Elevated levels of leukotriene C4 synthase mRNA distinguish a subpopulation of eosinophilic oesophagitis patients.

BACKGROUND: Cysteinyl leukotrienes contribute to Th2-type inflammatory immune responses. Their levels in oesophageal tissue, however, do not distinguish patients with eosinophilic oesophagitis (EoE) from controls. OBJECTIVE: We asked whether mRNA levels of leukotriene C4 synthase (LTC4 S), a key regulator of leukotriene production, could serve as a marker for EoE. METHODS: Digital mRNA expression profiling (nCounter(®) Technology) was performed on proximal and distal oesophageal biopsies of 30 paediatric EoE patients and 40 non-EoE controls. Expression data were confirmed with RT-qPCR. LTC4 S mRNA levels were quantified in whole blood samples. Leukotriene E4 was measured in urine. RESULTS: LTC4 S mRNA levels were elevated in proximal (2.6-fold, P < 0.001) and distal (2.9-fold, P < 0.001) oesophageal biopsies from EoE patients. Importantly, increased LTC4 S mRNA transcripts identified a subpopulation of EoE patients (28%). This patient subgroup had higher serum IgE levels (669 U/mL vs. 106 U/mL, P = 0.01), higher mRNA transcript numbers of thymic stromal lymphopoietin (TSLP) (1.6-fold, P = 0.009) and CD4 (1.4-fold, P = 0.04) but lower IL-23 mRNA levels (0.5-fold, P = 0.04). In contrast, elevated levels of IL-23 mRNA were found in oesophageal biopsies of patients with reflux oesophagitis. LTC4 S mRNA transcripts in whole blood and urinary excretion of leukotriene E4 were similar in EoE patient subgroups and non-EoE patients. CONCLUSION & CLINICAL RELEVANCE: Elevated oesophageal expression of LTC4 S mRNA is found in a subgroup of EoE patients, concomitant with higher serum IgE levels and an oesophageal transcriptome indicative of a more-pronounced allergic phenotype. Together with TSLP and IL-23 mRNA levels, oesophageal LTC4 S mRNA may facilitate diagnosis of an EoE subpopulation for personalized therapy.

Effect of strain on bone nodule formation by rat osteogenic cells in vitro.

The purpose of this study was to assess in vitro bone nodule formation by cells exposed to a range of microstrain, at a sub-optimal oscillation frequency for bone formation. Fetal rat calvarial cells experienced a Flexercell regimen within either FLEX I (deformable) or FLEX II (non-deformable) substrates. Cells in FLEX I plates were exposed to growth medium only; those in FLEX II plates were exposed to either growth medium only, or growth medium + 10(-7) M IGF-1. Cell numbers were assessed from 1 to 6 days. Other cells were exposed to the Flexercell regimen (-2 kPa, 0.05 Hz) for 1-3 (Group 1), 3-6 (Group 2), 1-9 (Group 3) or 10-15 (Group 4) days and were maintained, at other times, under standard conditions. After 21 days, nodules were counted within each well and within the compression, <999, 1000-4900, 5000-9999, 10,000-14,999 and 15,000-25,000 microstrain regions of the FLEX I membrane. Cyclic deformation inhibited cell numbers from 1 to 6 days, compared to control or IGF-1 groups (P<0.001). The number of nodules in Groups 2 and 4 were greater than Groups 1 or 3 (P<0.001), but not different from control or IGF-1 groups. Compression or tensile microstrain significantly affected nodule formation in all groups, with Group 4 producing more nodules than other groups in most microstrain regions. Thus, the number of bone nodules produced by osteogenic cell cultures exposed to cyclic deformation was significantly affected by the timing of initiation and the characteristics and magnitude of the deformation regimen.

Effects of sodium acetate on rat bone-nodule formation and mineralization in vitro.

Sodium acetate reportedly promotes bone atrophy by inducing resorption and inhibiting osteoprogenitor-cell proliferation, but little is known about its effects on bone-matrix deposition and mineralization by a population containing osteoprogenitor cells. The objective here was to assess the effects of 1-20 mM sodium acetate on the proliferation and differentiation of these cells and their resultant bone-nodule formation and mineralization in an in vitro assay. Exposure to 10 mM sodium acetate had no effect on cellular proliferation but significantly increased the production and mineralization of bone nodules (p < 0.01), suggesting that it affected osteoprogenitor differentiation and subsequent metabolism. However, 10 mM acetate did not increase net bone mass. Dilutions of 1-5 and 20 mM inhibited cellular proliferation and resultant bone-nodule formation and mineralization, significantly reducing the percentage bone area as compared to controls (p < 0.001). These data suggest that 1-5 and 20 mM sodium acetate significantly inhibit bone deposition, whereas 10 mM has no effects, which could contribute to iatrogenic metabolic bone disease in patients receiving either renal dialysis or total parenteral nutrition.

