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DNA, RNA and protein synthesis in healing wounds in young and old mice
Mechanisms of,4geing and Development, 3 (1974) 173-185
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© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
DNA, RNA AND PROTEIN Y O U N G A N D O L D MICE
SYNTHESIS
IN
HEALING
WOUNDS
IN
POUL HOLM-PEDERSEN*, A. M. FENSTAD and LARS E. A. FOLKE Division of Periodontology, School t2f Dentistry, University of Minnesota, Minneapolis, Minnesota 55455 (U.S.A.)
(Received April 18, 1974)
SUMMARY In the present study the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in the connective tissue of skin wounds was determined, and the number of fibroblasts present during the healing process was assessed. Six standardized incisions were made on the back of young and old mice utilizing an apparatus specially designed to produce identical wounds. Non-wounded young and old mice served as controls. At 4, 7, 14 and 21 days after surgery the mice received injections intraperitoneally of 3H-thymidine, 3H-uridine, and 14C-proline. In each mouse two wounds were examined histologically. The remaining four wounds were punched out and the epithelium was separated enzymatically from the connective tissue. After sequential separation of protein, RNA, and DNA, one aliquot from each sample was used for scintillation counting and another for quantitative determination of protein, R N A and DNA. After wounding, the number of fibroblasts increased markedly in both groups of animals. The fibroblast population showed a peak in the young animals at day 4, followed by a gradual decrease. In the old mice the initial increase of fibroblasts was of the same magnitude, but no subsequent decrease followed. D N A and R N A synthesis peaked at day 4 and protein synthesis at day 7 in young mice. The old mice showed a statistically significant increase in D N A , R N A and protein synthesis at 14 and 21 days following wounding. These findings suggest an altered fibroblastic function with age.
INTRODUCTION The properties of fibroblasts of different ages are of considerable interest with respect to the wound healing capacity of young and old individuals. In the past it has * Visiting Lasby Professor; present address: Royal Dental College, Vennelyst Boulevard, DK8000 Aarhus C, Denmark.
174 been generally accepted that fibroblasts maintained in tissue culture were potentially immortal. However, Hayflick and Moorhead 1 and Hayflick 2,3 showed that human fibroblasts could survive only a limited number of cell divisions when grown in vitro and that cells from young individuals had a higher average mitotic potential than cells from older individuals. Martin, Sprague and Epstein 4 subsequently showed that the number of times human fibroblasts could be cultured depended largely on the age of the individual from whom the cells were derived. More recently it was suggested by Pont6n, Westermark and Brunk 5 that the number of cell cycles rather than the chronological age is the important parameter for ageing in vitro. Evidence in favor of this hypothesis was also forwarded by Daniel and Young 6. The opinion, however, that the metabolic time, i.e., the total time a population of cells has been kept in vitro might be related to senescence rather than the number of cell divisions, has also been expressed by Hay, Menzies, Morgan and Strehler 7 and by McHale, Mouton and McHale s. Much work has been directed towards characterizing the biochemical and physiological changes which take place in senescent fibroblasts 9. Lately Houck, Sharma and Hayflick1° found that human diploid fibroblasts lost much of their ability to synthesize collagen during the final stages of their life span in vitro. Holliday and Tarrant 11 demonstrated that clonal ageing of human fibroblasts was accompanied by the appearance of at least two altered enzymes, and they suggested that defective proteins were synthesized in senescent fibroblasts. The effects of age upon the formation and metabolism of experimental granulation tissue, which was considered as a "fibroblast culture in vivo" was studied by Heikkinen, Aalto, Vihersaari and Kulonen 12. They found that the contents of DNA and RNA were greater in granulation tissue of younger rats, while the hydroxyproline and nitrogen contents were almost identical in young and old animals. However, protein synthesis and the hydroxylation of proline to hydroxyproline in vitro was significantly higher in the granulation tissue of young rats during the first 3-5 weeks of development. Since the amount of collagen formed remained the same during ageing, these findings indicate that the turnover of collagen in the granulation tissue is greater in young animals, while the older animals preserve the collagen previously formed to a greater extent. The observed retardation in protein synthesis in aged animals might be related to structural ageing of the D N A in the cells themselves 12. Progressive structural changes in the translating apparatus at the ribosomal level may be involved in this process ~3. Slow build-up of errors in protein synthesis has been suggested to be an important factor in ageingt4,15 the relative significance of nuclear and cytoplasmic changes varying between different organisms and tissues 11. Gelfant and Smith 16 suggested that cellular ageing results from a progressive conversion of cycling to noncycling cells in tissues capable of proliferation. According to this theory, the noncycling cells become blocked either in the G1 or G2 period of the cell cyclO 7,1s. They remain in these non-cycling stages until death or they can be released from the G1 and G2 blocks and start to proliferate in response to, for instance, wounding. The mitotic and functional potential of fibroblasts are, therefore, of considerable interest in the analysis of the impaired wound healing in old individuals 19. The
175 purpose of the present investigation was to determine and compare the synthesis of D N A , R N A and protein in the connective tissue o f skin wounds in young and old mice, and to assess the number of fibroblasts present during the healing process. MATERIALS AND METHODS Thirty-one young (6 weeks) and 24 old (18-24 months) CBA mice were utilized for this study. The mice were caged singly or in pairs under controlled conditions of lighting and temperature and provided with Purina lab chow and water ad libitum. The animals were anaesthetized with ether, and the dorsal hair was carefully removed with electric clippers. Four reference points were marked on the back, two in the cranial and two in the caudal part, 5 m m from the midline, on the left and the right side respectively (Fig. I). The distance between the anterior and the posterior points on each side was 3.5 cm. The skin was then folded in the midline, pulled up and
Fig. 1. Schematic drawing of a mouse showing the position of the four reference points on the dorsal skin and the three pairs of wounds.
176
Fig. 2. Apparatus designed for producing identical skin wounds. It consists of a table, an adjustable rod and a sliding knife holder with three No. 11 Bard-Parker blades. The elevated skin fold of the back of the mouse is suspended from the table by two pins (inserted through the anterior and posterior reference points on the dorsal skin) and gently compressed from the side by the rod. The subsequent perforation of the clamped skin fold with the sliding knives results in three pairs of identical incisional wounds.
two stainless steel pins were inserted through the anterior and posterior marks. The animal was subsequently transferred to an operating table specially designed to produce identical skin wounds (Fig. 2). The elevated skin fold was suspended by the pins and gently compressed from the side by an adjustable brass rod. In this way a 1.0 cm wide skin fold was clamped between the operating table and the brass rod. The cutting device consisted of three 5 mm wide knifeblades (Bard-Parker No. 11) which were mounted 2 mm apart on a sliding table. The subsequent perpendicular perforation of the skin fold resulted in three pairs of 5 mm long parallel incisions through the skin and the panniculus muscle. This design excluded the use and influence of sutures during the wound healing. Wounding was done at 4,7, 14 and 21 days prior to sacrifice. A sterile dressing, cyanoacrylate, was applied to the wounds. All the animals were operated on between the hours of 8 and 11 a.m. Five young and 5 old mice were left intact and served as controls. Three hours prior to sacrifice 0.5 #Ci/gram body weight (g bwt) of L-proline 14C (285 #Ci/mM, Radiochemical Centre Amersham) and 1.0 #Ci/g bwt uridine-53H (28 Ci/mM, Radiochemical Centre Amersham) were injected intraperitoneally. One hour prior to sacrifice 1.0 #Ci/g bwt of thymidine-methyl-3H (6.7 Ci/mM, New
177 TISSUE
l (nuclease free pronase) I SEPARATED (epithelium from connective tissue) I DICED (washed 3X 10% TeA) I LIPIDS EXTRACTED
TREATED ENZYMATICALLY
I
Ethanol 10% potassium acetate Ethanol Ethanol-chloroform 3:1 Ethanol-ether 3:1
DIGESTED (IN NaOH)
I
,
Aliquot for
I
NEUTRALIZED, (9N HCI)
Aliquot for protein counting
protein assay
ACIDIFIED (60% PCA)
J
[ SUPERNATE I Aliquot for RNA assay
I L HYDROLIZED
PRECIPITATE (washed 2X 6% PCA)
[ Aliquot for RNA counting
(6% PeA 70°C)
!
| SUPERNATE
I PRECIPITATE
l Aliquot for DNA assay
Ali!uot for DNA counting
Fig. 3. Flow chart of tissue extraction methods following administration of L-proline-a4C, uridine-53H and thymidine-methyl-3H.
England Nuclear) was administered in a similar manner. Each animal received all three isotopically labelled precursors. The animals were killed at 11.30 a.m. with an overdose of sodium pentobarbital. The dorsal skin was removed and pinned to a wooden board. Four of the six areas around the healing wounds were punched out with a No. 4 cork borer (7 mm diam.) and placed in 2.5 ml of 0.1 ~ nuclease free Pronase (Calbiochem) in 0.05 M TrisHC1 (pH 7.4) and incubated at 37 °C for two hours. The skin containing the two caudal wounds was pinned to a piece of cork and placed in Lavdowski's fixative and prepared for histological analysis. Following pronase digestion the epithelium and hair follicles were peeled from the underlying dermis, which was then diced with a razor blade and placed in respective test tubes containing cold 10 ~ trichloroacetic acid (TCA). The flow chart (Fig. 3) summarizes the scheme used to determine total and newly synthesized protein, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The diced tissue was washed three times with TCA and four times with lipid extractants (ethanol with 10 ~ potassium acetate, ethanol, ethanol-chloroform 3 :! and ethanol-ether 3 : 1). Digestion of the tissue was accomplished by incubation at 37°C for one hour in 1.0 ml of 1N sodium hydroxide (NaOH). A 50 #l aliquot of the digest was taken for the total protein assay 20. Another aliquot of 0.1 ml was placed in a scintillation vial containing 3.0 ml of ethanol and 10.0 ml scintillation fluid (4 gm/1 PPO and 100 rag/1 POPOP in toluene) for determination of 14C activity. 14C and 3H were differentially counted for
178 ten minutes at ambient temperature in a dual channel Packard spectometer. Counting efficiency for 3H and 14C were 15 ~ and 20 ~ respectively. Spill over of 14C into the 3H channel was approximately eight percent. The remainder of the digest was neutralized at 4°C with 0.1 ml of 9 N hydrochloric acid (HC1) and acidified with 0.1 ml of 60 perchioric acid (PCA). Separation of the supernate containing the hydrolysed RNA from the precipitate was done by centrifugation at 1700 × g for 30 minutes at 4°C. The 3H activity of a 0.1 ml aliquot of the supernate was determined as above. Another aliquot of 0.5 ml of the supernate was assayed for its RNA content according to the method of Volkin and Cohn 21. The precipitate was washed two times with 6 ~ PCA. The D N A was then hydrolysed in 6 ~ PCA at 70°C for one hour. Separation of the hydrolysed DNA from the protein was done with centrifugation at 1700 × g for 30 minutes at 20°C. A 0.1 ml aliquot of the supernate was taken for scintillation counting of tritium and 0.15 ml was utilized for the determination of total DNA by a procedure modified by Fenstad 22 after Giles and Meyers 2~. The specimens for the histological study were embedded in paraplast, serially sectioned at 3 #m and stained with hematoxylin-eosin. Every 10th section was selected and the number of fibroblasts per unit area was determined in the dermis and in the newly formed wound tissue. RESULTS
Synthesis of DNA The mean values for the ratio of newly synthesized DNA/total DNA (cpm/#g) in the connective tissue of intact skin and skin wounds from young and old mice are shown in Fig. 4. The DNA synthesis was greater in the intact skin of young control mice (.~ = 1.51 :t: 0.14 S.E. cpm/#g) as compared to the old control mice (X ---- 1.04 :[: 0.04 S.E. cpm/#g). After wounding, an increased synthesis of D N A was observed in both groups of animals. The D N A synthesis peaked at day 4 in the young mice, followed by a gradual decrease. Three weeks after wounding, the DNA synthesis almost equalled that observed in the intact dermis of the controls. The pattern observed in the old animals was entirely different. DNA synthesis was somewhat enhanced by the 4th day of healing. It remained almost unchanged on the 7th day but had increased substantially on the 14th day (100 ~). Of the time points studied the DNA synthesis reached its maximum on the 21st day of healing in the old animals. Analysis of variance for D N A synthesis showed no significant overall interaction between age of animals and time of healing (P ---- 0.20). However, D N A synthesis at 14 and 21 days of healing was significantly greater in the older animals (P -- 0.009) as assessed by the Wilcoxon rank sum procedure.
Synthesis of RNA The mean curves illustrating the ratio of newly synthesized RNA/total RNA (cpm//~g) in the connective tissue of intact skin and skin wounds of young and old mice are shown in Fig. 5. In non-wounded skin the ratios were 3.67 -I- 0.23 S.E. cpm/
179 C P M / ~ (J
DNA
.....
YOUNB
_ _
OLD
4.O 3.S 3.0 2.5
/"
2,0
°
•
.........
"t
1.5
1.0
O.5
0
4
7
14
21
DAYS
Fig. 4. Mean curves for D N A synthesis in the connective tissue of intact skin and skin wounds of young and old mice at different time intervals following wounding.
C PM/~(j
RNA
. . . . .
YOUNG
- -
OLD
10,O 9.0 8,O
7.0 6.0 5.0 4,O 3.0 2.0 1.O I
I
I
4
7
14
I
21
DAYS
Fig. 5. Mean curves for R N A synthesis in the connective tissue of intact skin and skin wounds of young and old mice at different time intervals following wounding.
180 #g and 3.91 ± 0.48 S.E. cpm/#g respectively for young and old mice. At day 4 after surgery, an increased R N A synthesis was observed in the wounds of the young animals. Thereafter, the R N A synthesis gradually decreased and the values approached those of normal skin by the 14th day of healing. In contrast to the young mice marked increase in R N A synthesis did not occur until 14 days after wounding in the old mice. By the 21st day the R N A synthesis was further enhanced reflecting the pattern seen for D N A synthesis. Analysis of variance demonstrated no significant overall age-time interaction for R N A synthesis (P ---- 0.10). However, the Wilcoxon rank sum test showed R N A synthesis to be significantly increased (P ~ 0.009) in the older animals at 14 and 21 days of healing.
Synthesis of Protein The mean curves for protein synthesis in the skin and wounds of young and old mice are shown in Fig. 6. The protein synthesis tended to be greater in the intact skin of young mice (X = 0.49 :L 0.11 S.E. cpm/#g) than of old mice (X ----0.32 ~ 0.05 S.E. cpm/#g). At day 7 it had increased in young mice, while it remained low in the old animals. At day 14, protein synthesis had decreased in young mice while a marked increase was observed in the wounds of the old mice. This was accentuated on day 21 in the old mice while the protein synthesis had returned to normal in the young mice after three weeks of healing.
CPM/~
PROTEIN
.....
YOUNG
_ _
OLD
2.0 1.8
1.6 14 1.2
i
10.
08
06
0.4
O.2
I
I
I
I
'4.
7
14.
21
DAYS
Fig. 6. Mean curves for protein synthesis in the connective tissue of intact skin and skin wounds of young and old mice at different time intervals following wounding.
181 Mean N of fibroblasts/
[ ] old mice [ ] young mice
.01 mm area
60
50
40
!
30
20
,'xx3
,-,,-q
10
4
7
of w o u ~
14
(days)
1
21
Fig. 7. M e a n n u m b e r o f fibroblasts per unit area of intact skin a n d skin w o u n d s o f y o u n g a n d old mice at different time intervals following wounding.
Analysis of variance of protein synthesis in young and old animals showed a statistically significant interaction (P < 0.005) between age and time period, indicating different response patterns across time and reflecting elevated protein synthesis in the older mice at the later times. The Wilcoxon rank sum procedure confirms that increase at 14 and 21 days of healing (P -----0.001). Fibroblasts The mean numbers of fibroblasts in normal and wounded skin of young and old mice are shown in Fig. 7. In non-wounded skin the number of fibroblasts per unit area was almost the same in young and old animals. After wounding the fibroblast population increased markedly in both groups on the 4th day of healing. By the 7th day the fibroblasts gradually decreased in young mice. The total number of fibroblasts approached the values of non-wounded skin at the termination of the experiment. The initial increase of fibroblasts, observed at day 4, remained the same, however, throughout the 21-day postoperative period in the old animals.
182 DISCUSSION Previous studies have shown that connective tissue wound healing is impailed in old animals (Holm-Pedersen19). These investigations indicated that the decreased healing potential of the tissues might be related to a slower rate of multiplication and/ or maturation of fibroblasts in old individuals. Another possibility might be that the number of cells involved in wound repair is decreased in older persons. In the present study the synthesis of DNA, RNA and protein in healing wounds of young and old mice were studied using a double isotope labelling technique, and the amounts of newly formed DNA, RNA and protein were correlated to the total amounts of DNA, RNA and protein. After wounding, an increased synthesis o f D N A , RNA and protein was observed in both groups. In the young animals the synthesis of DNA and RNA peaked within the first week of healing and the protein synthesis reached its highest values at the 7th day. In the old mice the synthesis of all three fractions increased substantially during the 2nd and 3rd weeks of healing and maximum synthesis was recorded on the 21st day of the preselected observation time points. These findings indicate that a "phase difference" exists in the development of the various metabolic activities in healing wounds of young and old animals. This confirms earlier observations on spongeinduced granulation tissue in vitro 12. Although delayed in old animals, the values for the maximum synthesis of DNA, RNA and protein were considerably higher than those obtained for the young animals during the healing stages studied. This may be a false interpretation, since it cannot be excluded that the DNA and RNA synthesis in young mice may have peaked before the 4th day and that the protein synthesis may have reached a maximum between the 7th and 14th day. The comparatively higher maximum values reported for DNA, RNA and protein synthesis in healing wounds of old animals could also be due to a greater incorporation of labelled precursors in these wounds, or a greater rate of destruction of labelled compounds in wounds of young animals. However, if there is a greater incorporation or destruction of labelled precursors in wounds of old and young animals respectively, it should apply to all time points studied, suggesting that the younger animals are more actively synthesizing these compounds at earlier healing stages. Another possibility might be that there is a a greater pool of unlabelled material in the young animals. Because the incorporation of label in the non-wounded skin of young and old mice is very similar, it would appear that the pool sizes are the same. However, the possibility exists that the pool of precursors could change following wounding. On the other hand, the observation of greater synthesis of DNA, RNA and protein in old mice confirms several previous reports that the amount of granulation tissue formed in response to injury is the same or even enhanced in old animals12,24-29; and, that the impairment of wound healing with advancing age is due to qualitative rather than quantitative differences of the granulation tissue formed28, 3°. An additional source of error in the results could conceivably occur during the pronase treatment for connective tissue/epithelium separation. This procedure was used to digest the collagen in the basement membrane, but the enzyme would also be
183 digesting cell membranes. If pronase has a predilection for cell membranes of one group o f animals over the other, a selective loss of counts could occur. During the early healing period a great number of inflammatory ceils were observed in the wound area. These cells inevitably will contribute to the total amount o f D N A in the tissue and influence the ratio of newly synthesized DNA/total DNA. This means that the DNA synthesis, when related to the total content of DNA, will appear lower than the actual synthesis taking place. This may be of particular concern in the analysis of young animals as Hohn-Pedersen, Nilsson and Brgmemark 31 have reported that the accumulation of inflammatory cells was greater in wounds of young than of old animals during the first 3-4 days of healing. The DNA synthesis in the wounds of young mice may, therefore, be substantially greater at day 4 than reflected in the values calculated on the basis of total DNA content. The histological study provided further information regarding the number and possible function of fibroblasts during the healing process. In the non-wounded dermis the number of fibroblasts per unit area was almost the same in young and old animals. After wounding the fibroblast population increased markedly in both groups of animals. The replication of fibroblasts showed a peak in young animals at day 4, followed by a decrease. In the old mice, a similar increase in the number of fibroblasts occurred but was not followed by a subsequent decrease. On the contrary, the initial increase in fibroblasts remained almost unchanged through the 21-day postoperative healing period (Fig. 7). This may have contributed to the higher values of DNA synthesis observed in the old animals during the third week of healing compared to the corresponding values of the young group of animals. Our findings suggest that the slower healing o f wounds in old animals is associated with an altered fibroblastic function with age. It might be argued that the reparative fibroblast population is not generated from the fibrocytes32 but that they arise from undifferentiated mesenchymal cells in the perivascular connective tissue adjacent to the wound 33-a5. Therefore, in the non-wounded dermis, an assessment of the number of mature fibroblasts or fibrocytes may not measure the actual number of progenitor cells which contribute to wound repair. However, the increased number of fibroblasts in the wounds of old animals during the second and third weeks of healing corresponds to the concomitant elevation of RNA and protein synthesis in these wounds. The structurally inferior wound tissue formed in old animals 2s,30 may, therefore, be related to an impaired quality of protein synthesis in old fibroblasts. Much effort has been expended to discover and identify the mechanisms of cellular ageing. Heikkinen e t al. ~2 suggested that retardation in protein synthesis in old animals might be related to structural ageing of the DNA ~3. According to Strehler36, 3v the sites at which cellular function can fail has been considered in relation to the flow of information from the primary genetic material, DNA, through RNA and ultimately into protein. Each of these components in the informational chain may be subject to constant damage. Johnson, Crisp and Strehler 38 have reported observations indicating a substantial decline in the number of ribosomal RNA cistrons in D N A of postmitotic cells in old animals and they considered genetic damage to be a factor in the age-dependent changes in DNA. Although no single mechanism may
184
account for the highly complex process of cellular ageing37,~9 evidence has been presented in favor of the hypothesis that ageing is determined by a genetic program 2. However, changes in the extracellular environment may also adversely affect fibroblast function. ACKNOWLEDGEMENTS
This investigation was Research Council and in part Health Service Grant No. DE The authors gratefully Schotzko.
supported by grant No. 512-1647 from the Danish by the National Institute of Dental Research, Public 03174-02. acknowledge the technical assistance of Mrs. Nancy
REFERENCES 1 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 585-621. 2 L. Hayflick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37 (1965) 614-636. 3 L. Hayflick, Aging under glass, Exp. Gerontol., 5 (1970) 291-303. 4 G. M. Martin, C. A. Sprague and C. J. Epstein, Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype, Lab. hlvest., 23 (1970) 86-92. 5 J. Pont6n, B. Westermark and V. Brunk, Aging of fibroblasts in long term tissue culture, Scand. J. Clin. Lab. hlvest., 29 (123) (1972) 26. 6 C. W. Daniel and L. J. T. Young, Influence of cell division on an aging process. Life-span of mouse mammary epithelium during serial propagation in vivo, Exp. Cell Res., 65 (1971) 27-32. 7 R. J. Hay, R. A. Menzies, H. P. Morgan and B. L. Strehler, The division potential of cells in continuous growth as compared to cells subcultivated after maintenance in stationary phase, Exp. Gerontol., 3 (1968) 35-44. 8 J. S. McHale, M. L. Mouton and J. T. McHale, Limited culture lifespan of human diploid cells as a function of metabolic time instead of division potential, Exp. Gerontol., 6 (1971) 89-93. 9 V. J. Cristofalo, Animal cell cultures as a model system for the study of aging. In B. L. Strehler (Ed.), Advances in Gerontological Research, Vol. 4, Academic Press, New York, 1972, pp. 45-79. 10 J. C. Houck, V. K. Sharma and L. Hayflick, Functional failures of cultured human diploid fibroblasts after continued population doublings, Proc. Soc. Exp. Biol. Med., 137 (1971) 331-333. 11 R. Holliday and G. M. Tarrant, Altered enzymes in ageing human fibroblasts, Nature, 238 (1972) 26-30. 12 E. Heikkinen, M. Aalto, T. Vihersaari and E. Kulonen, Age factor in the formation and metabolism of experimental granulation tissue, J. Gerontol., 26 (1971) 294-298. 13 H. P. von Hahn, Structural and functional changes in nucleoprotein during the ageing of the cell, Gerontologia, 16 (1970) 116-128. 14 L. E. Orgel, The maintenance of the accuracy of protein synthesis and its relevance to ageing, Proc. Nat. Acad. Sci. U.S., 49 (1963) 517-521. 15 L. E. Orgel, The maintenance of the accuracy of protein synthesis and its relevance to ageing: A correction, Proc. Nat. Acad. Sci. U.S., 67 (1970) 1476. 16 S. Gelfant and J. G. Smith, Aging: Noncycling cells an explanation, Science, 178 (1972) 357-361. 17 A. Macieira-Coelho, J. Pont6n and L. Philipson, The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro, Exp. Cell Res., 42 (1966) 673-684. 18 A. Macieira-Coelho, J. Pont6n and L. Philipson, Inhibition of the division cycle in confluent cultures of human fibrobalsts in vitro, Exp. Cell Res., 43 (1966) 20-29. 19 P. Holm-Pedersen, Studies on Healing Capacity in Young and Old Individuals, Munksgaard, Copenhagen, 1973. 20 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
185 21 E. Volkin and W. E. Cohn, Estimation of nucleic acids, Methods Biochem. Anal., 1 (1954) 287-305. 22 A. M. Fenstad, The influence of female sex hormones on the oral mucous membrane, Ph. D. Thesis, University of Minnesota, 1973. 23 K. W. Giles and A. Meyers, An improved diphenylamine method for the estimation of deoxyribonucleic acid, Nature, 206 (1965) 93. 24 R. J. Boucek, N. L. Noble, K.-Y. T. Kao and H. R. Elden, The effects of age, sex, and race upon the acetic acid fractions of collagen (human biopsy-connective tissue), J. Gerontol., 13 (1958)2-9. 25 W. Doberauer, Beeinflussung von Wundheilungsvorgangen durch das I.ebensalter, Gerontol. Clin., 4 (1962) 112-127. 26 J. C. Houck and R. A. Jacob, The effect of age on the chemistry of inflammation, J. Invest. Dermatol., 42 (1964) 377-371. 27 G. Junge-H/Jlsing and H. Wagner, Alterations in mucopolysaccharide (glycosaminoglycan) metabolism with age. In A. Engel and T. Larsson (Eds.), Aging of Connective and Skeletal Tissue, Nordiska Bokhandelns F6rlag, Stockholm, 1969, pp. 213-228. 28 P. Holm-Pedersen and B. Zederfeldt, Granulation tissue formation in subcutaneously implanted cellulose sponges in young and old rats, Scand. J. Plast. Reconstr. Surg., 5 (1971) 13-16. 29 P. Holm-Pedersen and B. Zederfeldt, Respiratory gas tensions and blood flow in wounded areas in young and old rats, Scand. J. Plast. Reconstr. Surg., 7 (1973) 91 96. 30 P. Holm-Pedersen and A. Viidik, Tensile properties and morphology of healing wounds in young and old rats, Scand. J. Plast. Reconstr. Surg., 6 (1972) 24-35. 31 P. Holm-Pedersen, K. Nilsson and P. I. Br~memark, The microvascular system of healing wounds in young and old rats, Advan. Microcirc., 5 (1973) 80-106. 32 T. Gillman and J. Penn, Studies on the repair of cutaneous wounds, Med. Proc., 2 (1956) 121-186. 33 R. A. MacDonald, Origin of fibroblasts in experimental healing wounds: autoradiographic studies using tritiated thymidine, Surgery, 46 (1959) 376-382. 34 H. C. Grillo, Origin of fibroblasts in wound healing: an autoradiographic study of inhibition of cellular proliferation by local X-irradiation, Ann. Surg., 157 (1963) 453-467. 35 R. Ross, N. B. Everett and R. Tyler, Wound healing and collagen formation. VI. The origin of the wound fibroblast studied in parabiosis, J. Cell Biol., 44 (1970) 645-654. 36 B. L. Strehler, Aging at the cellular level. In I. Rossman (Ed.), Clinical Geriatrics, Lippincroft, Philadelphia, 1971, pp. 49-81. 37 B. L. Strehler, Aging and the utilization of informational patterns, Proceedings 4th European Symposium on Basic Research in Gerontology, Varberg, 1973. 38 R. Johnson, C. Chrisp and B. Strehler, Selective loss of ribosomal RNA genes during the aging of post-mitotic tissues, Mech. Age. Dev., 1 (1972) 183-198. 39 H. P. von Hahn, Primary causes of ageing: A brief review of some modern theories and concepts, Mech. Age. Dev., 2 (1973) 245-250.
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© Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
DNA, RNA AND PROTEIN Y O U N G A N D O L D MICE
SYNTHESIS
IN
HEALING
WOUNDS
IN
POUL HOLM-PEDERSEN*, A. M. FENSTAD and LARS E. A. FOLKE Division of Periodontology, School t2f Dentistry, University of Minnesota, Minneapolis, Minnesota 55455 (U.S.A.)
(Received April 18, 1974)
SUMMARY In the present study the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in the connective tissue of skin wounds was determined, and the number of fibroblasts present during the healing process was assessed. Six standardized incisions were made on the back of young and old mice utilizing an apparatus specially designed to produce identical wounds. Non-wounded young and old mice served as controls. At 4, 7, 14 and 21 days after surgery the mice received injections intraperitoneally of 3H-thymidine, 3H-uridine, and 14C-proline. In each mouse two wounds were examined histologically. The remaining four wounds were punched out and the epithelium was separated enzymatically from the connective tissue. After sequential separation of protein, RNA, and DNA, one aliquot from each sample was used for scintillation counting and another for quantitative determination of protein, R N A and DNA. After wounding, the number of fibroblasts increased markedly in both groups of animals. The fibroblast population showed a peak in the young animals at day 4, followed by a gradual decrease. In the old mice the initial increase of fibroblasts was of the same magnitude, but no subsequent decrease followed. D N A and R N A synthesis peaked at day 4 and protein synthesis at day 7 in young mice. The old mice showed a statistically significant increase in D N A , R N A and protein synthesis at 14 and 21 days following wounding. These findings suggest an altered fibroblastic function with age.
INTRODUCTION The properties of fibroblasts of different ages are of considerable interest with respect to the wound healing capacity of young and old individuals. In the past it has * Visiting Lasby Professor; present address: Royal Dental College, Vennelyst Boulevard, DK8000 Aarhus C, Denmark.
174 been generally accepted that fibroblasts maintained in tissue culture were potentially immortal. However, Hayflick and Moorhead 1 and Hayflick 2,3 showed that human fibroblasts could survive only a limited number of cell divisions when grown in vitro and that cells from young individuals had a higher average mitotic potential than cells from older individuals. Martin, Sprague and Epstein 4 subsequently showed that the number of times human fibroblasts could be cultured depended largely on the age of the individual from whom the cells were derived. More recently it was suggested by Pont6n, Westermark and Brunk 5 that the number of cell cycles rather than the chronological age is the important parameter for ageing in vitro. Evidence in favor of this hypothesis was also forwarded by Daniel and Young 6. The opinion, however, that the metabolic time, i.e., the total time a population of cells has been kept in vitro might be related to senescence rather than the number of cell divisions, has also been expressed by Hay, Menzies, Morgan and Strehler 7 and by McHale, Mouton and McHale s. Much work has been directed towards characterizing the biochemical and physiological changes which take place in senescent fibroblasts 9. Lately Houck, Sharma and Hayflick1° found that human diploid fibroblasts lost much of their ability to synthesize collagen during the final stages of their life span in vitro. Holliday and Tarrant 11 demonstrated that clonal ageing of human fibroblasts was accompanied by the appearance of at least two altered enzymes, and they suggested that defective proteins were synthesized in senescent fibroblasts. The effects of age upon the formation and metabolism of experimental granulation tissue, which was considered as a "fibroblast culture in vivo" was studied by Heikkinen, Aalto, Vihersaari and Kulonen 12. They found that the contents of DNA and RNA were greater in granulation tissue of younger rats, while the hydroxyproline and nitrogen contents were almost identical in young and old animals. However, protein synthesis and the hydroxylation of proline to hydroxyproline in vitro was significantly higher in the granulation tissue of young rats during the first 3-5 weeks of development. Since the amount of collagen formed remained the same during ageing, these findings indicate that the turnover of collagen in the granulation tissue is greater in young animals, while the older animals preserve the collagen previously formed to a greater extent. The observed retardation in protein synthesis in aged animals might be related to structural ageing of the D N A in the cells themselves 12. Progressive structural changes in the translating apparatus at the ribosomal level may be involved in this process ~3. Slow build-up of errors in protein synthesis has been suggested to be an important factor in ageingt4,15 the relative significance of nuclear and cytoplasmic changes varying between different organisms and tissues 11. Gelfant and Smith 16 suggested that cellular ageing results from a progressive conversion of cycling to noncycling cells in tissues capable of proliferation. According to this theory, the noncycling cells become blocked either in the G1 or G2 period of the cell cyclO 7,1s. They remain in these non-cycling stages until death or they can be released from the G1 and G2 blocks and start to proliferate in response to, for instance, wounding. The mitotic and functional potential of fibroblasts are, therefore, of considerable interest in the analysis of the impaired wound healing in old individuals 19. The
175 purpose of the present investigation was to determine and compare the synthesis of D N A , R N A and protein in the connective tissue o f skin wounds in young and old mice, and to assess the number of fibroblasts present during the healing process. MATERIALS AND METHODS Thirty-one young (6 weeks) and 24 old (18-24 months) CBA mice were utilized for this study. The mice were caged singly or in pairs under controlled conditions of lighting and temperature and provided with Purina lab chow and water ad libitum. The animals were anaesthetized with ether, and the dorsal hair was carefully removed with electric clippers. Four reference points were marked on the back, two in the cranial and two in the caudal part, 5 m m from the midline, on the left and the right side respectively (Fig. I). The distance between the anterior and the posterior points on each side was 3.5 cm. The skin was then folded in the midline, pulled up and
Fig. 1. Schematic drawing of a mouse showing the position of the four reference points on the dorsal skin and the three pairs of wounds.
176
Fig. 2. Apparatus designed for producing identical skin wounds. It consists of a table, an adjustable rod and a sliding knife holder with three No. 11 Bard-Parker blades. The elevated skin fold of the back of the mouse is suspended from the table by two pins (inserted through the anterior and posterior reference points on the dorsal skin) and gently compressed from the side by the rod. The subsequent perforation of the clamped skin fold with the sliding knives results in three pairs of identical incisional wounds.
two stainless steel pins were inserted through the anterior and posterior marks. The animal was subsequently transferred to an operating table specially designed to produce identical skin wounds (Fig. 2). The elevated skin fold was suspended by the pins and gently compressed from the side by an adjustable brass rod. In this way a 1.0 cm wide skin fold was clamped between the operating table and the brass rod. The cutting device consisted of three 5 mm wide knifeblades (Bard-Parker No. 11) which were mounted 2 mm apart on a sliding table. The subsequent perpendicular perforation of the skin fold resulted in three pairs of 5 mm long parallel incisions through the skin and the panniculus muscle. This design excluded the use and influence of sutures during the wound healing. Wounding was done at 4,7, 14 and 21 days prior to sacrifice. A sterile dressing, cyanoacrylate, was applied to the wounds. All the animals were operated on between the hours of 8 and 11 a.m. Five young and 5 old mice were left intact and served as controls. Three hours prior to sacrifice 0.5 #Ci/gram body weight (g bwt) of L-proline 14C (285 #Ci/mM, Radiochemical Centre Amersham) and 1.0 #Ci/g bwt uridine-53H (28 Ci/mM, Radiochemical Centre Amersham) were injected intraperitoneally. One hour prior to sacrifice 1.0 #Ci/g bwt of thymidine-methyl-3H (6.7 Ci/mM, New
177 TISSUE
l (nuclease free pronase) I SEPARATED (epithelium from connective tissue) I DICED (washed 3X 10% TeA) I LIPIDS EXTRACTED
TREATED ENZYMATICALLY
I
Ethanol 10% potassium acetate Ethanol Ethanol-chloroform 3:1 Ethanol-ether 3:1
DIGESTED (IN NaOH)
I
,
Aliquot for
I
NEUTRALIZED, (9N HCI)
Aliquot for protein counting
protein assay
ACIDIFIED (60% PCA)
J
[ SUPERNATE I Aliquot for RNA assay
I L HYDROLIZED
PRECIPITATE (washed 2X 6% PCA)
[ Aliquot for RNA counting
(6% PeA 70°C)
!
| SUPERNATE
I PRECIPITATE
l Aliquot for DNA assay
Ali!uot for DNA counting
Fig. 3. Flow chart of tissue extraction methods following administration of L-proline-a4C, uridine-53H and thymidine-methyl-3H.
England Nuclear) was administered in a similar manner. Each animal received all three isotopically labelled precursors. The animals were killed at 11.30 a.m. with an overdose of sodium pentobarbital. The dorsal skin was removed and pinned to a wooden board. Four of the six areas around the healing wounds were punched out with a No. 4 cork borer (7 mm diam.) and placed in 2.5 ml of 0.1 ~ nuclease free Pronase (Calbiochem) in 0.05 M TrisHC1 (pH 7.4) and incubated at 37 °C for two hours. The skin containing the two caudal wounds was pinned to a piece of cork and placed in Lavdowski's fixative and prepared for histological analysis. Following pronase digestion the epithelium and hair follicles were peeled from the underlying dermis, which was then diced with a razor blade and placed in respective test tubes containing cold 10 ~ trichloroacetic acid (TCA). The flow chart (Fig. 3) summarizes the scheme used to determine total and newly synthesized protein, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The diced tissue was washed three times with TCA and four times with lipid extractants (ethanol with 10 ~ potassium acetate, ethanol, ethanol-chloroform 3 :! and ethanol-ether 3 : 1). Digestion of the tissue was accomplished by incubation at 37°C for one hour in 1.0 ml of 1N sodium hydroxide (NaOH). A 50 #l aliquot of the digest was taken for the total protein assay 20. Another aliquot of 0.1 ml was placed in a scintillation vial containing 3.0 ml of ethanol and 10.0 ml scintillation fluid (4 gm/1 PPO and 100 rag/1 POPOP in toluene) for determination of 14C activity. 14C and 3H were differentially counted for
178 ten minutes at ambient temperature in a dual channel Packard spectometer. Counting efficiency for 3H and 14C were 15 ~ and 20 ~ respectively. Spill over of 14C into the 3H channel was approximately eight percent. The remainder of the digest was neutralized at 4°C with 0.1 ml of 9 N hydrochloric acid (HC1) and acidified with 0.1 ml of 60 perchioric acid (PCA). Separation of the supernate containing the hydrolysed RNA from the precipitate was done by centrifugation at 1700 × g for 30 minutes at 4°C. The 3H activity of a 0.1 ml aliquot of the supernate was determined as above. Another aliquot of 0.5 ml of the supernate was assayed for its RNA content according to the method of Volkin and Cohn 21. The precipitate was washed two times with 6 ~ PCA. The D N A was then hydrolysed in 6 ~ PCA at 70°C for one hour. Separation of the hydrolysed DNA from the protein was done with centrifugation at 1700 × g for 30 minutes at 20°C. A 0.1 ml aliquot of the supernate was taken for scintillation counting of tritium and 0.15 ml was utilized for the determination of total DNA by a procedure modified by Fenstad 22 after Giles and Meyers 2~. The specimens for the histological study were embedded in paraplast, serially sectioned at 3 #m and stained with hematoxylin-eosin. Every 10th section was selected and the number of fibroblasts per unit area was determined in the dermis and in the newly formed wound tissue. RESULTS
Synthesis of DNA The mean values for the ratio of newly synthesized DNA/total DNA (cpm/#g) in the connective tissue of intact skin and skin wounds from young and old mice are shown in Fig. 4. The DNA synthesis was greater in the intact skin of young control mice (.~ = 1.51 :t: 0.14 S.E. cpm/#g) as compared to the old control mice (X ---- 1.04 :[: 0.04 S.E. cpm/#g). After wounding, an increased synthesis of D N A was observed in both groups of animals. The D N A synthesis peaked at day 4 in the young mice, followed by a gradual decrease. Three weeks after wounding, the DNA synthesis almost equalled that observed in the intact dermis of the controls. The pattern observed in the old animals was entirely different. DNA synthesis was somewhat enhanced by the 4th day of healing. It remained almost unchanged on the 7th day but had increased substantially on the 14th day (100 ~). Of the time points studied the DNA synthesis reached its maximum on the 21st day of healing in the old animals. Analysis of variance for D N A synthesis showed no significant overall interaction between age of animals and time of healing (P ---- 0.20). However, D N A synthesis at 14 and 21 days of healing was significantly greater in the older animals (P -- 0.009) as assessed by the Wilcoxon rank sum procedure.
Synthesis of RNA The mean curves illustrating the ratio of newly synthesized RNA/total RNA (cpm//~g) in the connective tissue of intact skin and skin wounds of young and old mice are shown in Fig. 5. In non-wounded skin the ratios were 3.67 -I- 0.23 S.E. cpm/
179 C P M / ~ (J
DNA
.....
YOUNB
_ _
OLD
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/"
2,0
°
•
.........
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1.5
1.0
O.5
0
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DAYS
Fig. 4. Mean curves for D N A synthesis in the connective tissue of intact skin and skin wounds of young and old mice at different time intervals following wounding.
C PM/~(j
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. . . . .
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- -
OLD
10,O 9.0 8,O
7.0 6.0 5.0 4,O 3.0 2.0 1.O I
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Fig. 5. Mean curves for R N A synthesis in the connective tissue of intact skin and skin wounds of young and old mice at different time intervals following wounding.
180 #g and 3.91 ± 0.48 S.E. cpm/#g respectively for young and old mice. At day 4 after surgery, an increased R N A synthesis was observed in the wounds of the young animals. Thereafter, the R N A synthesis gradually decreased and the values approached those of normal skin by the 14th day of healing. In contrast to the young mice marked increase in R N A synthesis did not occur until 14 days after wounding in the old mice. By the 21st day the R N A synthesis was further enhanced reflecting the pattern seen for D N A synthesis. Analysis of variance demonstrated no significant overall age-time interaction for R N A synthesis (P ---- 0.10). However, the Wilcoxon rank sum test showed R N A synthesis to be significantly increased (P ~ 0.009) in the older animals at 14 and 21 days of healing.
Synthesis of Protein The mean curves for protein synthesis in the skin and wounds of young and old mice are shown in Fig. 6. The protein synthesis tended to be greater in the intact skin of young mice (X = 0.49 :L 0.11 S.E. cpm/#g) than of old mice (X ----0.32 ~ 0.05 S.E. cpm/#g). At day 7 it had increased in young mice, while it remained low in the old animals. At day 14, protein synthesis had decreased in young mice while a marked increase was observed in the wounds of the old mice. This was accentuated on day 21 in the old mice while the protein synthesis had returned to normal in the young mice after three weeks of healing.
CPM/~
PROTEIN
.....
YOUNG
_ _
OLD
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i
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08
06
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Fig. 6. Mean curves for protein synthesis in the connective tissue of intact skin and skin wounds of young and old mice at different time intervals following wounding.
181 Mean N of fibroblasts/
[ ] old mice [ ] young mice
.01 mm area
60
50
40
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30
20
,'xx3
,-,,-q
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Fig. 7. M e a n n u m b e r o f fibroblasts per unit area of intact skin a n d skin w o u n d s o f y o u n g a n d old mice at different time intervals following wounding.
Analysis of variance of protein synthesis in young and old animals showed a statistically significant interaction (P < 0.005) between age and time period, indicating different response patterns across time and reflecting elevated protein synthesis in the older mice at the later times. The Wilcoxon rank sum procedure confirms that increase at 14 and 21 days of healing (P -----0.001). Fibroblasts The mean numbers of fibroblasts in normal and wounded skin of young and old mice are shown in Fig. 7. In non-wounded skin the number of fibroblasts per unit area was almost the same in young and old animals. After wounding the fibroblast population increased markedly in both groups on the 4th day of healing. By the 7th day the fibroblasts gradually decreased in young mice. The total number of fibroblasts approached the values of non-wounded skin at the termination of the experiment. The initial increase of fibroblasts, observed at day 4, remained the same, however, throughout the 21-day postoperative period in the old animals.
182 DISCUSSION Previous studies have shown that connective tissue wound healing is impailed in old animals (Holm-Pedersen19). These investigations indicated that the decreased healing potential of the tissues might be related to a slower rate of multiplication and/ or maturation of fibroblasts in old individuals. Another possibility might be that the number of cells involved in wound repair is decreased in older persons. In the present study the synthesis of DNA, RNA and protein in healing wounds of young and old mice were studied using a double isotope labelling technique, and the amounts of newly formed DNA, RNA and protein were correlated to the total amounts of DNA, RNA and protein. After wounding, an increased synthesis o f D N A , RNA and protein was observed in both groups. In the young animals the synthesis of DNA and RNA peaked within the first week of healing and the protein synthesis reached its highest values at the 7th day. In the old mice the synthesis of all three fractions increased substantially during the 2nd and 3rd weeks of healing and maximum synthesis was recorded on the 21st day of the preselected observation time points. These findings indicate that a "phase difference" exists in the development of the various metabolic activities in healing wounds of young and old animals. This confirms earlier observations on spongeinduced granulation tissue in vitro 12. Although delayed in old animals, the values for the maximum synthesis of DNA, RNA and protein were considerably higher than those obtained for the young animals during the healing stages studied. This may be a false interpretation, since it cannot be excluded that the DNA and RNA synthesis in young mice may have peaked before the 4th day and that the protein synthesis may have reached a maximum between the 7th and 14th day. The comparatively higher maximum values reported for DNA, RNA and protein synthesis in healing wounds of old animals could also be due to a greater incorporation of labelled precursors in these wounds, or a greater rate of destruction of labelled compounds in wounds of young animals. However, if there is a greater incorporation or destruction of labelled precursors in wounds of old and young animals respectively, it should apply to all time points studied, suggesting that the younger animals are more actively synthesizing these compounds at earlier healing stages. Another possibility might be that there is a a greater pool of unlabelled material in the young animals. Because the incorporation of label in the non-wounded skin of young and old mice is very similar, it would appear that the pool sizes are the same. However, the possibility exists that the pool of precursors could change following wounding. On the other hand, the observation of greater synthesis of DNA, RNA and protein in old mice confirms several previous reports that the amount of granulation tissue formed in response to injury is the same or even enhanced in old animals12,24-29; and, that the impairment of wound healing with advancing age is due to qualitative rather than quantitative differences of the granulation tissue formed28, 3°. An additional source of error in the results could conceivably occur during the pronase treatment for connective tissue/epithelium separation. This procedure was used to digest the collagen in the basement membrane, but the enzyme would also be
183 digesting cell membranes. If pronase has a predilection for cell membranes of one group o f animals over the other, a selective loss of counts could occur. During the early healing period a great number of inflammatory ceils were observed in the wound area. These cells inevitably will contribute to the total amount o f D N A in the tissue and influence the ratio of newly synthesized DNA/total DNA. This means that the DNA synthesis, when related to the total content of DNA, will appear lower than the actual synthesis taking place. This may be of particular concern in the analysis of young animals as Hohn-Pedersen, Nilsson and Brgmemark 31 have reported that the accumulation of inflammatory cells was greater in wounds of young than of old animals during the first 3-4 days of healing. The DNA synthesis in the wounds of young mice may, therefore, be substantially greater at day 4 than reflected in the values calculated on the basis of total DNA content. The histological study provided further information regarding the number and possible function of fibroblasts during the healing process. In the non-wounded dermis the number of fibroblasts per unit area was almost the same in young and old animals. After wounding the fibroblast population increased markedly in both groups of animals. The replication of fibroblasts showed a peak in young animals at day 4, followed by a decrease. In the old mice, a similar increase in the number of fibroblasts occurred but was not followed by a subsequent decrease. On the contrary, the initial increase in fibroblasts remained almost unchanged through the 21-day postoperative healing period (Fig. 7). This may have contributed to the higher values of DNA synthesis observed in the old animals during the third week of healing compared to the corresponding values of the young group of animals. Our findings suggest that the slower healing o f wounds in old animals is associated with an altered fibroblastic function with age. It might be argued that the reparative fibroblast population is not generated from the fibrocytes32 but that they arise from undifferentiated mesenchymal cells in the perivascular connective tissue adjacent to the wound 33-a5. Therefore, in the non-wounded dermis, an assessment of the number of mature fibroblasts or fibrocytes may not measure the actual number of progenitor cells which contribute to wound repair. However, the increased number of fibroblasts in the wounds of old animals during the second and third weeks of healing corresponds to the concomitant elevation of RNA and protein synthesis in these wounds. The structurally inferior wound tissue formed in old animals 2s,30 may, therefore, be related to an impaired quality of protein synthesis in old fibroblasts. Much effort has been expended to discover and identify the mechanisms of cellular ageing. Heikkinen e t al. ~2 suggested that retardation in protein synthesis in old animals might be related to structural ageing of the DNA ~3. According to Strehler36, 3v the sites at which cellular function can fail has been considered in relation to the flow of information from the primary genetic material, DNA, through RNA and ultimately into protein. Each of these components in the informational chain may be subject to constant damage. Johnson, Crisp and Strehler 38 have reported observations indicating a substantial decline in the number of ribosomal RNA cistrons in D N A of postmitotic cells in old animals and they considered genetic damage to be a factor in the age-dependent changes in DNA. Although no single mechanism may
184
account for the highly complex process of cellular ageing37,~9 evidence has been presented in favor of the hypothesis that ageing is determined by a genetic program 2. However, changes in the extracellular environment may also adversely affect fibroblast function. ACKNOWLEDGEMENTS
This investigation was Research Council and in part Health Service Grant No. DE The authors gratefully Schotzko.
supported by grant No. 512-1647 from the Danish by the National Institute of Dental Research, Public 03174-02. acknowledge the technical assistance of Mrs. Nancy
REFERENCES 1 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 585-621. 2 L. Hayflick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37 (1965) 614-636. 3 L. Hayflick, Aging under glass, Exp. Gerontol., 5 (1970) 291-303. 4 G. M. Martin, C. A. Sprague and C. J. Epstein, Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype, Lab. hlvest., 23 (1970) 86-92. 5 J. Pont6n, B. Westermark and V. Brunk, Aging of fibroblasts in long term tissue culture, Scand. J. Clin. Lab. hlvest., 29 (123) (1972) 26. 6 C. W. Daniel and L. J. T. Young, Influence of cell division on an aging process. Life-span of mouse mammary epithelium during serial propagation in vivo, Exp. Cell Res., 65 (1971) 27-32. 7 R. J. Hay, R. A. Menzies, H. P. Morgan and B. L. Strehler, The division potential of cells in continuous growth as compared to cells subcultivated after maintenance in stationary phase, Exp. Gerontol., 3 (1968) 35-44. 8 J. S. McHale, M. L. Mouton and J. T. McHale, Limited culture lifespan of human diploid cells as a function of metabolic time instead of division potential, Exp. Gerontol., 6 (1971) 89-93. 9 V. J. Cristofalo, Animal cell cultures as a model system for the study of aging. In B. L. Strehler (Ed.), Advances in Gerontological Research, Vol. 4, Academic Press, New York, 1972, pp. 45-79. 10 J. C. Houck, V. K. Sharma and L. Hayflick, Functional failures of cultured human diploid fibroblasts after continued population doublings, Proc. Soc. Exp. Biol. Med., 137 (1971) 331-333. 11 R. Holliday and G. M. Tarrant, Altered enzymes in ageing human fibroblasts, Nature, 238 (1972) 26-30. 12 E. Heikkinen, M. Aalto, T. Vihersaari and E. Kulonen, Age factor in the formation and metabolism of experimental granulation tissue, J. Gerontol., 26 (1971) 294-298. 13 H. P. von Hahn, Structural and functional changes in nucleoprotein during the ageing of the cell, Gerontologia, 16 (1970) 116-128. 14 L. E. Orgel, The maintenance of the accuracy of protein synthesis and its relevance to ageing, Proc. Nat. Acad. Sci. U.S., 49 (1963) 517-521. 15 L. E. Orgel, The maintenance of the accuracy of protein synthesis and its relevance to ageing: A correction, Proc. Nat. Acad. Sci. U.S., 67 (1970) 1476. 16 S. Gelfant and J. G. Smith, Aging: Noncycling cells an explanation, Science, 178 (1972) 357-361. 17 A. Macieira-Coelho, J. