine if the risk of cystoid macular edema is limited to or greatest in patients who are pseudophakic or aphakic.

Expression of laminin 5, fibronectin, and epithelium-associated integrins in recurrent aphthous ulcers.

Recurrent aphthous ulceration (RAU) is characterized by an ulcerated lesion that persists longer than traumatic ulcers of similar size. This delayed healing phase of the lesion was investigated for extracellular matrix components and matrix receptors (integrins). The hypothesis tested was that aphthous ulcers may lack key extracellular matrix components, or their receptors, that are necessary for the migration of marginal keratinocytes from the ulcer edge. We immunocytochemically stained biopsy specimens of RAUs and non-involved mucosal specimens from HIV+ and non-infected individuals to investigate the presence and distribution of molecules reported to be associated with reepithelialization of mucosal and cutaneous wounds. Fibronectin, laminin type 5 (kalinin), and integrin subunits beta 1, beta 4, alpha 6, and alpha v were consistently found at the margins of RAU, as they are in traumatic ulcers. The alpha 5 and beta 6 subunits were not always present. We also found alpha v in the intact stratified squamous epithelium adjacent to ulcers. Immunohistochemical stains showed distruption in the deposition of laminin 5 and an apparent lack of fibronectin at the edges of some ulcers. Although these tissue results do not determine which integrin subunits are paired with each other, they do show some alterations in their expression in RAU. Absence of one or more of these molecules at the migrating front may contribute to delayed epithelial regeneration. It is likely that the absence or inappropriate expression of keratinocyte integrins or their extracellular matrix receptors occurs after the causative factors (currently unknown) of the lesion are gone. The reason for the altered expression of these molecules may be related to the secretory products (including lymphokines and proteinases) of the lymphocytic infiltrate.

Protease repertoires of cells adherent to membranes recovered after guided tissue regeneration.

Guided tissue regeneration is a clinical procedure used to restore mineralized tissue that has been lost to periodontal disease or after tooth extraction. The procedure makes use of Gore-tex membranes or Gore-tex augmentation membranes (GTAM) to prevent migration of keratinocytes and gingival fibroblasts into healing wounds. To begin to characterize the regenerative cells associated with these membranes, human cells have been rescued from membranes retrieved after bone-inductive procedures. Cell lines were established from tissue adherent to Gore-tex membranes used to regenerate bone around periodontally compromised teeth, and from tissue adherent to GTAM used in edentulous ridge augmentation procedures or in conjunction with implant placement. Cell lines were screened for mineralized nodule formation in vitro prior to their subsequent analysis. All but one of the lines selected for this study formed mineralized nodules in vitro with cells from GTAM tending to form nodules more quickly than cells from Gore-tex. Zymograms and Western blots were used to compare protease profiles of these cells with those of human gingival fibroblasts, keratinocytes and periodontal ligament (PDL) cells. All cell types except for keratinocytes produced a 72 kD protease. In contrast, keratinocytes were the only cells that produced 92 kD gelatinase. In some cell lines, notably those removed from patients after short periods of regeneration, collagenase was the major protease detected on gelatin substrate gels. Some of these cell lines also produced additional proteases including a low molecular weight protease (30 kD) not seen in gingival fibroblasts, PDL cells or keratinocytes.

Development and histologic characterizations of an animal model of antigen-induced arthritis of the juvenile rabbit temporomandibular joint.

Children with juvenile rheumatoid arthritis or juvenile chronic arthritis often exhibit temporomandibular joint (TMJ) involvement accompanied by pain, dysfunction, and growth abnormalities. Despite the severe functional and developmental consequences of this disease, its pathogenesis remains poorly understood, but important insights may be provided by a suitable animal model of this disease. The purpose of this study was to develop and histologically characterize a juvenile animal model of antigen-induced arthritis of the TMJ. Arthritis was induced with an intra-articular administration of ovalbumin in previously sensitized 10-week-old male New Zealand white rabbits. Sham-treated and untreated rabbits were used as controls. The TMJs were retrieved en bloc at 5, 10, 15, 35, and 55 days post-challenge for histology and matrix histochemistry. Antigen-treated joints demonstrated severe arthritis, including mononuclear cell infiltration, synovial lining and villous hyperplasia, and pannus formation, as early as 5 days after challenge; the arthritis was maintained up to 55 days post-challenge. A decrease in the area of the TMJ disc that stained positively for glycosaminoglycans was observed throughout the experimental period. Loss of collagen staining was primarily localized to sites at the junction of the synovium with bone and fibrocartilage. The histopathologic features of this model of antigen-induced arthritis of the juvenile rabbit TMJ are similar to those observed previously in adult animal models of experimental arthritis and in human rheumatoid arthritis. This animal model will be useful for understanding the pathogenesis of juvenile rheumatoid arthritis of the TMJ, and for exploring the mechanisms for aberrant craniofacial growth.

