Effects of enamel matrix protein application on the viability, proliferation, and attachment of human periodontal ligament fibroblasts to diseased root surfaces in vitro.

OBJECTIVES: The purpose of this research was to examine the influence of enamel matrix proteins (EMP) on the viability, proliferation, and attachment of periodontal ligament fibroblasts (PDLF) to diseased root surfaces. MATERIALS AND METHODS: Primary cell cultures of PDFL were obtained from clinically healthy third molars or premolar teeth. Viability and proliferation rates were carried out over a 10-day period. A total of 80,000 cells were plated in 24-well plates followed by EMEM with 10% FBS (positive control) and EMEM plus EMP at 25, 50, 75, and 100 micro g/ml. Cells were harvested on days 1, 3, 5, 7, and 10 and viability was performed utilizing an MTS assay. PDLF proliferation rates were assessed by a CyQUANT GR dye assay. SEM analysis was used to examine the qualitative effects of cellular attachment to diseased root surfaces following EMP compared to nontreated controls. RESULTS: The results indicated that viability was negatively affected for higher doses over time while lower doses displayed viability effects similar to control. Proliferation, however, appeared to be ameliorated following exposure to EMP. The SEM analysis suggests that cellular attachment to diseased dentin was enhanced following EMP application. CONCLUSION: These in vitro studies support the concept that EMP may act as a suitable matrix for PDLF.

Distribution of plasma-membrane Ca2+ pump in mandibular condyles from growing and adult rabbits.

Chondrocytes may control the mineralization of the extracellular matrix of condylar cartilage by several mechanisms including the release of microvesicles involved in the initial nucleation, the creation or modification of the local matrix to help propagate or restrict mineralization, and the regulation of the ionic environment at the calcifying foci within the matrix. The plasma membrane Ca2+-Mg2+ ATPase (Ca2+ pump) is known to play a part in the vectorial efflux of calcium in a variety of cells including chondrocytes. The purpose here was to study the distribution of Ca2+-pump protein in mandibular condyles from growing and adult rabbits, and compare the expression of that protein in progressively differentiating chondrocytes whose final stage is associated with a mineralized extracellular matrix. Ca2+-pump antigen was identified immunohistochemically in six growing and six adult rabbit mandibular condyles with a Ca2+ pump-specific monoclonal antibody. The presence of Ca2+-pump antigen was established in hypertrophic chondrocytes, and in osteoblasts and osteoclasts of subchondral bone. Slot-blot analysis of nitrocellulose-immobilized chondrocyte homogenates showed that the amount of Ca2+ pump in growing cartilage was more than twice that in adult cartilage (p < 0.05). The demonstration of Ca2+-pump antigen in the hypertrophic chondrocytes of growing rabbit condyles is consistent with a role for the plasma-membrane Ca2+ pump in the calcification of mandibular condylar cartilage.

Human periodontal ligament fibroblast response to PDGF-BB and IGF-1 application on tetracycline HCI conditioned root surfaces.

