Isolation and Characterization of Epithelial and Connective Tissue Cells from Rat Palate VICTOR P. TERRANOVA and JOHN S. BRAND University of Rochester School of Medicine and Dentistry, Department of Radiation Biology and Biophysics, Rochester, New York 14642, USA
Epithelial and connective tissue cells were isolated from rat palate by sequential enzymatic digestion. Differences between the two populations were noted with respect to proline uptake and incorporation, % collagen synthesized, effects of parathyroid hormone and metabolism of D-valine. From these studies it can be concluded that the cell populations are viable and distinct with respect to the biochemical parameters examined. j Dent Res 57 (1):118-127 January 1978. The study of periodontal tissue at the biochemical level can be significantly advanced by isolation and subsequent study of the separate populations of cells that make up this tissue. There is much speculation as to which specific cell types are involved in periodontal disease. Characterization of the cells derived from normal tissue will provide the baseline for determination of the metabolic or biochemical aberrations which develop in periodontal disease. The periodontal unit-epithelium, connective tissue, and underlying alveolar bone has been studied extensively by histological methods.1 Biochemical differences between fibroblasts and epithelial cells have been examined predominantly in tissue culture. It is now possible to further advance the studv of the periodontium by isolation of the separate population of cells which make up this tissue. Viable cells, both epithelial and those of mesenchymal origin, can now be dispersed from many mammalian tissues. The isolation technique which has proven to be most effective is based on the enzymatic digestion of the extracellular matrix. This type of procedure has been applied to the preparation of isolated Received for publication January 19, 1977. Accepted for publication May 18, 1977. This work is supported in part by U.S.P.H.S. Training Grant 5-TOl-DE-00175 and NIH Career Development Award No. lKO4AM00101-01 to J. S. Brand and, in part, by U.S. E.R.D.A. Contract EY-76-C-023490 at the University of Rochester and assigned Report No. UR-3490-1009. * Worthington Biochemical Corp., Freehold, NJ. Sigma Chem. Co., St. Louis, Mo. M Gibco, Grand Island Biological Co., Grand Island, N.Y.
118
hepatocytes,2 intestinal epithelial cells,3 adipocytes,4 and lymphocytes,5 as well as cells from bone,6 cartilage,7 and other sources. The present study was undertaken to isolate separate populations of viable connective tissue and epithelial cells from normal oral tissue and to begin a biochemical characterization of these populations of cells. The information obtained using normal tissue will provide a baseline for quantifying changes in cell subpopulations and determining the nature of the metabolic aberrations which develop in periodontal disease. Materials and Methods HISTOLOGY.- Freshly excised palatal tissue (control) and palatal tissue removed at one hour intervals during a four hour incubation (test) were fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, sectioned (8 microns thick) and stained with hematoxylin and eosin. CELL ISOLATION. The cell isolation technique is a modification of the method described by Dziak and Brand6 for bone cells. Palatal tissue, freshly excised from decapitated young adult female rats was incubated at 37 C in a Hepes buffered isotonic salt solution, pH 7.4, containing crude collagenase* 3 mg/ml, crude hyaluronidase* 3 mg/ml and 0.5 mg/ml
elastase.* The buffer was 25 mM Hepes, 10 mM NaHCOM, 100 mM NaCl, 3 mM K2HPO4, 1 mM MgSO4, 1 mM CaCl9, 60 mM mannitol, glucose 5 mg/ml, bovine serum albumin (Fraction V) t 1 mg/ml, 5 jg/ml penicillin,+ 10 ,.g/ ml Neomycin+ and 5 jug/ml streptomycin.j Thirty milligrams of tissue per 1.0 ml buffer were incubated in 50 ml polypropylene beakers in a water bath and shaken at 90 oscillations per minute. At one-hour intervals up to four hours the cells were harvested as previously described6 and tissue digestion resumed. Cell counts were made with a haemocytometer. CELL VIABILITY STUDIES. The viability of J Dent Res January 1978
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the cells isolated by the above technlique wvas evaluated routinely using trypan bluie dye excltusion. To I X 106 cells/l100 X buiffer, 100 X 1S trypan l)lue was added. Cell counts for inclusion of dye were imiade 15 miinutes later. Intracellular spate was mcasuired by a double label procedure; 50 X 10f isolated cells were incubated at 37 C for 5 minutes in 2.0 ml of buffer containing 11mnM 14C polyethylene glycol (PIEG) * (0.5 ACi/ml) and 3H-F1 10j (0.3 gCi/ml). The cells were then sedimented for three imiinuites at 800 x .J after whiich 100 X of the supernatant wxas couinted in 10-ml scintillation cocktail (9.67 ml Toluene-Omnifluor+ + 0.33 ml BBS-3) .§ Tle suipernatant was coompletely removed with a micropipet and the side of the tule dried with absorbent paper. The pellet was thein re uspended in 1-ml fresh bufter, equilibrated for 5 mirnuites at 37 C, and centriftuged as before; 100 X of the supernatant was then counted. Total 14O was determined from tlhc CPM tritium and extracel* Amersham/Searle Corp., Arlington Heights, I1. t Mallinckrodt, St. Louis, Mo. t New England Nuclear, Boston, Ma. § Beckman Instruments Inc., Fullerton, Ca.
