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Increases in acid proteinase activity during epidermal wound healing
JOURNAL
OF SURGICAL
RESEARCH
35, 159- 162 ( 1983)
Increases in Acid Proteinase Activity during Epidermal Wound Healing’ J. IM, PH.D., AND JOHN E. HOOPES, M.D.
MICHAEL
Division qf Plustic Surgery, Department of Surgery, The Johns Hopkins University School qf Medicine, Baltimore, Maryland 21205 Submitted for publication
August 27, 1982
Incised wounds were made on the backs of young guinea pigs at 0, 1, 2, 3, 4, and 10 day intervals. Hemoglobin-hydrolizing acid proteinase activity was measured fluorometrically under optimal assay conditions by means of microanalytical methods. Acid proteinase activity was increased two to three times normal during epithelial migration and maturation during wound healing: no gradient of enzyme activity was found from the wound margin to the tip of the migrating epithelium. The results suggest that acid proteinase promotes epithelial migration by proteolytic action on serum protein and fibrin in wounds. It is also suggested that acid proteinase is required during epithelial maturation and differentiation.
INTRODUCTION
Epidermal cells are capable of phagocytosis and digestion of exogenous material using the lysosomal system [ 11, 141. Morphological studies have demonstrated that migrating epithelium ingests and digests strands of serum protein and fibrin lying in its path [ 121. The presence of lysosomes in the migrating epithelium has been revealed by ultrastructural study [lo] and by biochemical assays of acid phosphatase [5] and Bglycosidases [2, 31. In the present study, a quantitative assay of acid proteinase was performed in migrating and maturing (differentiating) epithelium during wound healing, since cathepsin D plays a major role in intracellular and extracellular protein catabolism in the epidermis [7]. The relative activities of acid proteinase, acid phosphatase, and @-glucuronidase were correlated with the different phases of epithelization. MATERIALS
AND
METHODS
Incised wounds and tissue preparation. A series of wounds were made on the backs of 6-week-old male guinea pigs under ether anesthesia. Three linear incisions, 1 cm in length, were created randomly on each animal at different time intervals. The wounds were exI This work was supported by NIH Research Grant AG02373.
posed to the air and were not treated in any fashion. All of the wounds and the unwounded control site in a given animal was biopsied at one time under ether anesthesia so as to obtain wound samples at 1, 2, 3, 4, 7, and 10 days postwounding. The skin specimens, 1 X 1 cm, were frozen immediately in liquid nitrogen, cross sectioned 20 pm in thickness perpendicular to the long axis of the incision in a cryostat (-3O”C), and lyophilized. The freezedried tissue sections were stored in vacuum tubes at -20°C until the enzyme assay. Unstained, freeze-dried sections demonstrated the histological integrity of the skin in terms of the original wound margin and the regenerating epithelium. The marginal epithelium at the original incision and the regenerating epithelium were isolated by microdissection [4]. The marginal epithelium was obtained within 0.5 mm of the incision. The entire length of the migrating and regenerating epithelium in a tissue section was utilized for the enzyme assays. Isolated epithelial segments were weighed on a quartz fiber microbalance [9] and transferred into micro test tubes (2.5 X 30 mm) for subsequent enzyme assays. Acid proteinase assay. Acid proteinase activity was assayed by a fluorometric method utilizing fluorescamine [l]. The assay procedure was based on the fact that peptides yield maximal fluorescence with fluorescamine at pH 7.0, whereas amino acids fluoresce more 159
0022-4804183 $1 SO Copyright All
rights
0
1983
by Academc
of rwrcduction
Press, Inc.
in any form
reserved.