Collagen synthesis from human PDL cells following orthodontic tooth movement.

The dynamic remodelling processes in the periodontal ligament (PDL) account for the reaction of PDL cells to different orthodontic force simulations. These occur mostly by degradation and synthesis of collagen types I, III, V, VI, XII and XIV. The purpose of this study was to quantify specific collagen types in the PDL from zones of tension and compression of experimental teeth. Such changes could then be correlated with the processes of orthodontic-stimulated tissue breakdown. Maxillary and mandibular premolars of three females and one male patient were orthodontically moved with a box loop for a total of 14 days, prior to tooth extraction. Teeth from the contralateral side of either the maxilla or the mandible served as the untreated controls. A total of seven experimental and seven control teeth were used in this investigation. PDL fibroblasts from the cervical third of the roots corresponding to the compression and tension zones of the experimental and control teeth, respectively, were scraped and cultured in vitro at 37 degrees C in a humidified incubator with 5 per cent CO2/95 per cent air. Collagen synthesis of types I, III, V and VI was quantified by using an ELISA. Application of orthodontic forces in the experimental teeth showed a significant increase (P < 0.05) of the synthesis of all collagen types in the compression as opposed to the tension zones. Collagen synthesis on the compression zone of experimental teeth was not significantly different in the mandible when compared with those of the maxilla. In addition, the proportional distribution of different types of collagen was also not significantly different in the PDL fibroblasts from either zone of experimental teeth of either the maxilla or the mandible. Collagen metabolism in response to orthodontic stimulation appears to be higher in the compression zones and lower in the tension zones. Contrary to what is traditionally assumed in the literature, such findings indicate that in addition to bone resorption, tissue remodelling is very active in zones of compression following the disappearance of the hyalinized areas. These findings constitute a model for future studies on collagen metabolism during orthodontic-stimulated tooth movement.

Vascular changes in the periodontal ligament after removal of orthodontic forces.

Vascular changes in the periodontal ligament after release of orthodontic force and their possible contribution to relapse of relocated teeth are poorly understood. This study documented the periodontal vascular changes after 2 weeks of tooth movement and during a 3-week period after release of orthodontic force. This study is the first comprehensive quantitative description of these events. Changes in blood vessel number and density were correlated with the direction of tooth movement (initially mesial in response to force but later distal because of relapse). Application and removal of orthodontic force produced significant changes in blood vessel number and density, which were not related to changes in tissue volume. The vascular changes were dependent on the site of evaluation and the size of the blood vessel. The periodontal vascular distribution and density can be summarized as follows: (1) increased after application of orthodontic force, (2) transient decrease subsequent to removal of force, (3) transient increase during reactivated distal drift, and (4) normalization. Normalization was achieved during an interval equivalent to the duration of orthodontic force, suggesting that the vasculature could modulate interstitial tissue pressures, resulting in relapse of relocated teeth. The role of the periodontal vasculature in alveolar remodeling and in modifying interstitial tissue fluid pressures coincident to human tooth movement requires further study.

A molecular mechanism of integrin regulation from bone cells stimulated by orthodontic forces.

The purpose of this paper is to discuss a molecular mechanism in the signal transduction pathways of the regulation of integrin genes taking place in bones cells as a result of orthodontic or mechanical stimulation. Human osteosarcoma (HOS) TE-85 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and grown to confluency in Flexercell type I dishes and orthodontic forces were applied to the cells via an intermittent strain of 3 cycles/minute using the Flexercell Strain Unite System for periods of 15 and 30 minutes, 2 and 24 hours and 3 days. Antibodies against beta 1 and alpha v integrins were immunolocalized in strained and unstrained cultures. Total RNA was extracted and cDNA probes were used to measure at various mRNA expression of beta 1 (1.2 kb) and alpha v (1.1 kb) integrins. A cDNA probe for cyclophylin (750 b) was used for controls of gene expression. Results showed that mechanical stimulation caused a reorganization of integrin distribution in comparison with non-stimulated controls. mRNA for beta 1 expression showed a marked increase at 30 minutes and 3 days, while mRNA levels for alpha v did not change with strain. The selective expression of integrins mRNA is indicative of a specific gene regulation by mechanical stimulation in the cells studied.