Increased interleukin-1 beta in the crevicular fluid of diseased implants.

Diseased implants can be distinguished from healthy implants by the presence of inflammation in the surrounding tissue. Inflammation correlates with elevated levels of interleukin-1 beta in gingival crevicular fluid. An analysis of interleukin-1 beta levels in diseased implants compared to those in matching healthy implants in 12 patients indicates that the level of interleukin-1 beta was approximately three times that at healthy sites. Interleukin-1 beta levels may provide a means of monitoring the health status of dental implants.

Clinical experience with the Baerveldt glaucoma drainage implant.

PURPOSE: To assess clinical outcomes in patients who were treated with the Baerveldt glaucoma drainage implant. METHODS: The authors performed a retrospective multicenter study of 100 patients (103 eyes) with medically uncontrollable glaucomas who underwent a one-stage implantation with either the 200-, 250-, 350-, or 500-mm2 Baerveldt implant. The authors defined surgical success as 5 mmHg less than intraocular pressure less than 22 mmHg without additional glaucoma surgery and without loss of light perception. RESULTS: With a mean follow-up of 13.6 +/- 0.9 months (range, 4-37 months), 74 eyes (71.8%) had successful outcomes. Cumulative life-table success rates were 90.3% at 3 months (n = 103), 72.6% at 6 months (n = 84), and 60.3% at 24 months (n = 34). Intraocular pressure (IOP) was reduced from a mean of 38.5 +/- 1.4 mmHg with 2.2 +/- 0.1 antiglaucoma medications to 15.1 +/- 0.8 mmHg (P < 0.0005) with 0.5 +/- 0.1 antiglaucoma medications (P < 0.0005). Visual acuity was improved or remained within one line of the preoperative visual acuity in 90 eyes (87.4%). Complications occurred in 74 eyes (71.8%). A significant portion of these complications (45%) was transient, resolving without any intervention. Only 8% were serious sight-threatening complications. The most common complications included shallow anterior chamber or hypotony (32%), choroidal effusion or hemorrhage (20.4%), corneal decompensation or edema (17.5%), hyphema (14.1%), and tube obstruction (12.6%). CONCLUSION: The Baerveldt implant is effective in lowering the IOP in patients with intractable glaucomas. Hypotony and other complications are common, which also have been reported in other nonvalved glaucoma drainage implants. However, the majority of these complications did not affect surgical outcome.

Characterization and identification of proteinases and proteinase inhibitors synthesized by temporomandibular joint disc cells.

The adult mammalian temporomandibular joint (TM) disc is a fibrocartilaginous tissue that undergoes normal developmental remodeling, requiring removal of the existing extracellular matrix and its replacement by new matrix macromolecules. This remodeling is probably mediated by matrix-degrading enzymes, but to date none has been demonstrated in association with the TMJ disc. We characterized, identified, and determined the regulation of proteinases and proteinase inhibitor (PIs) synthesized by TMJ disc cells in organ and cell cultures. TMJ discs were retrieved from 14-week-old male NZW rabbits and both tissue- and disc-derived cells were cultured in serum-free medium. The conditioned media were retrieved at 12-hour intervals and assayed for proteinases and PIs in gelatin- and casein-impregnated polyacrylamide gels. Three proteinases with gelatinolytic activities at 92 kDa, 72 kDa, and 42/57 kDa and one caseinolytic activity at 51/54 kDa were detected. All were inhibited by 1,10-1 phenanthroline, thus characterizing these enzymes as matrix metalloproteinases (MMPs), most likely 92-kDa gelatinase (proMMP-9), 72-kDa gelatinase (proMMP-2), procollagenase (proMMP-1), and prostromelysin (proMMP-3). The identity of the latter two MMPs was confirmed by Western blots. Two PIs and 30 kDa and 20 kDa, probably tissue inhibitors of metalloproteinase (TIMP) and TIMP-2, were observed on reverse zymograms. TPA, a protein kinase-C agonist, increased the expression of 92-kDa gelatinase and 30-kDa PI by both explanted discs and isolated disc cells. The profile of MMPs constitutively expressed by disc cells is similar to that of synovial fibroblasts but different from that of chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Histopathologic observations on human eyes following neodymium: YAG laser cyclophotocoagulation for glaucoma.