160
JOURNAL
OF SURGICAL
RESEARCH:
at pH 9.0 [ 131.Optimal assay conditions for acid proteinase were determined utilizing normal epidermal segmentsfrom guinea pigs. The assayreagent consisted of 0.05 M acetate buffer (pH 3.8) containing 1% of bovine hemoglobin. Linearity of enzyme activity was obtained for up to 5 pg of freeze-dried normal epidermis. The fluorescence reading was proportional to the incubation time for at least 3 hr at 37°C. The optimal pH in acetatebuffer was 3.8 (64% of maximal activity was detected at pH 3.2, and 44% at pH 4.5 and 5.0). Maximal enzyme activity was obtained at 1% of protein substrate; increased hemoglobin concentrations resulted in a substrate inhibition (a 50% inhibition at 4% of substrate). Microdissected epithelium (0.5- 1.5 pg) was incubated in 5 ~1 of the reagent mixture for 3 hr at 37°C. Standard tubes containing l-2 nmole of L-leucyl-L-alanine was carried throughout the procedure. After incubation, the reaction was stopped by adding 5 ~1 of 10%trichloroacetic acid to eachtest tube. After centrifugation for 10 min at 5OOOg,7 ~1 of the supernatant was added to 0.9 ml of 0.05 M phosphate buffer, pH 7.0. One hundred microliters of fluorescamine solution (7 mg of fluorescamine (Hoffmann LaRoche, Nutley, N. J.) dissolved in 25 ml of acetone) was then added to the supematant and buffer in each
VOL. 35, NO. 2, AUGUST
1983
test tube with immediate mixing. The fluorescence was read in a Farrand fluorometer equipped with Coming glass No. 5970 as the primary filter (390 nm) and Turner filter No. 1lo-828 as the secondary filter (475 nm). RESULTS
The average activity of hemoglobin-hydrolyzing acid proteinase was 95 f 7 (SEM, 12= 10) mmole/hr/kg dry weight in normal epidermis. Alterations in acid proteinase activity during wound healing are summarized in Table 1 and Fig. 1. Additional data on acid phosphatase and &glucuronidase activities, reported previously, are included for a comparative study [2, 51. The marginal epithelium demonstrated a twofold increase in acid proteinase activity on Day 2 and 3.5 times normal during Days 310 postwounding. The regenerating epithelium exhibited a similar response to that of the marginal epithelium. Acid proteinase activity was assayedin the proximal and distal halves of the migrating epithelium (Day 3). No distribution gradient of acid proteinase activity was detected within the regenerating ep ithelium; enzyme activity in the proximal and distal half was 226 + 27 (n = 3) and 257 * 42 (n = 3) mmole/hr/kg dry weight, re-
TABLE
1
ACID HYDROLASE ACXVITIES IN MARGINAL AND REGENERATING EPITHELIUM DURING WOUND HEALING Acid proteinase”
Acid phosphataw’,E
j3-Glucuronidase*’
Days after WOUIlding
0 I 2 3 4 7 10
Marginal 7 (lO)d ND 194 f 21’ (3) 315 + 29’ (4) 336 f 14’ (4) 337 f 31’ (4) 343 (2)
Regenerating
95 f
ND 212 + 17’ (5) 221 k 32’ (7) 245 k 24’ (7) 292 k 44f(7) 274 + 35’ (5)
Marginal 0.80 k 1.17 f 1.38 + 2.78 + 2.83 + 2.03 + 1.33
0.12 0.14 0.40 0.21’ 0.27’ 0.27’
Regenerating (10) (4) (6) (7) (8) (8)
(2)
0.41 f 0.08’ 0.73 + 0.16 1.29 + 0.29 1.63 1?:0.29’ 1.87 + 0.40’ 1.41
(4) (7) (7) (8) (7)
(2)
Marginal 13.5 k 9.6 f 9.6 k 12.1 f 14.6 k 15.1 + 16.6
1.5 (8) 2.4 (3) 2.4 (3) 2.1 (6) 2.6 (6) 2.1 (5)
Regenerating
31.4 * 33.2 + 30.4 f 17.3 + 19.3 +
14.5 6.5’ 5.7’ 3.0 2.5
(3) (7) (7) (7) (6)
’ Enzyme activity is expressedas mmoles per hr per kg dry weight. b Enzyme.activity is expressedas moles per hr per kg dry weight. ’ Data on acid phosphataseand ~-glucuronidase are-from Refs. [2, 51 and supplementary. ‘Each value representsa mean ? SEM determined from the number of difference animals (indicated in parenthesis), each assayed in quintuplicate. ND, Not determined. ‘P values for the differences between the control and experimental epithelium are ~0.05. ’ P values for the differences between the control and experimental epithelium are
IM AND HOOPES: ACID HYDROLASE IN EPIDERMAL WOUNDS
161
% NORMAL
I’ .
.’ ,’ .’