Glycosaminoglycan synthesis in the rat articular disk in response to mechanical stress.

The mechanism by which compressive mechanical stress affects glycosaminoglycan synthesis in the articular disk was investigated with a modified organ culture technique. Forty-eight male Sprague-Dawley rats were divided into three experimental groups and one control group of 12 animals each, aged 7 and 9 weeks. The experimental groups followed different regimens of stress applied for 25%, 75%, or 100% of the time during the total test period of 24 hours. Articular disks were stressed with flexible bottomed dishes (Flex I dishes, Flexcell Corp., McKeesport, Pa.) using the Flexercell Strain Unit (Flexcell Corp., McKeesport, Pa.) and incubated with [3H]-glucosamine for 24 hours. Samples were then collected, digested with Pronase-E, and after precipitation with cetylpyridinium chloride (CPC) and ethanol, the different glycosaminoglycans (GAGs) were separated by using cellulose acetate electrophoresis. The significant GAG types with stress were chondroitin6sulfate (C6S), hyaluronic acid (HA), and dermatan sulfate (DS). There was no significant relationship in the experimental groups between age and regimen of stress applied in either age. Higher stress regimens showed significantly higher proportions of C6S when compared with the controls, whereas HA appeared to decrease slightly and DS was not affected. Since C6S is the major component of hyaline cartilage, the results of this study suggest that compressive forces in the articular disk may stimulate the development of more cartilagenous-like properties with respect to GAG content.

The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells.

Mechanical stimulation of the skeleton alters the metabolism of bone cells, but the effects of mechanical strain on the cytoskeleton of osteoblasts are poorly understood. While changes in the distribution of the cytoskeleton in mechanically strained cells have been reported, little is known about the pathways by which these changes are transduced into cell functions. Human osteosarcoma (HOS) TE-85 cells were cultured in Dubelcco's modified Eagle's medium/F-12 and grown to confluency in Flexercell type I dishes in a humidified incubator with 5% CO2 and 95% air. Intermittent strain (3 cycles/min) was applied to the cells for periods of 15 and 30 min, 2, 4 and 24 h, and 3, 5, 7, 10, 14, 20 and 28 days. Unstrained cells were used as controls. The distribution of beta 1 integrin was studied immunocytochemically. Total RNA was isolated at every period of time and Northern blots were used to study the effects of strain on the levels of beta 1-integrin expression. The results indicated that mechanical strain increased the synthesis of beta 1 integrin. Northern blots showed that beta 1 mRNA expression was increased significantly (p < 0.005) at 30 min and 3 days of strain application. Strain also affected beta 1 distribution markedly in 24-h cultures. The response of HOS cells to mechanical strain demonstrates that the cytoskeleton of the osteoblast adapts to strain through the stimulation of specific cytoskeletal and receptor proteins. These results suggest a pathway through which mechanical strain is transmitted to the osteoblastic-like cells.

Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone.

Second-messenger systems have been implicated to transmit mechanical stimulation into cellular signals; however, there is no information on how mechanical stimulation is affected by such systemic factors as parathyroid hormone (PTH). Regulation of adenylyl cyclase and phosphatidylinositol pathways in rat dentoalveolar bone cells by mechanical strain and PTH was investigated. Two different cell populations were isolated after sequential enzyme digestions from dentoalveolar bone (group I and group II) to study potential differences in response. Mechanical strain was applied with 20 kPa of vacuum intermittently at 0.05 Hz for periods of 0.5, 1, 5, 10, and 30 minutes and 1, 3, and 7 days using the Flexercell system. Levels of cAMP, measured by RIA, and levels of inositol 1,4,5-triphosphate (IP3) and protein kinase C activity (PKC), measured by assay systems, increased with mechanical strain. When PTH was added to the cells, there was a significant increase in levels of all the intracellular signals, which appeared to potentiate the response to mechanical strain. IP3 levels (0.5 minute) peaked before those of PKC activity (5 minutes), which in turn peaked before those of cAMP (10 minutes). Group II cells showed higher levels of cAMP and IP3 than the group I cells. This suggests that the former may ultimately play the predominant roles in skeletal remodeling in response to strain. Immunolocalization of the cytoskeleton proteins vimentin and alpha-actinin, focal contact protein vinculin, and PKC showed a marked difference between strained and nonstrained cells. However, the addition of PTH did not cause any significant effect in cytoskeleton reorganization. Staining of PKC and vimentin, alpha-actinin, and vinculin suggests that PKC participates actively in the transduction of mechanical signals to the cell through focal adhesions and the cytoskeleton, although only PKC seemed to change with short time periods of strain. In conclusion, dentoalveolar osteoblasts responded to mechanical strain initially through increases in levels of IP3, PKC activity, and later cAMP, and this response was potentiated when PTH was applied together with mechanical strain.