The purpose of this study was to investigate the effect of 2 growth factors, platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor-1 (IGF-1), alone or in combination, on the adherence of human periodontal ligament fibroblast (PDL) to tetracycline HCl (TTC) conditioned and nonconditioned periodontally involved root surfaces. There were 80 root dentine chips from 80 patients, ranging from 35 to 70 years of age, each with one periodontally involved tooth requiring extraction. A root dentine chip was obtained from the subgingival surface opposite to the periodontal pocket of each extracted tooth. The dentine chips were randomly distributed into one of 8 groups. In group 1, PDL fibroblasts were cultured and allowed to attach on the dentine surface. In group 2, PDL fibroblasts were cultured on a PDGF-BB pre-treated dentine surface and in group 3, they were cultured on a IGF-1 pre-treated dentine surface. In group 4, PDL fibroblasts were cultured on a dentine surface pretreated with a combination of PDGF-BB and IGF-1. In group 5, PDL fibroblasts were cultured and allowed to attach on the TTC conditioned dentine surfaces. In groups 6 and 7, surface of dentine chips were conditioned with TTC and then were treated with PDGF-BB or IGF-1 respectively, followed by placement of PDL fibroblast and cultured. In group 8, dentine surfaces were conditioned with TTC and then pre-treated with a combination of PDGF-BB and IGF-1 before the fibroblasts were cultured. After 24 h of incubation, the media was removed and samples were fixed and processed for SEM at magnifications of x34, x750, x2000. Photographing and evaluation of samples was performed at x750 in which fibroblast adherence was measured by counting cells within a standard test area. The results of the non-TTC conditioned root surfaces demonstrated a significant increase in fibroblasts adherence in the PDGF-BB and combination PDGF-BB/IGF-I treatment groups (groups 2, 4) when compared to the control (group 1) as well as the TTC control (group 5). The combination of PDGF-BB/IGF-1 (group 4) did not significantly improve the adhesion of cells compared to PDGF-BB alone (group 2), but did significantly improve adhesion when compared to IGF-1 alone (group 3). There were no significant differences in cell morphology between the growth factor groups (groups 2, 3, 4) and control (group 1). In general, the cells demonstrated a flat, stellate-shaped morphology. The results of the TTC conditioned root surfaces, showed a statistically significant increase of cellular adherence in the PDGF-BB group (group 6) when compared to the TTC control (group 5), similar to the non-TTC group (group 2). However, the morphology of the cells in groups 5, 6, 7, and 8 demonstrated generally a rounded or oval shape with only an occasional cell exhibiting a flat form. In the experimental system of this study, the inclusion of PDGF-BB on the surface of dentine chips increased the number of adhering PDL cells, and the addition of TTC conditioning had little effect except to change the morphology of adhering cells.

Porous polysulfone coated with platelet-derived growth factor-BB stimulates proliferation of human periodontal ligament fibroblasts.

The purpose of this study was to investigate if the treatment of porous polysulfone (PPSF) with various concentrations of platelet-derived growth factor-BB (PDGF-BB) would stimulate the proliferation of the adherent human periodontal ligament fibroblasts (HPDLF) in culture. Sterilized PPSF cylinders were immersed in an Eagle's minimum essential medium supplemented with 0.5% fetal bovine serum and 1% penicillin/streptomycin containing either 0, 10, 20, or 50 ng/ml of PDGF-BB for 24 hours. After 24 hours, the PPSF cylinders were removed and allowed to dry then placed in culture plates for each time point. Pooled HPDLF (8 x 10(4)) and 3H-thymidine in medium were pipetted into each well to cover the treated and control PPSF cylinders and plates were then incubated. At 1, 4, and 10 days the PPSF cylinders were removed and macromolecular precipitation was performed. Incorporation of 3H-thymidine was measured and a 2-way ANOVA with repeated measures was performed. In addition, determination of binding and release was performed using I125-PDGF-BB treated PPSF at 0, 2, 12, and 24 hours, and at 4 and 10 days. Results showed that the effects on HPDLF were significant for dose (P = 0.0012; F = 5.74) and time (P = 0.0001; F = 40.83). At 4 days, the percent increases above the control for the doses 10, 20, and 50 ng/ml were 192%, 310%, and 162% respectively. In conclusion, our findings suggest that treating PPSF with PDGF-BB results in a significant increase in the proliferation of HPDLF cells adherent to PPSF.

An immunohistochemical study of collagen types III, VI and IX in rabbit craniomandibular joint tissues following surgical induction of anterior disk displacement.

The purpose of this study was to determine the effect of surgical induction of anterior disk displacement (ADD) on type-III, VI and IX collagens of the rabbit craniomandibular joint (CMJ) tissues using an immunohistochemical technique. The right joint was exposed surgically, all discal attachments were severed except for the posterior discal attachment (bilaminar zone). The disk was then repositioned anteriorly and sutured to the zygomatic arch. The left joint served as a sham-operated control. Ten additional joints were used as non-operated controls. Deeply anesthetized rabbits were perfused with 2% buffered formalin 2 weeks (10 rabbits) or 6 weeks (10 rabbits) following surgery. The articular disk, bilaminar zone, mandibular condyle and articular eminence were excised. The last two were decalcified in EDTA. All tissues were then sectioned at 10 microns in a cryostat. Sections were incubated with monoclonal antibodies directed against type-III, VI or IX collagens. Following incubation in the appropriate FITC-labelled secondary antibodies, all sections were studied under the fluorescence microscope. The results showed a reduction in immunostaining for type-VI and IX collagens in the condylar cartilage, disk and articular eminence at 2 weeks, followed by an increase in their immunostaining at 6 weeks and the appearance of a de novo type-III collagen in the condylar cartilage and the articular eminence. It is concluded that surgical induction of ADD in the rabbit CMJ leads to alterations in its type-III, VI and IX collagens.