lul.ar fliid (ECI) was determiined from- the 14C PEG. Intracelluilar fluid JICF) per 106 tells wxas obtairned by subtracting ECF from total 1-.0 and dividing by 50. For an additional measurement of total H.,O xvet wxeights of cell saniples were determined by sedimiienting the cells at 800 X g for 3 minutes in a tarecd conical centrifuge tube. The supernatant xxas carefLully decanted off and the sides of the tuibe dried. Tuibes wxere then dried in a 37 C oven and weighed. This procecdure wx as repeated uLntil a constant weight was obtained. Total H.,O equals wet weight minus dry weight. Total cell protein w,as measur ed by the Toennier anld Fe'ng mnethod.8 Proline uptake was deternmined bv incubating 2 X 106 isolated cells in 1.0 nml buffer wvith 0.2 mnM 1IC proline (0.1 gc/nil)* for x ariotis titre poinits up to fouir hoiirs. The cells anid inCLIbation imiediumiii were then carefully transfer-red into smiiall silic oniized glass test tuLbs arid centriftuged at 1,500 X g for 2 minu tes. TIe supernatant wx as rermoved and the resuItincg cell pellet washed with 1.0-nil cold bufTer anid recentrifuged. After discardinig the
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supernatant the pellet xx as sLuspended in. 200 X distilled 110 and placed in an oil bath at 100 C for 10 nminuites to lyse the cells; 50 X of 25% tricholoroacetic acid (TCA) were then added and the samiiple centrifuged at 10,000 X g for 5 nminuites; 100 X of the resultinig suiperniatant nivle tliein counted in 9.67 Ixiv TolueneOninifluori anid 0.33 nil BBS-3.§ (This is free '4C-proline.) To the resultinlg cell pellet, 200 X of g% TCA were added and the tube vortexed vigorouslv. After 10 imlitlties, thie mixture is spuni at 10,000 X g for 5 minutes and the supernatant discarded. To the pellet xas added 0.5 nml 6 N I-ICl. This reaction mixture xas hydrolyzed in glass tubes sealed xxith teflon-coated rubber-lined caps for 16 hours at 110 C and then- dried uinder vacuuim to remove the HCI. The hydrolysate was then dissolved in 25 X distilled H.O to which 50 X 0.1M NaHC03 anid 150 X dansyl chloride 09 mg dansyl chloride per ml acetone) xx ere added. The reat tion was stopped after 10 minuites by the addition of 25 X 0.4 M atetic acid; 25 X of the reactionmixtuLre xx-as spotted oni 500 miicron thlick silica gel plates anid developed in a ben-
1978
zene, pyridine, acethc acid bu-ffer (80 nil: 20 nil:2 ml).9 Standards of proline anad hydroxyproline (10 mIIM 14C-proline and 10 mM N1-11yvdroxxprol ine) were ruiii simultaneously and corresponddiny areas on the thin layer plates sctraped and couinted in scinitillation fluid to deteriijine (1) the aniount of prolinc incorporated into protein and 29) the amoun1t of proteini bound proliine hydroxxlated. To calculate per ccnt recoverx, 25 X of the danisylation Tmixttiire xN-ere spotted at thc top of the plate and subsequiently scraped anid counted. Thus total proline incorporated equals proline incorporated plu-s hb-droxylated protein bound proline corrected to 100%c recoverv. To stLudy the effectt of PTH on- proline ulptake and incorporation into protein, prior to isolationi the tissue xas preincubated xxith PTH at a concentration of 1 unit/nil buffer (xxithout enzyme) for onc half hoLur; cointrols had no PTH. Eniyines xxere then added and the cells x ere harvested fronii the first and last txxo houirs of digcstilon. Cell proteini hxydrolx sates xxvere again assayyed for con-tenit of labeled proline and hydroxyproline.
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ISOLATED RAT PALATAL CELLS
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FTm 3. Cross section of
rat
121
palate after 1.5 hours of diges-
tioil (orig marg 500 X )
Itx orcler to IntoIIS\[. 14CD V\IINEF ' inisure no epithelial cell containination in the connective tissue cell population for these experiinents, about three fouirths of the conniective tissue ccll laver w-as dissected awax fromll the palates befoie incubatioin x-ith enzyne. The reIainiaing tissue w\cas then trcateci as described al)ove, discarding the cells fromii the first 2 honii s of dig£:(estion. 'Thus, two preparations of (-(,lls were obtained; coninectixte tissue inculbated for 2 hiours and epithelitni inlclllatedl for fouir houLrs wxith the first 2-hour cells reiovcd. Whole palates which xx ere not dissected xw ere also used in separate experiments. Connective tiSSue cells anld epithelial cells xw re inc uhated at 37 C ,at a concentration of 4 bdhuffer xwith 1 .0 Ci (arricr X 10" cells pernl free "1C-D-valine (specific activitx 09.8 mCi/ inV!.* At ton-lhour intcrx als, 1 .0 mlI of cell sulspenlsion Nx-xas renioxcld ft o-ni the inc ubator, placed in glass test tuhes onl ice, auid iimiediately precipitated with 0.5 nil 25% ice cold TCA. The test tubes wxx rc then cectr ifi-ed at 1.5.000 X for 10 minuites; 25 N of the siliper* Calbiochem., Los Angeles, Ca.
inttant ere 1eactecd xwith dansxl chloride as de-
sitribecl abovxe anid 50 N samiples of tlhis reaction iiiixtLrre were counted, wvith an-d wsithostt a solubJilizer acddecl, in a liquiid scintillationi c ouinter. Results
dealIIIsTros o(-. IHistt-)dogicallv xx can onstrate a sequential rnioxoval of cclls frhost the rat palate. The connective tissue la1 er closest to the palatal bone is the first area frotio xhich cells aire liberated. Firiure I shoxxws a c ross section through normal rat palate before inctnbation with eavnines. In I,igrtic 2, after onie h-ouir of inlculbatioIl xNith cnzixiiues, thec conncctix c tissue layer closest to the palatal botie apperns rcntaAec of c ells atC dlilooser and a simiall pen persed (approxititately 100,, of the total cell vield). Fi-iure 3 Shosxx s a cross section of rat palate after 1.5 hostrs of incubation xxith enznie. Fifty percent of the connec tive tiSsLe laxver has been dig-estcd. After two hours of digestion, 00%- to 100% of the connectixe tissue ell lax7er has been removed (Fia 4). Dutring the Incxt txx cO Iours of incubationi, the epithelial
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TERRANOVA & BRAND
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cells are liberated and all that remains of the palate is the cornified epithelial cell layer which cloes inot digest further (Fig 5). This proc educe yielded approximiiately 9 X 106 cells per palate, 40% of xwhich xwas connective tissue ( ells and 60%. ep)Lthelial c ells. MlF\%LSUREMtENTS.--In-
ICE AND PROTF.IN
tracellular fluid space (ICF) measuriemients for 106 connective tissute cells gave 0.23 ± 0.04 lul. Epithelial cells hlad an ICF of 0.53 ± 0.10Iil/ 103 cells. Total cell piotein measurements yielded 170 9- 10 gg protein per 106 connective tissuie c els and 235 15 ug protein per 106 cpithelial cells. CELL VIIAIi'rY. -Routinely 90 to 95% of botlh epithelial and connlective tissue cell populationis excluded trvpan- blue dy e. The ability of the cells to actively accumulate proline cas also used as a criterion of their viability. Proline uptake per 106 cells was calculated for cells incubated with 0.2 MM 14C-proline for various tiine points. In Figure 6, epithelial cells shmocsN a slightly higher uptake of free proline over connective tissuie cells. However, due to the dlifference in ICF space nmeasuirements the
epithelial cells have a losser distribution ratio than do the connective tissue cells (Fig 7). Both epithelial and connective tissue cells for all time points do show distribution ratios well over 1.0 and thus are actively accumulating proline. Control data for total incorporation of 14C-proline (Fig 8) shoxx that during the four houir incubation period both the epithelial cell population anid connective tissue cell population incorporate proline into protein at approximately the same rate after 30 minutes. Data for liydroxylatioui of piotein bound proline (i.e., hydroxyproline) (Fig 9), showv that epithelial cells hxdroxvlate 20 pAl/hr/106 cells imore prolinie than do connective tissue cells. Comiibining the control data for proline and hvdroxvproline, the per cent of protein miade by the cell that is collageni, over the fourhour incubation, is 26% for connective tissue cells and 43%SJ for epithelial cells, assuming an armino atici content of 20 mole %- for collagen aind an axveragSe prolinie conrtent of 5 miiole % for
ionco(llagen protein.