,’ ,,a---.- “...* Acid
proteinase
Acid
phosphatese
,O”
B-Glucuronidase
0
2 DAYS
4
6
AFTER
8
10
WOUNDING
FIG. 1. Alterations in acid phosphatase, acid proteinase, and &glucuronidase activity in regenerating epithelium during wound healing. 0 Cl, Acid phosphatase; 0 ----- 0, acid proteinase; 0 0, @glucuronidase.
spectively. In general, the marginal epithelium exhibited a slightly higher activity of acid proteinase than the regenerating epithelium, but the difference may not be statistically significant.
of phagocytosis in migrating epithelium [ 121, suggestthat acid proteinase promotes cell migration by means of a proteolytic action on serum protein and fibrin embedded in the wound exudate. Acid proteinase appears to be required in epithelial differentiation [8] as indicated by continued increasesin its activity DISCUSSION during the cell maturation phase of epitheliHemoglobin-hydrolyzing acid proteinase of zation. skin is primarily attributable to cathepsin D The curves representing the alterations in activity [6]. The present data on proteolytic acid phosphatase,acid proteinase, and &$,icactivity measured by the hydrolysis of he- uronidase activity reflect their different funcmoglobin at pH 3.8 represent cathepsin D ac- tions in epithelization during wound healing. tivity in epithelial tissue. Cathepsin D may &Glucuronidase is associated with the cell play a major role in intracellular and extra- proliferation and migration phaseswhile acid cellular protein catabolism [7] and phagocytic phosphatesare involved in the cell maturation activity [ 11, 141in the epidermis. Increasesin phase of epithelial wound healing. Acid proacid proteinase activity assayedin this inves- teinase plays a significant role throughout all tigation, together with morphological evidence three phasesof epithelization.
162
JOURNAL OF SURGICAL RESEARCH: VOL. 35, NO. 2, AUGUST 1983
REFERENCES 1. Hirsch, H. E., and Parks, M. E. The quantitative histochemistry of acid proteinase in the nervous system: Localization in neurons. J. Neurochem. 21: 453, 1973. 2. Im, M. J., and Hoopes, J. E. Enzyme activities in regenerating epithelium during wound healing. II. flGlucuronidase. J. Surg. Res. 12: 406, 1972. 3. Im, M. J., and Hoopes, J. E. Enzyme activities in regenerating epithelium during wound healing. III. /3-Galactosidaseand &lucosidase. J. Surg. Res. 13: 82, 1972. 4. lm, M. J., and Hoopes, J. E. Measurement of epithelial production in healing skin wounds. J. Surg. Res. 24: 52, 1978. 5. Im, M. J., and Hoopes, J. E., and Sohn, Y. T. Enzyme activities in regenerating epithelium during wound healing. I. Acid Phosphatase.J. Surg. Res. 12: 402, 1912. 6. Lazarus, G. S., and Dingle, J. T. Cathepsin D of rabbit skin: An immunoenzymic study. J. Invest. Dermatol. 62: 6 I, 1974. 7. Lazarus,G. S., and Poole, A. R. Immunocytochemical localization of cathepsin D in rabbit skin. Arch. Dermatol. 111: 1150, 1975.
8. Lazarus, G. S., Hatcher, V. B., and Levine, N. I. Lysosomesand the skin. J. Invest. Dermatol. 65: 259, 1975. Lowry, 0. H., and Passonneau,J. V. A Flexible System 9. of Enzymatic Analysis. New York Academic Press, 1972. 10. Martinez, I. R. Fine structural studies of migrating epithelial cells following incision wounds. In H. I. Maibach and D. T. Rovee (Eds.), Epidermal Wound Healing. Chicago: Year Book Medical Publishers, 1972. 11. Nordquist, R. E., Olson, R. L., and Everett, M. A. The transport, uptake and storageof ferritin in human epidermis. Arch. Dermatol. 94: 482, 1966. 12. Odland, G., and Ross, R. Human wound repair. I. Epidermal regeneration. J. Cell Biol. 39: 135, 1968. 13. Udenfiiend, S., Stein, E., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. Fluorescamine: A reagent for assay of amino acids, peptides, protein, and primary amines in the picomole range. Science 178: 871, 1972. 14. Wolff, K., and Konrad, K. Phagocytosisof latex beads of epidermal keratinocytes in vivo. J. Ultrastruct. Res. 39: 262, 1972.
