Formation of afibrillar acellular cementum-like layers induced by alkaline phosphatase activity from periodontal ligament explants maintained in vitro.

Fibroblasts of the periodontal ligament, by their alkaline phosphatase (ALP) activity, are considered to play a role in the formation of acellular cementum. As a means of exploring this hypothesis, periodontal ligament explants from rat incisors were cultured in direct contact with bovine dentin slices in the presence of 10 mmol/L beta-glycerophosphate. Periosteal and pericardial tissue explants were maintained under similar conditions.

The influence of the organic matrix on demineralization of bovine root dentin in vitro.

The effect of matrix degradation on the rate of demineralization of dentin lesions was investigated. It was hypothesized that the demineralized matrix would inhibit the demineralization of the underlying mineralized dentin. Bovine root dentin specimens were alternately demineralized and incubated with either a bacterial collagenase or buffer (control).

Immunolocalisation of collagenase in rabbit periosteal tissue explants and extraction of the enzyme. The effect of the cytokines IL-1 alpha and EGF.

The effect of interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on incorporation of endogenously produced collagenase in the extracellular matrix of soft connective tissue was studied in an in vitro model system using periosteal explants obtained from rabbit calvariae. Immunohistochemical analysis indicated the highest level of collagenase in explants cultured for 72 hours with IL-1 alpha in combination with EGF. Most enzyme appeared to be associated with the extracellular matrix, but labeling was also found in numerous fibroblast-like cells.

Failure of calcitriol treatment in a patient with malignant osteopetrosis.

In an attempt to stimulate bone resorption, a 10-week-old infant with malignant infantile osteopetrosis was treated with high doses of calcitriol, a potent bone resorption stimulatory agent, combined with a low calcium diet to prevent hypercalcaemia. Although calcitriol administration was initiated at this very young age, our patient did not show any clinical, radiological, or histological improvement. Despite reports of positive results of this treatment in the literature, our patient did not reveal any signs of bone resorption.

A quantitative enzyme histochemical analysis of the distribution of alkaline phosphatase activity in the periodontal ligament of the rat incisor.

The spatial distribution of alkaline phosphatase (ALP) activity was examined in the periodontal ligament of the continuously growing rat incisor. With the indoxyl-tetrazolium salt method, enzyme activity was demonstrated in undecalcified cryosections, and the amount of reaction product was quantified. ALP activity appeared to be distributed heterogeneously.

Use of frozen biologic material for combined light and electron microscopy.

A simple and rapid method that enables the use of unfixed frozen material for light and electron microscopic purposes is described. At the light microscopic (LM) level, unfixed cryostat sections were used for enzyme histochemistry. When electron microscopic (EM) inspection was needed, tissue blocks, which were stored at -80 degrees C, were fixed at 4 degrees C and prepared for EM according to standard procedures.

Interleukin-1 alpha and epidermal growth factor synergistically enhance the release of collagenase by periosteal connective tissue in vitro.

The effects of recombinant human interleukin-1 alpha (IL-1 alpha) and murine epidermal growth factor (EGF) on the release of collagenase were studied in an in vitro model system using periosteal explants from rabbit calvariae. Following an incubation period of 72 h it was shown that IL-1 alpha in combination with EGF (IL-1 alpha + EGF) induced a synergistic increase in the amount of collagenase released by periosteal explants. This increase appeared to be at least 10-fold.

The release of tissue inhibitor of metalloproteinases by calvarial bone explants and its immunolocalization.

Tissue inhibitors of metalloproteinases (TIMPs) play an important role in the regulation of the activity of matrix metalloproteinases (MMPs) such as collagenase, stromelysin and gelatinase. Although it has been shown that upon culturing bone tissue releases relatively large amounts of TIMP, little is known as to the source of the inhibitor. In an attempt to investigate this in more detail calvarial bone explants from young rabbits were cultured in serum-free medium.

Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases.

The site of action of cysteine-proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP-inhibitor trans-epoxy-succinyl-L-leucylamido (4-guanidino)-butane (E-64) and the MMP-inhibitor N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide (Cl-1) in an in vitro model system of PTH-stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor-treated explants than in controls.

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A.

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils.

Selective inhibition of cysteine proteinases by Z-Phe-AlaCH2F suppresses digestion of collagen by fibroblasts and osteoclasts.

Effects of the selective inhibitor of cathepsins B and L, Z-Phe-AlaCH2F were studied on the degradation of fibrillar collagen by fibroblasts and osteoclasts in cultured rabbit calvariae at the electron microscopic level. Periosteal fibroblasts from inhibitor-treated explants showed a dose-dependent increase of the volume fraction of vacuoles containing cross-banded collagen fibrils. This was a 7-fold increase over control fibroblasts and the ratio of intracellular and extracellular collagen increased from 2 to 43.

Morphologic features of bone in human osteopetrosis.

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells.

Interleukin 1 increases the production of collagenase but does not influence the phagocytosis of collagen fibrils.

The aim of the present study was to determine whether interleukin 1 alpha (Il-1), a cytokine known to have a stimulatory effect on collagenase production, also influences the phagocytosis and intracellular digestion of collagen fibrils by fibroblasts. Mouse long bones and calvariae both with surrounding periosteum were cultured for 24 or 48 hours in media containing varying concentrations of the cytokine. The periostea were subjected to morphometric analysis in order to assess the volume density of phagocytosed collagen fibrils in fibroblasts.

Formation of acellular root cementum in relation to dental and non-dental hard tissues in the rat.

After the periodontium of the rat was wounded, the formation of acellular extrinsic fiber cementum (AEFC) did not appear to be restricted to the hard dental tissues (pre-existing cementum, dentin, and enamel). Layers resembling AEFC were also deposited along the inner wall of the alveolar bone. At the time of observation (six weeks after being wounded), cells other than fibroblast-like cells could not be distinguished close to the newly formed AEFC-like layers.

Elastic and collagenous fibers in the temporomandibular joint capsule of the rabbit and their functional relevance.

This study deals with the form and function of the temporomandibular joint capsule and compares the histological structure of the capsule and kinetics of condylar and disc movement in rabbit and man. The morphology of the capsule of the jaw joint of the rabbit was studied by means of histological sections of isolated capsules, celloidin sections of decalcified heads, and cryostat sections containing the joint, fixed in jaw open and closed positions. General staining methods and selective methods for elastin and collagen were used.

Metalloproteinases are not involved in the phagocytosis of collagen fibrils by fibroblasts.

The effect of various metalloproteinase-inhibiting compounds on collagen phagocytosis by fibroblasts was studied in cultured periosteal tissue. Evidence is presented indicating that neither anti-collagenase nor anti-stromelysin interfere with the uptake of collagen fibrils from the extracellular space and their intracellular digestion. Similar results were obtained with tissue inhibitor of metalloproteinases (TIMP).

Effects of the proteinase inhibitors leupeptin and E-64 on osteoclastic bone resorption.

To determine the possible involvement of cysteine-proteinases in bone matrix degradation by osteoclasts, the effects of the proteinase inhibitors leupeptin and E-64 were studied in an in vitro system using mouse bone explants. It was observed that in explants treated with the drugs, the amount of demineralized matrix opposing the ruffled border of the osteoclasts increased about 20-fold within 6 hours. This suggests that demineralization had proceeded whereas matrix degradation had been retarded.

Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5-nitrosalicylaldehyde.

The histochemical fluorescence method using 5-nitrosalicylaldehyde for the demonstration of cathepsin B activity has been used. Precipitation of the fluorescent final reaction product was analysed continuously during incubation for cathepsin B activity. Unfixed cultured human fibroblasts as well as cryostat sections of mouse metacarpal bone explants were used.

The role of microtubules in the phagocytosis of collagen by fibroblasts.

The effects of the anti-microtubular agents, colchicine and vinblastine, on the phagocytosis of collagen by fibroblasts were assessed quantitatively in cultured mouse bone explants. It was found that in the absence of the microtubular system the volume density of lysosomal vacuoles containing cross-banded collagen fibrils in periosteal cells did not differ from that seen in controls. In contrast, cytochalasin B which interferes with the microfilament system prevented the accumulation of collagen-containing vacuoles in the cytoplasm.

Experimental gingivitis in relation to susceptibility to periodontal disease. II. Phase-contrast microbiological features and some host-response observations.

In the present investigation, a number of histological and immunohistochemical characteristics of periodontal tissues as well as the phase-contrast microscopy of dental plaque were studied after experimentally-induced gingival inflammation in relation to susceptibility to periodontal disease. The study included a younger (mean age 34.1 years) and an older age group (mean age 48 years) with a reduced but healthy periodontium.