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( ori g i liag 5 00 X ). Downloaded from jdr.sagepub.com at The University of Melbourne Libraries on June 5, 2016 For personal use only. No other uses without permission.
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FIG 5. ---Cross sectioni of rat palate after 4 hours of digestion (orig mag 500 X).
The resuilts of the PTH experiments indicate that the PTH decreases protein syinthesis in the connective tissue cell population as can be seen in Figure 9. Proline incorporation over the four-hour incubation is decreased by 20 pM protein bound hydroxyproline per hiour per 106 cells. Total incorporatin of 4C proline at the en-d of the fouir-hour incubation is decreased by 28%. As a further control, and as could be expected, no effect of PTII is seen on the epithelial cell popuilation (Fig 9) subjected to preincubationl xvitlh PTH. Combining the PTH data for proline and hydroxyprolinie the epithelial cells still produice 43% collagen and 57cr-$S noncollagen protein. Data for the connective tissue cell population show that these cells nie making protein 27% of which is collageni aind 73%0 of wxhich is noncollagen protein. L-C -D-VALINF rETABOI SIS. -Gilbert and Migion"O developed a inuitrienlt media that provides for normal groxwtlh in culture of epithelial cells xvhile selectively inihibiting fibroblast proliferation. In this mediumrll D-valine is substituted for L-valine and onily the epithelial cells having a D-amino acid oxidase can utilize the * Beckman Instruments Inc., Fullerton, Ca.
D-valine. D-Amino acid oxidase catalyzes the oxidative deamination of D-valine to 2-ketoisovaleric acid. Although the cell does inot possess a specific transport mechanism for D-amiino acids, D-valine is free to enter by passive diffusion. D-Valine, once inside the cell and deamiiiated to 2-ketoisovaleric acid can then be fuirther metabolized. l)ansylation of '4C-D-valine will give danxx lcated 14C-D-valine (1 -dimethylaiiminonaph'C-D-valine ) Which is solnIchalene1 5 SUlfoux 1? 1)e in toluene and will couint in the ahseIIce of a soluibilizer. 2-Ketoisovaleric acid or any futrher metabolic proclucts, lacking an- aml-ino group, wxill not react with dansyl chloricle and hence are n-ot soluble in toluene but can be solubilized by the addition of BBS-3.* Thus, the pernent of Dx-valinie remaining in the buiffer is ecIlIal to the cOulnts per minute xvithout BBS3 dixvided by the counts per minute pltus BBS-3. Figure I 0 shoxxNrs that connective tissue cells do not imetabolize D-valine, whereas epithelial cells shoxv a continuotuis breakdowxn of D-xaline through a six-houir incubation. Discussion The resuilts presented in this paper indi-
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j Dent Res January 1978
TERRANO VA & BRAND
K) FIG 6. Uptake of proline by epithelial and connective tissue cells. Each point represents the mean with its upper and lower limits; n -10.
K-Q
HOURS cate that by sequential digestion of keraterized oral tissue two morphologically and biochemically distinct populations of cells can be obtained. In the studies wxith D-valine, we have developed a method for a quantitative assay of the metabolism of any labeled amino acid with the exception of the basic amino acids which have in addition to the alpha amino group, a nitrogen which will react with dansyl chloride. Using this technique, we have demonstrated that rat oral epithelial cells contain a D-amino acid oxidase and thus provided additional evidence that we have two distinct populations of cells. This evidence strongly supports what was observed histologically, namely, a sequential digestion in which the connective tis-
cells are dispersed during the first two hours and the epithelial cells during the last two hours. Further biochemical evidence supports the observation that we have isolated distinct cell populations. The distribution ratios for both populations demonstrate that the isolated cells are able to accumulate proline against a concentration gradient. This requires that the plasma membrane is intact and is capable of active transport. The amount of free proline accumulated by the epithelial cells at steady state (1.7 nAI/106 cells) is slightly higher than that found for connective tissue cells (1.40 nM/106 cells). However, from ICF measurements the epithelial cells have nearly doubled the volume of connective tissue cells which are
sue
FIG 7.
Distribution ratio-
nM Free Proline/,al Cell H20
Extracellular Proline Concentration over a 4-hour incubation of epithelial and connective tissue cells. Each point represents the mean with upper and lower limits determined from upper and lower limits of ICF measurements; n-10.
2
HOURS
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ISOLATED RAT PALATAL CELLS
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1000 In
FIG 8. Incorporation of proline into protein by 106 epithelial and connective tissue cells. Points are the mean with upper and lower limits of 10 experiments.
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114
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300
200
A
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0600 - CONNECTIVE T
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FIG 9.-Effect of PTH on connective tissue cells and epithelial cells as determined by pM proline incorporated per 106 cells and pM of hydroxylated protein bound proline per 106 cells. Results are from thin layer chromatography resolution of dansylated amino acids. Points represent the mean ± 1 SEM of 5 experiments.
400
N
300
X
2001
HOURS Downloaded from jdr.sagepub.com at The University of Melbourne Libraries on June 5, 2016 For personal use only. No other uses without permission.
126
TERRANOVA & BRAND
f Dent Res January 1978
100 FIG 10. Metabolism of Dvaline by epithelial cells over a 6-hour incubation; % D-valine remaining in the media is an indication of metabolism of the D-amino acid by epithelial cells. Points are the mean + 1 SEM, of 4 experiments.