OF SURGICAL
RESEARCH
35, 159- 162 ( 1983)
Increases in Acid Proteinase Activity during Epidermal Wound Healing’ J. IM, PH.D., AND JOHN E. HOOPES, M.D.
MICHAEL
Division qf Plustic Surgery, Department of Surgery, The Johns Hopkins University School qf Medicine, Baltimore, Maryland 21205 Submitted for publication
August 27, 1982
Incised wounds were made on the backs of young guinea pigs at 0, 1, 2, 3, 4, and 10 day intervals. Hemoglobin-hydrolizing acid proteinase activity was measured fluorometrically under optimal assay conditions by means of microanalytical methods. Acid proteinase activity was increased two to three times normal during epithelial migration and maturation during wound healing: no gradient of enzyme activity was found from the wound margin to the tip of the migrating epithelium. The results suggest that acid proteinase promotes epithelial migration by proteolytic action on serum protein and fibrin in wounds. It is also suggested that acid proteinase is required during epithelial maturation and differentiation.
INTRODUCTION
Epidermal cells are capable of phagocytosis and digestion of exogenous material using the lysosomal system [ 11, 141. Morphological studies have demonstrated that migrating epithelium ingests and digests strands of serum protein and fibrin lying in its path [ 121. The presence of lysosomes in the migrating epithelium has been revealed by ultrastructural study [lo] and by biochemical assays of acid phosphatase [5] and Bglycosidases [2, 31. In the present study, a quantitative assay of acid proteinase was performed in migrating and maturing (differentiating) epithelium during wound healing, since cathepsin D plays a major role in intracellular and extracellular protein catabolism in the epidermis [7]. The relative activities of acid proteinase, acid phosphatase, and @-glucuronidase were correlated with the different phases of epithelization. MATERIALS
AND
METHODS
Incised wounds and tissue preparation. A series of wounds were made on the backs of 6-week-old male guinea pigs under ether anesthesia. Three linear incisions, 1 cm in length, were created randomly on each animal at different time intervals. The wounds were exI This work was supported by NIH Research Grant AG02373.
posed to the air and were not treated in any fashion. All of the wounds and the unwounded control site in a given animal was biopsied at one time under ether anesthesia so as to obtain wound samples at 1, 2, 3, 4, 7, and 10 days postwounding. The skin specimens, 1 X 1 cm, were frozen immediately in liquid nitrogen, cross sectioned 20 pm in thickness perpendicular to the long axis of the incision in a cryostat (-3O”C), and lyophilized. The freezedried tissue sections were stored in vacuum tubes at -20°C until the enzyme assay. Unstained, freeze-dried sections demonstrated the histological integrity of the skin in terms of the original wound margin and the regenerating epithelium. The marginal epithelium at the original incision and the regenerating epithelium were isolated by microdissection [4]. The marginal epithelium was obtained within 0.5 mm of the incision. The entire length of the migrating and regenerating epithelium in a tissue section was utilized for the enzyme assays. Isolated epithelial segments were weighed on a quartz fiber microbalance [9] and transferred into micro test tubes (2.5 X 30 mm) for subsequent enzyme assays. Acid proteinase assay. Acid proteinase activity was assayed by a fluorometric method utilizing fluorescamine [l]. The assay procedure was based on the fact that peptides yield maximal fluorescence with fluorescamine at pH 7.0, whereas amino acids fluoresce more 159
0022-4804183 $1 SO Copyright All
rights
0
1983
by Academc
of rwrcduction
Press, Inc.
in any form
reserved.