PURPOSE: Although Nd:YAG laser cyclophotocoagulation has been extensively used for nearly a decade in treatment of severe glaucoma, there have been remarkably few reports (each of them extremely brief) of histopathological examination of glancomatous human eyes that had been so treated. We undertook this study to provide further details regarding the nature of the lesions produced in this type of ciliary ablation. METHODS: We chose three representative cases in which a glaucomatous human eye had been subjected to Nd: YAG cyclophotocoagulation, and was subsequently enucleated. To better understand the temporal evolution of the lesions, we selected eyes that were enucleated 1 day, 20 days, and 3 months, respectively, after they had been treated with noncontact Nd: YAG laser cyclophotocoagulation. RESULTS AND CONCLUSIONS: (1) energy levels ranging from 4.4 Joules to 5.6 Joules were effective in producing appropriate lesions; (2) direction of the laser beam 1 to 1.5 mm behind the limbus caused severe destructive lesions of the pars plicata: (3) toward the periphery of the individual treatment sites, the stroma and ciliary muscle continued to exhibit severe degeneration, as did the epithelium lining the valleys between the crests of the ciliary processes; but in those peripheral zones of individual treatment sites, the epithelium lining the crests of the ciliary processes survived and appears normal; (4) bleb-like separations of the ciliary epithelium from the adjacent stroma, particularly along the posterior aspect of the ciliary body lesions, are a prominent early feature of Nd:YAG cyclophotocoagulation; (5) the pigmented epithelium is more vulnerable to laser energy than is the nonpigmented epithelium of the ciliary body; (6) the destruction of the ciliary epithelium is permanent; (7) deeply pigmented persons have more melanocytes in the ciliary body muscle and stroma than do more lightly pigmented individuals, a circumstance that renders the tissues more vulnerable to laser energy; (8) the ciliary muscle was always severely damaged; (9) no scleral injury was observed other than evanescent, focal areas of edema of the deep sclera; and (10) except in the episclera, inflammatory cells were strikingly few in number, a circumstance providing support for the clinical observation that eyes treated with laser cyclophotocoagulation exhibit less of an inflammatory response than do those treated with cyclocryotherapy.

Detection of intraocular pressure change in the eye using sonoelastic Doppler ultrasound.

We report the first use of sonoelastic Doppler ultrasound for in vivo and in vitro clinical studies of intraocular pressure (IOP). The method involves low-amplitude and low-frequency sonic excitation of the eye, and detection of the resulting vibration using Doppler ultrasound. A dependence of the frequency of resonance of the eye on the IOP has been observed in both in vitro and in vivo experiments. Preliminary in vitro experiments have been performed using eviscerated and enucleated human and pig eyes. As little as 4 mm Hg change in IOP has been found detectable in these experiments. Preliminary in vivo experiments also showed promising results in this regard. We present a simple model for the resonances of the eye, a method to detect the amplitude of vibration using Doppler ultrasound and results from the in vitro and in vivo experiments.

Intracameral tissue plasminogen activator to treat blocked glaucoma implants.

I report the use of tissue plasminogen activator to open blocked silicone tubes of Baerveldt glaucoma implants in two cases. A dose of 25 micrograms was used in both cases; lysis of fibrin clots occurred within 15 to 45 minutes.

A study of macrophages, macrophage-related cells, and endothelial adhesion molecules in recurrent aphthous ulcers in HIV-positive patients.

The purpose of this immunohistochemical study was to investigate the presence and distribution of macrophages (CD11c+ and CD68+) and macrophage-related dendritic cells (factor XIIIa+ and CD36+) in early and late aphthous ulcers associated with HIV infection. To substantiate a mechanism by which these cells may move from the vascular compartment to tissue spaces, we also investigated expression of ELAM (endothelial leukocyte adhesion molecule), ICAM-1 (intercellular adhesion molecule), and CD18 (leukocyte function antigen). Numerous CD11c+ and CD68+ macrophages were seen in early lesions, though larger numbers of CD68+ cells were present in older lesions. No significant increases in factor XIIIa+ dendrocytes were seen in either early or late lesions, though dendrocytes appeared enlarged. CD36+ cells and CD18+ leukocytes were more numerous in early than in late aphthous ulcers. ELAM and ICAM expression was most intense on endothelial cells in early aphthous ulcers, with staining intensity fading toward the lesion periphery. Control specimens showed weaker ELAM and ICAM staining than did the ulcer specimens. Keratinocytes did not express ICAM. By virtue of their numbers, macrophages and macrophage subtypes appear to have a significant role in both the early and late stages of this disease. Although factor XIIIa-expressing dendrocytes may not have been more numerous in the ulcers, they appeared to be "activated" because of their prominence in the lesions and their occasional co-expression of CD68 antigen (KP1+). They may have a minor role in antigen processing, phagocytosis, and fibroplasia. ELAM and ICAM expression by endothelial cells provides a mechanism by which macrophages and other leukocytes can be recruited to the site of the lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced detection of normal retinal nerve-fiber striations using a charge-coupled device and digital filtering.

We used a 1024 x 1024 pixel, 15-microns, 16-bit-encoding, multi-pin-phase charge-coupled device (CCD) to obtain images of the normal human retinal nerve fiber layer. This device, which operates at room temperature, offers significantly better signal-to-noise ratio, linearity, and dynamic range than do photographic film, video imaging techniques, or commercially available CCDs. We demonstrate the use of a nonlinear digital filter, together with filter windows, that enhances fine detail of NFL striations, while suppressing noise, in limited areas of the CCD images. High-sensitivity imaging of this type, together with appropriate digital processing, may prove useful in diagnosing and following nerve-fiber-layer damage due to glaucoma.

Ophthalmic contact lens with internal fixation light for examination of the optic nerve head.

Stereoscopic slitlamp examination of the optic nerve head in one-eyed patients is difficult because there is currently no way to provide the patient with a fixation target. This situation is often aggravated in glaucoma patients by a miotic and fixed pupil. We have developed a funduscopic contact lens which incorporates a fixation target, allowing the patient to view the target with the eye being examined. The device has been successfully tested on normal volunteers.

Sulfated glucuronyl glycolipids and gangliosides in the optic nerve of humans.

Pathologically delayed visual evoked potentials may be present in patients with neuropathy associated with IgM M-proteinemia, which is directed against myelin-associated glycoprotein and sulfated glucuronyl glycolipids (SGGLs), but there are no reports of these antigens in the optic nerve. We recently examined human optic nerve and occipital lobe tissues for the occurrence of SGGLs using the technique of immunostaining on thin-layer chromatographic plates and found them in the optic nerve, but not the occipital lobe. SGGLs in the optic nerve may represent target antigens for CNS involvement by the M-protein in patients with neuropathy. We also studied the ganglioside composition of the optic nerve and found it different from that of the brain. Human optic nerve is characterized by an abundance of the b-series gangliosides, including GD1b, GT1b, and GQ1b. GD1a, which is usually a major component of brain gangliosides, is only a minor species of the optic nerve ganglioside fraction.

Immune-related disease and normal-tension glaucoma. A case-control study.

We reviewed the charts of 67 patients with the diagnosis of normal-tension glaucoma listed in the Bascom Palmer Eye Institute computer database. These patients were matched with respect to age, race, and sex with an equal number of patients having ocular hypertension. All medical diagnoses in the charts for both groups were tabulated and classified as either immune-related or non-immune-related. Twenty (30%) patients with normal-tension glaucoma had one or more immune-related disease(s) compared with five (8%) patients in the comparison group (P = .00134, McNemar statistic with continuity correction).