60
20
HOURS accumulating proline to a higher intracellular concentration. One might expect that the connective tissue cell population would incorporate more proline into protein than the epithelial cells. On histological ground, this should also be expected in that the cells of the connective tissue are embedded in a matrix of collagen fibers. There is also a wealth of literature concerning collagen biosynthesis in the fibroblast.'1 From the results obtained this was not the case. Total proline incorporated into protein per 106 cells was found to be about the same in epithelial cells (860 pM) as in connective tissue cells (820 pM). The observation becomes more apparent if one examines the amount of hydroxvlation of protein bound proline. Epithelial cells hydroxylate protein bound proline at a 30% higher rate than do connective tissue cells. The amounts of proline and hydroxyproline incorporated converted to percent collagen synthesized indicates that the epithelial cells, over a four hour incubation, have synthesized 60% more collagen than the connective tissue cells. From studies presently underway it appears that the basal cell layer of the epithelium is the most metabolically active. Grant et al.'2 have shown that the collagen component of basement membranes contains higher quantities of hydroxyproline and hydroxylysine than other interstitial collagen. This leads one to speculate that most of this collagen is synthesized by the basal layer epithelial cells and is basement membrane col-
lagen. Studies will be undertaken to define the type of collagen produced by each cell type. Smith and Johnson14 have shown that periosteal cells from rat skeletal tissue respond to PTH. Vaes1 found that in a bone tissue culture system, bone cells respond to PTH whereas cultured fibroblasts show no effect to added hormone. Peck et al.16"17 demonstrated that primary cultures of isolated bone cells are most responsive to PTH. The effect of hormone diminished with repeated subculturing. The results of the PTH experiments indicate that this hormone decreases protein synthesis in the connective tissue cell population. PTH decreases the rate of proline hydroxylation by 30% and decreases the rate of proline incorporated by 22%. Thus, what we are observing is equivalent to a 30% decrease in collagen synthesis. This apparent decrease in collagen synthesis could be due in part to a PTH induced inhibition of prolyl hydroxylase.1' The effect is not strictly limited to inhibiting collagen synthesis or prolyl hydroxylase since PTH causes a comparable decrease in synthesis of both collagen and noncollagen protein by connective tissue cells. Since it has previously been shown that PTH has no effect on fibroblasts the effect observed must be on cells of osseous or preosseous origin, probably periosteal cells. From histological observations, these are the cells that are liberated during the first hour of incubation with enzyme. It is of interest that the most metabolically active population of epithelial cells is
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ISOLATED RAT PALATAL CELLS
the basal layer and apparently the most metabolically active segment of the connective tissue is the periosteal cell layer. As a further control to demonstrate PTH effect, epithelial cells subjected to preincubation with and without PTH showed no response to hormone with respect to incorporation of proline into protein and hydroxylation of protein bound proline. Further experiments are planned to separate these two populations into subpopulations by the use of ficoll gradients. Thus, the inhomogeneity of the connective tissue cell populations could be resolved and the epithelial cell population could be examined for connective tissue cell contaminates.
Conclusions It was demonstrated that sequential isolation techniques yielded separate populations of cells from the epithelium and underlying connective tissue of rat palate. These two populations exhibit distinct biochemical differences. The correlation observed between the morphology and the biochemical evidence suggests that the technique of isolation and identification, described here for cells from the rat palate, can be applied to biochemical study of specific cell populations from normal and diseased oral tissues. The authors wish to thank Dr. H. A. Zander, Department of Periodontology, Eastman Dental Center, Rochester, NY, for his help in preparing the histological sections.
References 1. MELCHER, A.H., and BOWEN, W.H. (eds.): Biology of the Periodontium, Academic
Press, N,Y., 1969. 2. BERRY, M.N., and FRIEND, D.S.: High-Yield Preparation of Isolated Rat Liver Parenchymal Cells. J Cell Bio 43:506-520, 1969. 3. KIMMICH, G.A.: Preparation and Properties of Mucosal Epithelial Cells Isolated from Small Intestine of the Chicken, Biochem 9: 3659-3668, 1970. 4. RODBELL, M.: Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis, I Biol Chem 293: 375-380, 1964. 5. WILSON, J.K.V.; LUBEROFF, D.E.; PITTS, A.; and PRETLOw, T.G.: A Method for the Separation of Lymphocytes and Plasma Cells from the Human Palatine Tonsil Using Sedirmentation in an Isokinetic Gradient of Ficoll in Tissue Culture Medium, Immunology 28: 161-170, 1975. 6. DZIAK, R., and BRAND, J.S.: Calcium Transport in Isolated Bone Cells. I. Bone Cell Iso-
127
lation Procedure, J Cell Physiol 84:75-84, 1974. 7. WIEBKIN, O.W., and MUIR, H.: The Inhibition of Sulphate Incorporation in Isolated Adult Chondrocytes by Hyaluronic Acid, FEBS Letters 37:42-46, 1973. 8. TOENNIER, G., and FENG, F.: Measurement and Characterization of Protein by Color Reactions, Ann Biochem 11:411-417, 1965. 9. MORSE, D., and HORECKER, B.L.: ThinLayer Chromatographic Separation of DNSAmino Acids, Anal Biochem 14:429-433, 1966. 10. GILBERT, S.F., and MIGION, B.R.: D-Valine as a Selective Agent for Normal Human and Rodent Epithelial Cells in Culture, Cell 5: 11-17, 1975. 1 1. TRAUB, W., and PIEZ, K.: The Chemistry and Structure of Collagen, Adv Prot Chem25:243-352, 1971. 12. GRANT, M.E.; KEFALIDES, N.A.; and PROCKop, D.J.: The Biosynthesis of Basement Membrane Collagen in Embryonic Chick Lens. I. Delays Between the Synthesis of Polypeptide Chains and the Secretion of Collagen by Matrix-Free Cells, J Biol Chem 247:3539-3544, 1972. 13. GRANT, M.E.; KELAFIDES, N.A.; and PROCKop, D.J.: The Biosynthesis of Basement Membrane Collagen in Embryonic Chick, Lens. II. Synthesis of a Precursor Form by Matrix-Free Cells and a Time-Dependent! Conversion to a Chains in Intact Lens, J Biol Chem 247:3545-3551, 1972. 14. SMITH, D.M., and JOHNSTON, C.C.: Cyclic 3',5'-Adenosine Monophosphate Levels in Separated Bone Cells, Endocrin 96:12611269, 1975. 15. VAES, G.: The Role of Lysosomes and of Their Enzymes in the Development of Bone Resorption Induced by Parathyroid Hormone. Parathyroid Hormone and Thyrocalcitonin, TALMAGE, R.V. and BELANGER, L.F. (eds) Excerpta Medica Foundation, N.Y., 1968. 16. PECK, W.A.; BIRGE, S.J.; and FEDAK, S.A.: Bone Cells: Biochemical and Biological Studies after Enzymatic Isolation, Science 146:1476-1477, 1964. 17. PECK, W.A.; CARPENTER, J.; MESSINGER, K.; and DEBRA, D.: Cyclic 3'5' Adenosine Monophosphate in Isolated Bone Cells: Response to Low Concentrations of Parathyroid Hormone, Endocrinology 92:692-697, 1976. 18. LUBEN, R.A.; WONG, G.L.; and COHN, D.V.: Biochemical Characterization with Parathormone and Calcitonin of Isolated Bone Cells: Provisional Identification of Osteoclasts and Osteoblasts, Endocrinology 99:
526-534, 1976.
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Epithelial and connective tissue cells were isolated from rat palate by sequential enzymatic digestion. Differences between the two populations were noted with respect to proline uptake and incorporation, % collagen synthesized, effects of parathyroid hormone and metabolism of D-valine. From these studies it can be concluded that the cell populations are viable and distinct with respect to the biochemical parameters examined. j Dent Res 57 (1):118-127 January 1978. The study of periodontal tissue at the biochemical level can be significantly advanced by isolation and subsequent study of the separate populations of cells that make up this tissue. There is much speculation as to which specific cell types are involved in periodontal disease. Characterization of the cells derived from normal tissue will provide the baseline for determination of the metabolic or biochemical aberrations which develop in periodontal disease. The periodontal unit-epithelium, connective tissue, and underlying alveolar bone has been studied extensively by histological methods.1 Biochemical differences between fibroblasts and epithelial cells have been examined predominantly in tissue culture. It is now possible to further advance the studv of the periodontium by isolation of the separate population of cells which make up this tissue. Viable cells, both epithelial and those of mesenchymal origin, can now be dispersed from many mammalian tissues. The isolation technique which has proven to be most effective is based on the enzymatic digestion of the extracellular matrix. This type of procedure has been applied to the preparation of isolated Received for publication January 19, 1977. Accepted for publication May 18, 1977. This work is supported in part by U.S.P.H.S. Training Grant 5-TOl-DE-00175 and NIH Career Development Award No. lKO4AM00101-01 to J. S. Brand and, in part, by U.S. E.R.D.A. Contract EY-76-C-023490 at the University of Rochester and assigned Report No. UR-3490-1009. * Worthington Biochemical Corp., Freehold, NJ. Sigma Chem. Co., St. Louis, Mo. M Gibco, Grand Island Biological Co., Grand Island, N.Y.
118
hepatocytes,2 intestinal epithelial cells,3 adipocytes,4 and lymphocytes,5 as well as cells from bone,6 cartilage,7 and other sources. The present study was undertaken to isolate separate populations of viable connective tissue and epithelial cells from normal oral tissue and to begin a biochemical characterization of these populations of cells. The information obtained using normal tissue will provide a baseline for quantifying changes in cell subpopulations and determining the nature of the metabolic aberrations which develop in periodontal disease. Materials and Methods HISTOLOGY.- Freshly excised palatal tissue (control) and palatal tissue removed at one hour intervals during a four hour incubation (test) were fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, sectioned (8 microns thick) and stained with hematoxylin and eosin. CELL ISOLATION. The cell isolation technique is a modification of the method described by Dziak and Brand6 for bone cells. Palatal tissue, freshly excised from decapitated young adult female rats was incubated at 37 C in a Hepes buffered isotonic salt solution, pH 7.4, containing crude collagenase* 3 mg/ml, crude hyaluronidase* 3 mg/ml and 0.5 mg/ml
elastase.* The buffer was 25 mM Hepes, 10 mM NaHCOM, 100 mM NaCl, 3 mM K2HPO4, 1 mM MgSO4, 1 mM CaCl9, 60 mM mannitol, glucose 5 mg/ml, bovine serum albumin (Fraction V) t 1 mg/ml, 5 jg/ml penicillin,+ 10 ,.g/ ml Neomycin+ and 5 jug/ml streptomycin.j Thirty milligrams of tissue per 1.0 ml buffer were incubated in 50 ml polypropylene beakers in a water bath and shaken at 90 oscillations per minute. At one-hour intervals up to four hours the cells were harvested as previously described6 and tissue digestion resumed. Cell counts were made with a haemocytometer. CELL VIABILITY STUDIES. The viability of J Dent Res January 1978
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ISOLATED RAT PALATAL CELLS
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119
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the cells isolated by the above technlique wvas evaluated routinely using trypan bluie dye excltusion. To I X 106 cells/l100 X buiffer, 100 X 1S trypan l)lue was added. Cell counts for inclusion of dye were imiade 15 miinutes later. Intracellular spate was mcasuired by a double label procedure; 50 X 10f isolated cells were incubated at 37 C for 5 minutes in 2.0 ml of buffer containing 11mnM 14C polyethylene glycol (PIEG) * (0.5 ACi/ml) and 3H-F1 10j (0.3 gCi/ml). The cells were then sedimented for three imiinuites at 800 x .J after whiich 100 X of the supernatant wxas couinted in 10-ml scintillation cocktail (9.67 ml Toluene-Omnifluor+ + 0.33 ml BBS-3) .§ Tle suipernatant was coompletely removed with a micropipet and the side of the tule dried with absorbent paper. The pellet was thein re uspended in 1-ml fresh bufter, equilibrated for 5 mirnuites at 37 C, and centriftuged as before; 100 X of the supernatant was then counted. Total 14O was determined from tlhc CPM tritium and extracel* Amersham/Searle Corp., Arlington Heights, I1. t Mallinckrodt, St. Louis, Mo. t New England Nuclear, Boston, Ma. § Beckman Instruments Inc., Fullerton, Ca.
lul.ar fliid (ECI) was determiined from- the 14C PEG. Intracelluilar fluid JICF) per 106 tells wxas obtairned by subtracting ECF from total 1-.0 and dividing by 50. For an additional measurement of total H.,O xvet wxeights of cell saniples were determined by sedimiienting the cells at 800 X g for 3 minutes in a tarecd conical centrifuge tube. The supernatant xxas carefLully decanted off and the sides of the tuibe dried. Tuibes wxere then dried in a 37 C oven and weighed. This procecdure wx as repeated uLntil a constant weight was obtained. Total H.,O equals wet weight minus dry weight. Total cell protein w,as measur ed by the Toennier anld Fe'ng mnethod.8 Proline uptake was deternmined bv incubating 2 X 106 isolated cells in 1.0 nml buffer wvith 0.2 mnM 1IC proline (0.1 gc/nil)* for x ariotis titre poinits up to fouir hoiirs. The cells anid inCLIbation imiediumiii were then carefully transfer-red into smiiall silic oniized glass test tuLbs arid centriftuged at 1,500 X g for 2 minu tes. TIe supernatant wx as rermoved and the resuItincg cell pellet washed with 1.0-nil cold bufTer anid recentrifuged. After discardinig the
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TERRANOVA & BRAND
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supernatant the pellet xx as sLuspended in. 200 X distilled 110 and placed in an oil bath at 100 C for 10 nminuites to lyse the cells; 50 X of 25% tricholoroacetic acid (TCA) were then added and the samiiple centrifuged at 10,000 X g for 5 nminuites; 100 X of the resultinig suiperniatant nivle tliein counted in 9.67 Ixiv TolueneOninifluori anid 0.33 nil BBS-3.§ (This is free '4C-proline.) To the resultinlg cell pellet, 200 X of g% TCA were added and the tube vortexed vigorouslv. After 10 imlitlties, thie mixture is spuni at 10,000 X g for 5 minutes and the supernatant discarded. To the pellet xas added 0.5 nml 6 N I-ICl. This reaction mixture xas hydrolyzed in glass tubes sealed xxith teflon-coated rubber-lined caps for 16 hours at 110 C and then- dried uinder vacuuim to remove the HCI. The hydrolysate was then dissolved in 25 X distilled H.O to which 50 X 0.1M NaHC03 anid 150 X dansyl chloride 09 mg dansyl chloride per ml acetone) xx ere added. The reat tion was stopped after 10 minuites by the addition of 25 X 0.4 M atetic acid; 25 X of the reactionmixtuLre xx-as spotted oni 500 miicron thlick silica gel plates anid developed in a ben-
1978
zene, pyridine, acethc acid bu-ffer (80 nil: 20 nil:2 ml).9 Standards of proline anad hydroxyproline (10 mIIM 14C-proline and 10 mM N1-11yvdroxxprol ine) were ruiii simultaneously and corresponddiny areas on the thin layer plates sctraped and couinted in scinitillation fluid to deteriijine (1) the aniount of prolinc incorporated into protein and 29) the amoun1t of proteini bound proliine hydroxxlated. To calculate per ccnt recoverx, 25 X of the danisylation Tmixttiire xN-ere spotted at thc top of the plate and subsequiently scraped anid counted. Thus total proline incorporated equals proline incorporated plu-s hb-droxylated protein bound proline corrected to 100%c recoverv. To stLudy the effectt of PTH on- proline ulptake and incorporation into protein, prior to isolationi the tissue xas preincubated xxith PTH at a concentration of 1 unit/nil buffer (xxithout enzyme) for onc half hoLur; cointrols had no PTH. Eniyines xxere then added and the cells x ere harvested fronii the first and last txxo houirs of digcstilon. Cell proteini hxydrolx sates xxvere again assayyed for con-tenit of labeled proline and hydroxyproline.