160
JOURNAL
OF SURGICAL
RESEARCH:
at pH 9.0 [ 131.Optimal assay conditions for acid proteinase were determined utilizing normal epidermal segmentsfrom guinea pigs. The assayreagent consisted of 0.05 M acetate buffer (pH 3.8) containing 1% of bovine hemoglobin. Linearity of enzyme activity was obtained for up to 5 pg of freeze-dried normal epidermis. The fluorescence reading was proportional to the incubation time for at least 3 hr at 37°C. The optimal pH in acetatebuffer was 3.8 (64% of maximal activity was detected at pH 3.2, and 44% at pH 4.5 and 5.0). Maximal enzyme activity was obtained at 1% of protein substrate; increased hemoglobin concentrations resulted in a substrate inhibition (a 50% inhibition at 4% of substrate). Microdissected epithelium (0.5- 1.5 pg) was incubated in 5 ~1 of the reagent mixture for 3 hr at 37°C. Standard tubes containing l-2 nmole of L-leucyl-L-alanine was carried throughout the procedure. After incubation, the reaction was stopped by adding 5 ~1 of 10%trichloroacetic acid to eachtest tube. After centrifugation for 10 min at 5OOOg,7 ~1 of the supernatant was added to 0.9 ml of 0.05 M phosphate buffer, pH 7.0. One hundred microliters of fluorescamine solution (7 mg of fluorescamine (Hoffmann LaRoche, Nutley, N. J.) dissolved in 25 ml of acetone) was then added to the supematant and buffer in each
VOL. 35, NO. 2, AUGUST
1983
test tube with immediate mixing. The fluorescence was read in a Farrand fluorometer equipped with Coming glass No. 5970 as the primary filter (390 nm) and Turner filter No. 1lo-828 as the secondary filter (475 nm). RESULTS
The average activity of hemoglobin-hydrolyzing acid proteinase was 95 f 7 (SEM, 12= 10) mmole/hr/kg dry weight in normal epidermis. Alterations in acid proteinase activity during wound healing are summarized in Table 1 and Fig. 1. Additional data on acid phosphatase and &glucuronidase activities, reported previously, are included for a comparative study [2, 51. The marginal epithelium demonstrated a twofold increase in acid proteinase activity on Day 2 and 3.5 times normal during Days 310 postwounding. The regenerating epithelium exhibited a similar response to that of the marginal epithelium. Acid proteinase activity was assayedin the proximal and distal halves of the migrating epithelium (Day 3). No distribution gradient of acid proteinase activity was detected within the regenerating ep ithelium; enzyme activity in the proximal and distal half was 226 + 27 (n = 3) and 257 * 42 (n = 3) mmole/hr/kg dry weight, re-
TABLE
1
ACID HYDROLASE ACXVITIES IN MARGINAL AND REGENERATING EPITHELIUM DURING WOUND HEALING Acid proteinase”
Acid phosphataw’,E
j3-Glucuronidase*’
Days after WOUIlding
0 I 2 3 4 7 10
Marginal 7 (lO)d ND 194 f 21’ (3) 315 + 29’ (4) 336 f 14’ (4) 337 f 31’ (4) 343 (2)
Regenerating
95 f
ND 212 + 17’ (5) 221 k 32’ (7) 245 k 24’ (7) 292 k 44f(7) 274 + 35’ (5)
Marginal 0.80 k 1.17 f 1.38 + 2.78 + 2.83 + 2.03 + 1.33
0.12 0.14 0.40 0.21’ 0.27’ 0.27’
Regenerating (10) (4) (6) (7) (8) (8)
(2)
0.41 f 0.08’ 0.73 + 0.16 1.29 + 0.29 1.63 1?:0.29’ 1.87 + 0.40’ 1.41
(4) (7) (7) (8) (7)
(2)
Marginal 13.5 k 9.6 f 9.6 k 12.1 f 14.6 k 15.1 + 16.6
1.5 (8) 2.4 (3) 2.4 (3) 2.1 (6) 2.6 (6) 2.1 (5)
Regenerating
31.4 * 33.2 + 30.4 f 17.3 + 19.3 +
14.5 6.5’ 5.7’ 3.0 2.5
(3) (7) (7) (7) (6)
’ Enzyme activity is expressedas mmoles per hr per kg dry weight. b Enzyme.activity is expressedas moles per hr per kg dry weight. ’ Data on acid phosphataseand ~-glucuronidase are-from Refs. [2, 51 and supplementary. ‘Each value representsa mean ? SEM determined from the number of difference animals (indicated in parenthesis), each assayed in quintuplicate. ND, Not determined. ‘P values for the differences between the control and experimental epithelium are ~0.05. ’ P values for the differences between the control and experimental epithelium are
IM AND HOOPES: ACID HYDROLASE IN EPIDERMAL WOUNDS
161
% NORMAL
I’ .
.’ ,’ .’