The effect of growth on collagen and glycosaminoglycans in the articular disc of the rat temporomandibular joint.

Newly synthesized collagen and glycosaminoglycans (GAG) were studied in the temporomandibular discs of male Sprague-Dawley rats of 3-13 weeks of age. Each age group had eight animals and [14C]glycine or [3H]glucosamine were used to determine the proportion of newly synthesized type III to type I collagens or the proportion of different types of newly synthesized GAGs during 4 h of labelling in organ culture. Separation of newly synthesized collagen bands from rat disc by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a peak in type III at the ages of 7 and 8 weeks. Type III collagen synthesis and the rate of mandibular growth were strongly related through all ages studied. GAG chains were separated by cellulose-acetate electrophoresis. Calculation of disintegrations/min per mg of wet disc tissue for each GAG peak showed that hyaluronic acid (HA), chondroitin-6-sulphate and keratan sulphate/chondroitin-4-sulphate (KS/C4S) were the predominant molecules synthesized in the disc. There was also a steady increase in newly synthesized HA and C6S synthesis up to 6 and 7 weeks respectively. Proportions of newly synthesized C4S/KS, HA and C6S were significantly higher than those of other GAGs with respect to ageing. From these observations it appears that the articular disc shows more of the characteristics of cartilage, as evidenced by the increased amounts of C6S and KS/C4S during the mandibular growth spurt at the ages of 6 and 7 weeks, similar to that of type III collagen. There were also increased amounts of HA, suggesting that during 5-7 weeks of age the rat disc is undergoing more active remodelling. This study provides baseline data for further analysis of the effects of mechanical loading and trauma on articular disc responses.

Taiwan, R.O.C.: perspectives in school health.

Continuous economic development and evolving social systems have created unique challenges for school health personnel in Taiwan. Due in part to economic growth and an improving health care system, average life expectancy has increased markedly for males and females since 1950. Traditional leading causes of death such as pneumonia, tuberculosis, and gastroenteritis have been replaced by accidents, neoplasms, and cardiovascular diseases. Schoolchildren suffer from various disorders that include hepatitis B, dental caries, and health problems related to scholastic pressure such as myopia, anxiety, and depression. However, new problems such as violence, substance use, and teen-age pregnancy may develop in the future. In response to the challenge, an extensive school health program has emerged that emphasizes school health instruction, school health services, and a healthful school environment. Increased attention has focused on teacher preparation in health education, and a cooperative approach to school health promotion emphasizes school, community, and interagency cooperation. Prompted by the establishment in 1980 of the Graduate Institute of Health Education, National Taiwan Normal University, school-based research initiatives have increased dramatically. Eighteen recommendations for further improvement are offered.

Continuous stressing of mouse interparietal suture fibroblasts in vitro.

The morphological and biochemical response of sutural fibroblasts in vitro to continuous force was examined. Cells from mouse interparietal sutures were grown and subcultured on glass slides. Titanium disks coated with collagen were allowed to attach to the cellular multilayers. Four of the glass slides were then placed at an angle of 75 degrees for a period of three days so that continuous stress would be created, while four others were left flat. Also, four glass slides were left flat with no disk. Following the incubation period, the dishes were labeled with 14C-glycine for 15 h. The cells and medium were then collected for collagen extraction followed by SDS-polyacrylamide gel electrophoresis. Dried gels impregnated with fluor were exposed to x-ray films that were then scanned densitometrically for collagen types I and III. It was found that the proportion of newly-synthesized type III collagen increased significantly with the application of continuous stress. A second set of experimental and control glass slides was fixed in glutaraldehyde and post-fixed in osmium tetroxide. Following critical-point drying and coating, the glass slides were examined under a scanning electron microscope. The scanning images showed the formation of a ligament-like structure between the disk and the glass slide. Moreover, mitotic activity, as evidenced by spheroidal cells, was stimulated in the areas previously adjacent to the disc, which had since moved away. This system offers a standardized continuous force system that can stress cells in a ligament-like structure and thus provides an in vitro model analogous to clinical orthodontic and orthopedic stress.

Effect of force level on synthesis of type III and type I collagen in mouse interparietal suture.

Nine-week-old Swiss male white mice were divided into groups killed after time intervals of force application of six h, and one, three, five, seven, ten, 14, 21, and 28 days. Each group had 45 animals: three control, three sham-operated, and three experimental animals for each of the five force levels: 50 g, 35 g, 25 g, 15 g, and 5 g. The experimental animals had helical springs placed surgically in their calvaria for expansion of the interparietal suture. The sham-operated animals received inactive springs. Control animals were at the same age as the experimental and sham-operated animals. After death, the amount of sutural expansion was measured, and the calvaria with the implanted springs were explanted into Trowell-type organ culture dishes. [14C]-glycine was added for two h after 60 min of culture for all explants. The rate of suture expansion was directly proportional to the force value of the tensile stress, and a maximum 2.0-mm expansion was achieved for all force levels by the 28th day. Sutural collagen was solubilized by limited pepsin digestion, and radiolabeled types I and III alpha-chains were separated by SDS-PAGE, visualized fluorographically, and measured densitometrically. All the experimental and sham-operated animals responded with a rapid rise followed by an almost equally rapid fall in the proportion of newly-synthesized type III collagen before becoming stabilized for the rest of the experimental period at a level that was significantly higher than that of the control and sham-treated animals of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of sutural growth rate on collagen phenotype synthesis.

We determined the ratio of newly-synthesized type III collagen to the total of type I and type III collagen in mouse interparietal sutural tissue at selected ages between birth and adulthood (36 weeks old). We incubated mouse calvaria explants in Trowell-type organ culture dishes for one h and then added [14C]-glycine for two h. We dissected the interparietal sutural tissues for collagen solubilization by limited pepsin digestion. Fluorographic visualization of separated radiolabeled collagens, after SDS-PAGE, found the ratio of collagen type III alpha-chains to the total type I and type III alpha-chains to be age-dependent. The proportion of type III alpha-chains at birth was quite high, but there was a significant drop (p less than 0.05) during the first two days of life, probably because of the sudden environmental change from in utero. The proportion of type III alpha-chains rose significantly from day 2 to day 4, reaching a maximum and then dropping significantly to about the same proportion as at birth by day 7. A further significant drop took place during the second week of life, with the proportion stabilized at around 3.5% at two weeks to ten weeks of age. A final significant drop during the eleventh week of life led to no detectable synthesis of type III collagen after 12 weeks of age. The changes in the collagen phenotype ratio did not relate to changes in body weight during growth and development, which suggests that the interparietal suture may have an independent maturing pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen and prostaglandin synthesis in force-stressed periodontal ligament in vitro.

A periodontal organ culture system capable of receiving orthodontic type forces was developed. Histological, radioautographical, collagen, and prostaglandin synthetic data demonstrated the vitality of the organ over a 24-hour period of culture. Significant increases in the proportion of type III collagen synthesized during periods of active stress were found, but no alterations in relative levels of prostaglandins synthesized during periods of force application were discernible.

A radioautographic study of the effect of age on the protein-synthetic and bone-deposition activity in interparietal sutures of male white mice.

Groups of male white mice were killed at 4, 5, 6, 7, 8, 9 and 10 weeks of age 2 h after intraperitoneal injection with [3H]-proline. Radioautographic analysis of sections of the interparietal suture demonstrated significantly greater protein-synthetic activity in the para-osseous zones relative to the middle zone (p less than 0.01) and a plateau of lower protein-synthetic activity by 7-8 weeks of age (p less than 0.05). Groups of mice were selected at 4, 6, 8 and 10 weeks of age. Each mouse was injected intraperitoneally with [3H]-proline three times at one-week intervals. Sutural growth rate was determined from incremental lines revealed by radioautographs prepared from serial paraffin sections of the interparietal suture and demonstrated a stabilization of growth by 8 weeks of age. This, together with the grain counting data, suggested that a mouse of 7-8 weeks would provide a suitable model for experimental studies in sutural remodelling response without masking effects by normal growth.

Effect of ascorbic acid on protein synthesis and collagen hydroxylation in continuous flow organ cultures of adult mouse periodontal tissues.

A continuous flow organ culture system (CFCS) was used to determine the effect of ascorbic acid on the synthesis of collagen and noncollagenous protein by bone of the alveolar process and periodontal ligament in organ cultures of adult mouse periodontium. For the last 24 h of 2 day cultures, 5 microCi/ml 3H-proline was added to the medium. Highly purified collagenase was used to separate the collagenous and noncollagenous proteins and the incorporation of isotope into each fraction measured. Collage synthesized in the presence of less than 10 micrograms/ml ascorbic acid was found to be highly under-hydroxylated (pro:hypro ap. acts. 2.3-3.1) in both tissues. When the ascorbic acid levels were between 25 and 100 micrograms/ml, the synthesis of collagenous proteins was selectively stimulated and hydroxylation significantly improved (pro:hypro sp. acts 1.72-1.89). The effect of ascorbic acid was not related to tissue viability since tissues cultured initially in the absence of ascorbic acid were able to recover completely when compared to controls given ascorbic acid continuously. The proportion of radioactivity in collagen and noncollagenous protein, collagen hydroxylation, and percentage of collagen synthesized as type III (av.23%) in bone of the alveolar process was similar to that found in vivo. However, in the periodontal ligament in vitro the proportion of noncollagenous protein synthesized was increased from 70% to 87% and the percentage of type III collagen increased from 14% to 26% compared to in vivo results.

The effect of oxygen partial pressure on protein synthesis and collagen hydroxylation by mature periodontal tissues maintained in organ cultures.

Mature periodontal tissues from adult-mouse first mandibular molars were cultured in a continuous-flow organ-culture system which allowed the regulation of both ascorbic acid concentration and pO(2) (oxygen partial pressure). Protein synthesis was measured by analysing the incorporation of [(3)H]proline into collagenous and non-collagenous proteins during the last 24h of a 2-day culture. At low pO(2) [16.0kPa (approx. 120mmHg)] approx. 60% of protein-incorporated [(3)H]proline was found in collagenous proteins. However, it was evident that this collagen was considerably underhydroxylated. At high pO(2) [56.0kPa (approx. 420mmHg)], both the amount of collagen deposited in the tissues and the degree of hydroxylation were increased considerably. In contrast, no significant effect on non-collagenous protein was observed. Tissues cultured at low pO(2) for the first 48h were unable to respond to a subsequent increase in pO(2) during the last 24h. Analysis of pepsin-solubilized collagen alpha-chains labelled with [(14)C]glycine demonstrated the synthesis of both type-I and type-III collagens by explants cultured for 48h at high pO(2). Type-III collagen comprised 20-30% of the radioactivity in alpha-chains in both the periodontal ligament and the tissues of the alveolar process. The pattern of protein synthesis in the alveolar tissues at high pO(2) was similar to that observed in these tissues in vivo. However, in the cultured periodontal ligament the proportions of non-collagenous proteins and type-III collagens were increased in comparison with the tissue in vivo.

A continuous-flow culture system for organ culture of large explants of adult tissue: effect of oxygen tension on mouse molar periodontium.

A modified continuous-flow culture system (CFCS) was developed to maintain large explants of periodontium from adult mouse in organ culture. The culture medium was stored in a reservoir outside of the incubator, pumped via polyvinyl tubing into small glass culture chambers that were placed in the oxygenator and then collected in a waste flask. Medium was analyzed for pO2, pCO2 and pH during the culture period. Three-molar and single-molar explants of periodontium were maintained for 48 hr in the CFCS at two different pO2 ranges: 100 to 120 mm Hg and 400 to 420 mm Hg. [3H]Proline was added 24 hr prior to sacrifice. Light-microscope morphological and radioautographic observations suggested that cell viability and incorporation of [3H]proline, probably into newly synthesized protein, increased with an increase in pO2 and was related to a pO2 gradient extending from the periphery to the center of the explants.