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FiG 2.- Cross section of rat palate after 1 hour of incubation in buLffer containing enzymes (orig inag 200 X ). Downloaded from jdr.sagepub.com at The University of Melbourne Libraries on June 5, 2016 For personal use only. No other uses without permission.
ISOLATED RAT PALATAL CELLS
Vol, 57 No. I
FTm 3. Cross section of
rat
121
palate after 1.5 hours of diges-
tioil (orig marg 500 X )
Itx orcler to IntoIIS\[. 14CD V\IINEF ' inisure no epithelial cell containination in the connective tissue cell population for these experiinents, about three fouirths of the conniective tissue ccll laver w-as dissected awax fromll the palates befoie incubatioin x-ith enzyne. The reIainiaing tissue w\cas then trcateci as described al)ove, discarding the cells fromii the first 2 honii s of dig£:(estion. 'Thus, two preparations of (-(,lls were obtained; coninectixte tissue inculbated for 2 hiours and epithelitni inlclllatedl for fouir houLrs wxith the first 2-hour cells reiovcd. Whole palates which xx ere not dissected xw ere also used in separate experiments. Connective tiSSue cells anld epithelial cells xw re inc uhated at 37 C ,at a concentration of 4 bdhuffer xwith 1 .0 Ci (arricr X 10" cells pernl free "1C-D-valine (specific activitx 09.8 mCi/ inV!.* At ton-lhour intcrx als, 1 .0 mlI of cell sulspenlsion Nx-xas renioxcld ft o-ni the inc ubator, placed in glass test tuhes onl ice, auid iimiediately precipitated with 0.5 nil 25% ice cold TCA. The test tubes wxx rc then cectr ifi-ed at 1.5.000 X for 10 minuites; 25 N of the siliper* Calbiochem., Los Angeles, Ca.
inttant ere 1eactecd xwith dansxl chloride as de-
sitribecl abovxe anid 50 N samiples of tlhis reaction iiiixtLrre were counted, wvith an-d wsithostt a solubJilizer acddecl, in a liquiid scintillationi c ouinter. Results
dealIIIsTros o(-. IHistt-)dogicallv xx can onstrate a sequential rnioxoval of cclls frhost the rat palate. The connective tissue la1 er closest to the palatal bone is the first area frotio xhich cells aire liberated. Firiure I shoxxws a c ross section through normal rat palate before inctnbation with eavnines. In I,igrtic 2, after onie h-ouir of inlculbatioIl xNith cnzixiiues, thec conncctix c tissue layer closest to the palatal botie apperns rcntaAec of c ells atC dlilooser and a simiall pen persed (approxititately 100,, of the total cell vield). Fi-iure 3 Shosxx s a cross section of rat palate after 1.5 hostrs of incubation xxith enznie. Fifty percent of the connec tive tiSsLe laxver has been dig-estcd. After two hours of digestion, 00%- to 100% of the connectixe tissue ell lax7er has been removed (Fia 4). Dutring the Incxt txx cO Iours of incubationi, the epithelial
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122
TERRANOVA & BRAND
J Dent Res januarj, 1978
cells are liberated and all that remains of the palate is the cornified epithelial cell layer which cloes inot digest further (Fig 5). This proc educe yielded approximiiately 9 X 106 cells per palate, 40% of xwhich xwas connective tissue ( ells and 60%. ep)Lthelial c ells. MlF\%LSUREMtENTS.--In-
ICE AND PROTF.IN
tracellular fluid space (ICF) measuriemients for 106 connective tissute cells gave 0.23 ± 0.04 lul. Epithelial cells hlad an ICF of 0.53 ± 0.10Iil/ 103 cells. Total cell piotein measurements yielded 170 9- 10 gg protein per 106 connective tissuie c els and 235 15 ug protein per 106 cpithelial cells. CELL VIIAIi'rY. -Routinely 90 to 95% of botlh epithelial and connlective tissue cell populationis excluded trvpan- blue dy e. The ability of the cells to actively accumulate proline cas also used as a criterion of their viability. Proline uptake per 106 cells was calculated for cells incubated with 0.2 MM 14C-proline for various tiine points. In Figure 6, epithelial cells shmocsN a slightly higher uptake of free proline over connective tissuie cells. However, due to the dlifference in ICF space nmeasuirements the
epithelial cells have a losser distribution ratio than do the connective tissue cells (Fig 7). Both epithelial and connective tissue cells for all time points do show distribution ratios well over 1.0 and thus are actively accumulating proline. Control data for total incorporation of 14C-proline (Fig 8) shoxx that during the four houir incubation period both the epithelial cell population anid connective tissue cell population incorporate proline into protein at approximately the same rate after 30 minutes. Data for liydroxylatioui of piotein bound proline (i.e., hydroxyproline) (Fig 9), showv that epithelial cells hxdroxvlate 20 pAl/hr/106 cells imore prolinie than do connective tissue cells. Comiibining the control data for proline and hvdroxvproline, the per cent of protein miade by the cell that is collageni, over the fourhour incubation, is 26% for connective tissue cells and 43%SJ for epithelial cells, assuming an armino atici content of 20 mole %- for collagen aind an axveragSe prolinie conrtent of 5 miiole % for
ionco(llagen protein.