,’ ,,a---.- “...* Acid
proteinase
Acid
phosphatese
,O”
B-Glucuronidase
0
2 DAYS
4
6
AFTER
8
10
WOUNDING
FIG. 1. Alterations in acid phosphatase, acid proteinase, and &glucuronidase activity in regenerating epithelium during wound healing. 0 Cl, Acid phosphatase; 0 ----- 0, acid proteinase; 0 0, @glucuronidase.
spectively. In general, the marginal epithelium exhibited a slightly higher activity of acid proteinase than the regenerating epithelium, but the difference may not be statistically significant.
of phagocytosis in migrating epithelium [ 121, suggestthat acid proteinase promotes cell migration by means of a proteolytic action on serum protein and fibrin embedded in the wound exudate. Acid proteinase appears to be required in epithelial differentiation [8] as indicated by continued increasesin its activity DISCUSSION during the cell maturation phase of epitheliHemoglobin-hydrolyzing acid proteinase of zation. skin is primarily attributable to cathepsin D The curves representing the alterations in activity [6]. The present data on proteolytic acid phosphatase,acid proteinase, and &$,icactivity measured by the hydrolysis of he- uronidase activity reflect their different funcmoglobin at pH 3.8 represent cathepsin D ac- tions in epithelization during wound healing. tivity in epithelial tissue. Cathepsin D may &Glucuronidase is associated with the cell play a major role in intracellular and extra- proliferation and migration phaseswhile acid cellular protein catabolism [7] and phagocytic phosphatesare involved in the cell maturation activity [ 11, 141in the epidermis. Increasesin phase of epithelial wound healing. Acid proacid proteinase activity assayedin this inves- teinase plays a significant role throughout all tigation, together with morphological evidence three phasesof epithelization.
162
JOURNAL OF SURGICAL RESEARCH: VOL. 35, NO. 2, AUGUST 1983
REFERENCES 1. Hirsch, H. E., and Parks, M. E. The quantitative histochemistry of acid proteinase in the nervous system: Localization in neurons. J. Neurochem. 21: 453, 1973. 2. Im, M. J., and Hoopes, J. E. Enzyme activities in regenerating epithelium during wound healing. II. flGlucuronidase. J. Surg. Res. 12: 406, 1972. 3. Im, M. J., and Hoopes, J. E. Enzyme activities in regenerating epithelium during wound healing. III. /3-Galactosidaseand &lucosidase. J. Surg. Res. 13: 82, 1972. 4. lm, M. J., and Hoopes, J. E. Measurement of epithelial production in healing skin wounds. J. Surg. Res. 24: 52, 1978. 5. Im, M. J., and Hoopes, J. E., and Sohn, Y. T. Enzyme activities in regenerating epithelium during wound healing. I. Acid Phosphatase.J. Surg. Res. 12: 402, 1912. 6. Lazarus, G. S., and Dingle, J. T. Cathepsin D of rabbit skin: An immunoenzymic study. J. Invest. Dermatol. 62: 6 I, 1974. 7. Lazarus,G. S., and Poole, A. R. Immunocytochemical localization of cathepsin D in rabbit skin. Arch. Dermatol. 111: 1150, 1975.
8. Lazarus, G. S., Hatcher, V. B., and Levine, N. I. Lysosomesand the skin. J. Invest. Dermatol. 65: 259, 1975. Lowry, 0. H., and Passonneau,J. V. A Flexible System 9. of Enzymatic Analysis. New York Academic Press, 1972. 10. Martinez, I. R. Fine structural studies of migrating epithelial cells following incision wounds. In H. I. Maibach and D. T. Rovee (Eds.), Epidermal Wound Healing. Chicago: Year Book Medical Publishers, 1972. 11. Nordquist, R. E., Olson, R. L., and Everett, M. A. The transport, uptake and storageof ferritin in human epidermis. Arch. Dermatol. 94: 482, 1966. 12. Odland, G., and Ross, R. Human wound repair. I. Epidermal regeneration. J. Cell Biol. 39: 135, 1968. 13. Udenfiiend, S., Stein, E., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M. Fluorescamine: A reagent for assay of amino acids, peptides, protein, and primary amines in the picomole range. Science 178: 871, 1972. 14. Wolff, K., and Konrad, K. Phagocytosisof latex beads of epidermal keratinocytes in vivo. J. Ultrastruct. Res. 39: 262, 1972.