In situ hybridization and monoclonal antibody analysis of plasma membrane Ca-pump mRNA and protein in submandibular glands of rabbit, rat and man.

The degree of supersaturation of saliva with calcium (Ca) is related to the mineral phase of enamel in erupted teeth, the incidence of caries, and the formation of calculus. The mechanisms for regulating salivary Ca concentration are therefore of relevance to dentistry. Sections of rabbit, rat and human submandibular gland (SMG) were processed for immuno-histochemistry with a specific anti-plasma membrane Ca-pump antibody, 5F10. Western blots confirm that the molecular weight of the proteins identified by our antibody (135 kDa) is consistent with an appropriate molecular weight for PMCA antigen (135-150 kDa). Tissue sections were also processed for in situ hybridization to study the distribution of the PMCA mRNA isoforms. In mammals, the PMCA1 gene is reported to code for a PMCA protein with a role in maintaining the intracellular Ca levels in both epithelial and non-epithelial cells. Other genes including the PMCA2 and PMCA4 genes may code for PMCA proteins specific to Ca transporting tissues. Our studies demonstrate cytoplasmic labeling of PMCA mRNA with hPMCA-1 and hPMCA-4 specific cDNA probes in humans, and rPMCA-1 and rPMCA-2 specific oligonucleotide probes in rats. Labeling of PMCA protein and all mRNA isoforms was found in the cytoplasm of the interlobular and intralobular ducts (except for intercalated ducts). The demonstrated presence of PMCA in SMGs of rabbit, rat, and man, may suggest a role for PMCA in the regulation of intracellular Ca and in a mechanism for regulating and maintaining the high concentration of Ca in salvia.

Alteration in fibronectin of the rabbit craniomandibular joint tissues following surgical induction of anterior disk displacement: immunohistochemical study.

The purpose of this study was to determine the effect of surgical induction of anterior disk displacement (ADD) on fibronectin amount and distribution in the rabbit craniomandibular joint (CMJ) tissues using an immunohistochemical technique. The right CMJ was exposed surgically, and all discal attachments were severed except for the posterior attachment. The disk was then repositioned anteriorly and sutured to the zygomatic arch. The left CMJ served as a sham-operated control. Ten additional joints were used as nonoperated controls. Deeply anesthetized rabbits were perfused with 2% buffered formalin 2 weeks (10 rabbits) or 6 weeks (10 rabbits) following surgery. Disks, bilaminar zones, condyles and articular eminences were excised. Condyles and articular eminences were decalcified in EDTA. All tissues were sectioned at 10 microns in a cryostat. Sections were incubated with monoclonal antibodies directed against fibronectin. Following incubation in the appropriate FITC-labeled secondary antibodies, tissue sections were studied under the fluorescence microscope. The results showed that at 2 weeks following induction of ADD, there was a reduction in fibronectin immunostaining in the condyle, articular eminence and articular disk. Depletion of fibronectin in these tissues was followed by restoration of its immunostaining at 6 weeks. Also, there was a progressive increase in fibronectin immunostaining in the bilaminar zone at 2 and 6 weeks. It was concluded that surgical induction of ADD in rabbit CMJ leads to alteration in the amount and distribution of fibronectin.

Changes in the innervation of rabbit craniomandibular joint tissues associated with experimental induction of anterior disk displacement: histochemical and immunohistochemical studies.

We have previously reported that surgical induction of anterior disk displacement (ADD) in a rabbit craniomandibular joint (CMJ) leads to histopathological changes consistent with osteoarthritis. This paper reports the changes that were noted in the innervation of rabbit CMJ tissues following surgical induction of ADD. The right joint of 30 rabbits was exposed surgically and the discal attachments were severed except for the posterior discal attachment (bilaminar zone). The disk was then displaced anteriorly and sutured to the zygomatic arch. The left joints was used as sham-operated control. CMJ tissues were then removed after fixation and processed for histochemical localization of nerve fibers using the silver impregnation technique and immunohistochemical localization of neurofilaments using monoclonal antibodies. The results showed an absence of nerve fibers in the control and experimental disks and their presence in the control and experimental bilaminar zones. The bilaminar zone adhesions to the experimental condyles were also innervated. The spread of nerve fibers into the pathological fibrous adhesions surrounding the arthritic condyles in this animal model of ADD may indicate a possible mechanism of nociception in this disease.

Histopathological changes in rabbit craniomandibular joint associated with experimentally induced anterior disk displacement (ADD).

Several studies have shown that anterior disk displacement (ADD) of human temporomandibular joint (TMJ) can lead to cellular and extracellular alterations in the disk proper, bilaminar zone (BZ), condyle, articular eminence and synovial membrane. Due to lack of an animal model for this disease, it is not known whether the mechanical displacement of the disk could lead to the observed histopathological changes. The purpose of this experiment was to investigate the histopathological changes that occur in the rabbit craniomandibular joint (CMJ) following surgical induction of ADD. The right CMJ was exposed surgically and the discal attachments were severed except for the BZ attachments. Then the disk was displaced anteriorly and sutured to the zygomatic arch. The left joint served as surgical control. The CMJs were removed after 24 h, 1 week, 2 weeks or 6 weeks and stained with H&E or modified Masson stain. The results showed neovascularization, cell clustering and fibrillation of the displaced disk. The BZ showed marked fibrosis. The condyle showed subchondral hemorrhage and fibrosis followed by osteoarthritic changes in the articular cartilage. The articular eminence showed chondrocytic clustering and an increase in the amount of chondroid bone. Synovial membrane exhibited marked hyperplasia. We concluded that surgical induction of ADD in the rabbit CMJ leads to cellular and extracellular alterations in the disk proper, BZ, condyle, articular eminence and synovial membrane similar to those described previously in human ADD. It appears that the mechanical trauma resulting from ADD could lead to a cascade of reparative and degenerative changes of the affected joints similar to those described for osteoarthritis.

Structural and functional changes of cementum surface following exposure to a modified Nd:YAG laser.

A modified Nd:YAG laser was evaluated for its effect on root cementum topography and fibroblastic attachment. Fifteen extracted human teeth were curetted, sectioned, and divided into 60 areas representing 4 groups. Group I were non-lased controls, while groups II, III, and IV were lased with the same power (4 watts, 1 second), but at 3 different laser-target distances (1, 3, and 5 mm), thus delivering 3 different energy levels. Following lasing, 20 areas (5 per group) were examined under SEM for detection of any structural changes. Human gingival fibroblasts were cultured on both experimental and control samples of the remaining 40 areas. Photomicrographs at x 500 were obtained and the number of attached fibroblasts were counted. Results showed that lased cemental surfaces exhibited changes in surface topography which ranged from what appeared to be an apparent fusion of the surface of the covering smear layer (lowest energy level), to cracking and fissuring of the lased surface (highest energy level). When fibroblasts were cultured on the specimens, the results demonstrated the presence of a monolayer of cells on the control surfaces and on the surfaces lased with the lowest energy level (5 mm distance). Specimens lased at the mid-energy level (3 mm) showed decreased numbers of attached cells, but not significantly different from the controls. On the other hand lasing the cementum surface at the highest energy level (1 mm distance) caused a significant decrease in the number of the attached cells as compared to the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair of temporomandibular joint disc perforation using a synovial membrane flap in Macaca fascicularis monkeys: light and electron microscopy studies.

Previous studies have demonstrated that experimentally produced perforations in the discs of Macaca fascicularis monkeys lead to osteoarthrosis. Synovial membrane hyperplasia also was demonstrated in monkey and human joints with disc perforations. The hypothesis was advanced that a synovial flap obtained from within the affected joint would be the most appropriate tissue to repair chronic disc perforation. To test this hypothesis, four adult M fascicularis monkeys were anesthetized and 4- to 6-mm perforations were made in the posterolateral aspects of the avascular discs bilaterally. The wounds were sutured leaving the perforations open, and the animals were fed their normal diet. After 4 weeks, one joint in each monkey was reopened and a repair was performed using a double-layered flap from the synovial lining of the superior and inferior recesses. Four weeks after repair, the animals were killed and the temporomandibular joints (TMJs) were removed en bloc and decalcified. The joints were sectioned into lateral, middle, and medial sections and were photographed using a stereomicroscope and then processed for light and electron microscopy. The same processing was done to four intact joints that were used as controls. Eight weeks following perforation, the joint components showed degenerative changes consistent with osteoarthritis. Close to the perforations the disc showed loss of collagen, vacuolation of extracellular matrix, accumulation of dense proteoglycan-like material, and the appearance within the disc of type A or macrophage-like cells of the synovium. The discal tissue away from the perforation showed high cellularity and vascularity. The temporal and condylar surfaces showed denudation, fibrillation, osteophytes, and chondrocytic clustering, all characteristics of osteoarthrosis. The surgically repaired discs were intact and the articular surfaces showed no degenerative changes. Discal collagen was restored and appearance of myofibroblasts and elastogenesis were a consistent feature of the repaired disc. The vascularity of the condylar cartilage of the repaired joints appeared similar to that of embryonic cartilage. The reversibility of the degenerative alterations following discal repair in this experimental model should provide the basis for a rational and useful method for surgical repair of TMJ disc perforation using intraarticular synovial tissue.

Histopathological and immunocytochemical studies of the effect of raised occlusal vertical dimension on the condylar cartilage of the rabbit.

The objective of this study was to evaluate the effect of raising the vertical dimension of occlusion on the condylar cartilage of young adult rabbits. Ten rabbits of approximately the same age were divided into two equal control and experimental groups. The vertical dimension of occlusion of the experimental group was raised bilaterally 1.5 mm using composite resin. No procedure was done for the control group. Animals were sacrificed during a six week period, and changes in condylar cartilage of experimental animals (compared to controls) were evaluated histomorphometrically. In addition, alterations of collagen type I and II were detected using immunohistochemical techniques. The results demonstrated an increase in the volume of the experimental condylar cartilage, which was attributed to an increase in the cartilage zone. Immunohistochemical examination of the hyperplastic cartilage showed no evidence for the production of type I collagen. These changes in condylar cartilage were considered adaptive and may lead to change in condylar shape. Further studies are needed to show if these adaptive changes would progress into osteoarthritis.

Structural and functional changes in root dentin following exposure to KTP/532 laser.

The use of a new, modified Nd:YAG laser called the KTP/532 laser was evaluated within root canals to determine whether it would modify dentin permeability or alter the scanning electron microscopic appearance of canal dentin. Energies and exposure times were chosen which did not permit periodontal temperatures to increase above 5 degrees C (1 W x 1 s-5 W x 0.5 s). Half of the root canals were covered with smear layer and half were treated with EDTA/NaOCI to remove the smear layer. The results showed that this laser did not change the permeability of the smear layer-covered dentin, although scanning electron microscopic examination revealed modifications to the surface of smear layer. Lasing of etched dentin produced modest increases in root permeability which were associated with enlargement and cracking of tubule orifices.

Morphological study of lectin binding in the rabbit temporomandibular joint disc.

Glycoconjugates of the extracellular matrix are important for the normal mechanical functions of connective tissue structures such as the temporomandibular joint disc. Since lectins are known to bind to sugar residues with high affinity, a variety of lectins were used to study the presence and distribution of glycoconjugates in the temporomandibular joint disc. Discs were removed from 6 to 8-month-old rabbits and either sectioned in a cryostat and processed for light microscopy or fixed in 2% glutaraldehyde and processed for electron microscopy. The frozen sections were incubated with fluorescein- or peroxidase-conjugated lectin solutions. Ultrathin sections mounted on grids were incubated with lectins combined with a colloidal gold marker system for electron microscopical study. Our results indicate that Canavalia ensiformis agglutinin (ConA) showed little or no binding to the discal tissue. Triticum vulgaris agglutinin (WGA) and Maclura pomifera (MPA) were bound strongly to both the synovium and the extracellular matrix and WGA also bound to the territorial matrix of chondrocyte-like cells. Glycine max and Arachis hypogoea agglutinins (SBA and PNA), were localized in the synovium and extracellular matrix but to a lesser degree than WGA and MPA. WGA, MPA, Griffonia simplicifolia II and Ulex europaeus were bound by discal fibroblasts. WGA was also localized in lysosomes of synovial A-cells (macrophages). The electron microscopical studies with lectins and colloidal gold marker systems indicated that some areas of the disc may be fibrocartilagenous as had been suggested by earlier immunohistochemical studies using monoclonal antibodies to characteristic glycosaminoglycans (GAGs) in cartilage.

Histopathological study of human TMJ perforated discs with emphasis on synovial membrane response.

Fifteen perforated TMJ discs from human cadavers were studied histologically to examine the synovial membranes and to compare the findings with previous experimental results in monkeys. There were four with perforations in the bilaminar zone (these four discs were displaced anteriorly), three in the medial third of the disc, and eight in the lateral third of the disc. Histopathologically, there was an increase in vascularity and strong methyl pyronine-positive cellularity around the margins of the perforations. A young, loose, collagenous tissue lined the lateral margins of the perforated discs. Increased fibrous tissue content of the synovial subintimal territorial matrix and osteochondroid metaplasia were also seen. Severe synovial hyperplasia was visible in all joint recesses, but was greatest within those associated with displaced discs. There was patchy distribution of acidic glycoproteins, especially in the lateral parts of the perforated discs. As in the animal studies, human TMJ disc perforation was associated with a vigorous synovial reaction that was seen to form lateral bridges along the margins.

Synovial chondromatosis associated with experimental osteoarthritis in adult monkeys.

This report describes the synovial response to temporomandibular joint disc perforation in an experimental animal model. Histologic examination revealed the presence of marked synovial membrane hypertrophy in all experimental joint recesses. Gradual transformation of synovial islands from a cellular to a metaplastic stage, with chondroid or even osteochondroid-containing tissue also was seen. The cartilage was arranged in a lobular pattern with intervening fibrous septae within the synovial islands. These synovial islands were observed in the anterior recess of three joints and in the posterior recess of another joint. The experimental findings suggest a relation between synovial chondromatosis and osteoarthritis.

Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction.

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.

Fine structural study of postcastrational adrenocortical carcinomas in female CE-mice.

Postcastrational adrenocortical carcinomas in the CE/Ki inbred strains of mice and the adrenals of noncastrated CE/Ki mice were studied using light and electron microscopic techniques. Most of the tumors appeared as large nodules of cells separated by septae comprised of collagen and blood sinusoids. The majority of tumor cells (Type 1) showed few or no lipid droplets (sudanophobic), polymorphic hyperchromatic nuclei, lack of SER, abundant RER and free ribosomes, prominent Golgi complexes, and few mitochondria with scant internal membranes. Clusters of Type 1 cells were surrounded by a basal lamina. In contrast, Type 2 cells revealed abundant and dilated tubules of SER, large number of lipid droplets and mitochondria with tubulovesicular cristae. These results suggest that Type 2 cells were probably active in steroid hormone synthesis and secretion while Type 1 cells were highly anaplastic and apparently non-steroid-secreting cells.

Continuous in vitro measurement of mouse salivary gland enzymes.

A perifusion chamber technique is described that permits continuous measurement of the in vitro release of salivary enzymes from slices of mouse salivary glands responding to various stimuli. Kinetics of amylase secretion differed from those of esterase, which suggests that the secretory processes differ for these two enzymes.