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it all .--
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FTG 4. G---ross section of rqt palate after 2 hours of digestion
( ori g i liag 5 00 X ). Downloaded from jdr.sagepub.com at The University of Melbourne Libraries on June 5, 2016 For personal use only. No other uses without permission.
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ISOLATED RAT PALATAL CELLS
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FIG 5. ---Cross sectioni of rat palate after 4 hours of digestion (orig mag 500 X).
The resuilts of the PTH experiments indicate that the PTH decreases protein syinthesis in the connective tissue cell population as can be seen in Figure 9. Proline incorporation over the four-hour incubation is decreased by 20 pM protein bound hydroxyproline per hiour per 106 cells. Total incorporatin of 4C proline at the en-d of the fouir-hour incubation is decreased by 28%. As a further control, and as could be expected, no effect of PTII is seen on the epithelial cell popuilation (Fig 9) subjected to preincubationl xvitlh PTH. Combining the PTH data for proline and hydroxyprolinie the epithelial cells still produice 43% collagen and 57cr-$S noncollagen protein. Data for the connective tissue cell population show that these cells nie making protein 27% of which is collageni aind 73%0 of wxhich is noncollagen protein. L-C -D-VALINF rETABOI SIS. -Gilbert and Migion"O developed a inuitrienlt media that provides for normal groxwtlh in culture of epithelial cells xvhile selectively inihibiting fibroblast proliferation. In this mediumrll D-valine is substituted for L-valine and onily the epithelial cells having a D-amino acid oxidase can utilize the * Beckman Instruments Inc., Fullerton, Ca.
D-valine. D-Amino acid oxidase catalyzes the oxidative deamination of D-valine to 2-ketoisovaleric acid. Although the cell does inot possess a specific transport mechanism for D-amiino acids, D-valine is free to enter by passive diffusion. D-Valine, once inside the cell and deamiiiated to 2-ketoisovaleric acid can then be fuirther metabolized. l)ansylation of '4C-D-valine will give danxx lcated 14C-D-valine (1 -dimethylaiiminonaph'C-D-valine ) Which is solnIchalene1 5 SUlfoux 1? 1)e in toluene and will couint in the ahseIIce of a soluibilizer. 2-Ketoisovaleric acid or any futrher metabolic proclucts, lacking an- aml-ino group, wxill not react with dansyl chloricle and hence are n-ot soluble in toluene but can be solubilized by the addition of BBS-3.* Thus, the pernent of Dx-valinie remaining in the buiffer is ecIlIal to the cOulnts per minute xvithout BBS3 dixvided by the counts per minute pltus BBS-3. Figure I 0 shoxxNrs that connective tissue cells do not imetabolize D-valine, whereas epithelial cells shoxv a continuotuis breakdowxn of D-xaline through a six-houir incubation. Discussion The resuilts presented in this paper indi-
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j Dent Res January 1978
TERRANO VA & BRAND
K) FIG 6. Uptake of proline by epithelial and connective tissue cells. Each point represents the mean with its upper and lower limits; n -10.
K-Q
HOURS cate that by sequential digestion of keraterized oral tissue two morphologically and biochemically distinct populations of cells can be obtained. In the studies wxith D-valine, we have developed a method for a quantitative assay of the metabolism of any labeled amino acid with the exception of the basic amino acids which have in addition to the alpha amino group, a nitrogen which will react with dansyl chloride. Using this technique, we have demonstrated that rat oral epithelial cells contain a D-amino acid oxidase and thus provided additional evidence that we have two distinct populations of cells. This evidence strongly supports what was observed histologically, namely, a sequential digestion in which the connective tis-
cells are dispersed during the first two hours and the epithelial cells during the last two hours. Further biochemical evidence supports the observation that we have isolated distinct cell populations. The distribution ratios for both populations demonstrate that the isolated cells are able to accumulate proline against a concentration gradient. This requires that the plasma membrane is intact and is capable of active transport. The amount of free proline accumulated by the epithelial cells at steady state (1.7 nAI/106 cells) is slightly higher than that found for connective tissue cells (1.40 nM/106 cells). However, from ICF measurements the epithelial cells have nearly doubled the volume of connective tissue cells which are
sue
FIG 7.
Distribution ratio-
nM Free Proline/,al Cell H20
Extracellular Proline Concentration over a 4-hour incubation of epithelial and connective tissue cells. Each point represents the mean with upper and lower limits determined from upper and lower limits of ICF measurements; n-10.
2
HOURS
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ISOLATED RAT PALATAL CELLS
125
1000 In
FIG 8. Incorporation of proline into protein by 106 epithelial and connective tissue cells. Points are the mean with upper and lower limits of 10 experiments.
c4
K)
0 0.25 0.5
1.0
2.0 HOURS
3.0
4.0
600- EPITHELIUM 500 400 -
114
0,0 CONTROL
~~~A,A =PT H
300
200
A
100
0600 - CONNECTIVE T
500 kr4
0,0o- CONTROL A,A PTH
FIG 9.-Effect of PTH on connective tissue cells and epithelial cells as determined by pM proline incorporated per 106 cells and pM of hydroxylated protein bound proline per 106 cells. Results are from thin layer chromatography resolution of dansylated amino acids. Points represent the mean ± 1 SEM of 5 experiments.
400
N
300
X
2001
HOURS Downloaded from jdr.sagepub.com at The University of Melbourne Libraries on June 5, 2016 For personal use only. No other uses without permission.
126
TERRANOVA & BRAND
f Dent Res January 1978
100 FIG 10. Metabolism of Dvaline by epithelial cells over a 6-hour incubation; % D-valine remaining in the media is an indication of metabolism of the D-amino acid by epithelial cells. Points are the mean + 1 SEM, of 4 experiments.
60
20
HOURS accumulating proline to a higher intracellular concentration. One might expect that the connective tissue cell population would incorporate more proline into protein than the epithelial cells. On histological ground, this should also be expected in that the cells of the connective tissue are embedded in a matrix of collagen fibers. There is also a wealth of literature concerning collagen biosynthesis in the fibroblast.'1 From the results obtained this was not the case. Total proline incorporated into protein per 106 cells was found to be about the same in epithelial cells (860 pM) as in connective tissue cells (820 pM). The observation becomes more apparent if one examines the amount of hydroxvlation of protein bound proline. Epithelial cells hydroxylate protein bound proline at a 30% higher rate than do connective tissue cells. The amounts of proline and hydroxyproline incorporated converted to percent collagen synthesized indicates that the epithelial cells, over a four hour incubation, have synthesized 60% more collagen than the connective tissue cells. From studies presently underway it appears that the basal cell layer of the epithelium is the most metabolically active. Grant et al.'2 have shown that the collagen component of basement membranes contains higher quantities of hydroxyproline and hydroxylysine than other interstitial collagen. This leads one to speculate that most of this collagen is synthesized by the basal layer epithelial cells and is basement membrane col-
lagen. Studies will be undertaken to define the type of collagen produced by each cell type. Smith and Johnson14 have shown that periosteal cells from rat skeletal tissue respond to PTH. Vaes1 found that in a bone tissue culture system, bone cells respond to PTH whereas cultured fibroblasts show no effect to added hormone. Peck et al.16"17 demonstrated that primary cultures of isolated bone cells are most responsive to PTH. The effect of hormone diminished with repeated subculturing. The results of the PTH experiments indicate that this hormone decreases protein synthesis in the connective tissue cell population. PTH decreases the rate of proline hydroxylation by 30% and decreases the rate of proline incorporated by 22%. Thus, what we are observing is equivalent to a 30% decrease in collagen synthesis. This apparent decrease in collagen synthesis could be due in part to a PTH induced inhibition of prolyl hydroxylase.1' The effect is not strictly limited to inhibiting collagen synthesis or prolyl hydroxylase since PTH causes a comparable decrease in synthesis of both collagen and noncollagen protein by connective tissue cells. Since it has previously been shown that PTH has no effect on fibroblasts the effect observed must be on cells of osseous or preosseous origin, probably periosteal cells. From histological observations, these are the cells that are liberated during the first hour of incubation with enzyme. It is of interest that the most metabolically active population of epithelial cells is
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ISOLATED RAT PALATAL CELLS
the basal layer and apparently the most metabolically active segment of the connective tissue is the periosteal cell layer. As a further control to demonstrate PTH effect, epithelial cells subjected to preincubation with and without PTH showed no response to hormone with respect to incorporation of proline into protein and hydroxylation of protein bound proline. Further experiments are planned to separate these two populations into subpopulations by the use of ficoll gradients. Thus, the inhomogeneity of the connective tissue cell populations could be resolved and the epithelial cell population could be examined for connective tissue cell contaminates.
Conclusions It was demonstrated that sequential isolation techniques yielded separate populations of cells from the epithelium and underlying connective tissue of rat palate. These two populations exhibit distinct biochemical differences. The correlation observed between the morphology and the biochemical evidence suggests that the technique of isolation and identification, described here for cells from the rat palate, can be applied to biochemical study of specific cell populations from normal and diseased oral tissues. The authors wish to thank Dr. H. A. Zander, Department of Periodontology, Eastman Dental Center, Rochester, NY, for his help in preparing the histological sections.
References 1. MELCHER, A.H., and BOWEN, W.H. (eds.): Biology of the Periodontium, Academic
Press, N,Y., 1969. 2. BERRY, M.N., and FRIEND, D.S.: High-Yield Preparation of Isolated Rat Liver Parenchymal Cells. J Cell Bio 43:506-520, 1969. 3. KIMMICH, G.A.: Preparation and Properties of Mucosal Epithelial Cells Isolated from Small Intestine of the Chicken, Biochem 9: 3659-3668, 1970. 4. RODBELL, M.: Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis, I Biol Chem 293: 375-380, 1964. 5. WILSON, J.K.V.; LUBEROFF, D.E.; PITTS, A.; and PRETLOw, T.G.: A Method for the Separation of Lymphocytes and Plasma Cells from the Human Palatine Tonsil Using Sedirmentation in an Isokinetic Gradient of Ficoll in Tissue Culture Medium, Immunology 28: 161-170, 1975. 6. DZIAK, R., and BRAND, J.S.: Calcium Transport in Isolated Bone Cells. I. Bone Cell Iso-
127
lation Procedure, J Cell Physiol 84:75-84, 1974. 7. WIEBKIN, O.W., and MUIR, H.: The Inhibition of Sulphate Incorporation in Isolated Adult Chondrocytes by Hyaluronic Acid, FEBS Letters 37:42-46, 1973. 8. TOENNIER, G., and FENG, F.: Measurement and Characterization of Protein by Color Reactions, Ann Biochem 11:411-417, 1965. 9. MORSE, D., and HORECKER, B.L.: ThinLayer Chromatographic Separation of DNSAmino Acids, Anal Biochem 14:429-433, 1966. 10. GILBERT, S.F., and MIGION, B.R.: D-Valine as a Selective Agent for Normal Human and Rodent Epithelial Cells in Culture, Cell 5: 11-17, 1975. 1 1. TRAUB, W., and PIEZ, K.: The Chemistry and Structure of Collagen, Adv Prot Chem25:243-352, 1971. 12. GRANT, M.E.; KEFALIDES, N.A.; and PROCKop, D.J.: The Biosynthesis of Basement Membrane Collagen in Embryonic Chick Lens. I. Delays Between the Synthesis of Polypeptide Chains and the Secretion of Collagen by Matrix-Free Cells, J Biol Chem 247:3539-3544, 1972. 13. GRANT, M.E.; KELAFIDES, N.A.; and PROCKop, D.J.: The Biosynthesis of Basement Membrane Collagen in Embryonic Chick, Lens. II. Synthesis of a Precursor Form by Matrix-Free Cells and a Time-Dependent! Conversion to a Chains in Intact Lens, J Biol Chem 247:3545-3551, 1972. 14. SMITH, D.M., and JOHNSTON, C.C.: Cyclic 3',5'-Adenosine Monophosphate Levels in Separated Bone Cells, Endocrin 96:12611269, 1975. 15. VAES, G.: The Role of Lysosomes and of Their Enzymes in the Development of Bone Resorption Induced by Parathyroid Hormone. Parathyroid Hormone and Thyrocalcitonin, TALMAGE, R.V. and BELANGER, L.F. (eds) Excerpta Medica Foundation, N.Y., 1968. 16. PECK, W.A.; BIRGE, S.J.; and FEDAK, S.A.: Bone Cells: Biochemical and Biological Studies after Enzymatic Isolation, Science 146:1476-1477, 1964. 17. PECK, W.A.; CARPENTER, J.; MESSINGER, K.; and DEBRA, D.: Cyclic 3'5' Adenosine Monophosphate in Isolated Bone Cells: Response to Low Concentrations of Parathyroid Hormone, Endocrinology 92:692-697, 1976. 18. LUBEN, R.A.; WONG, G.L.; and COHN, D.V.: Biochemical Characterization with Parathormone and Calcitonin of Isolated Bone Cells: Provisional Identification of Osteoclasts and Osteoblasts, Endocrinology 99:
526-534, 1976.
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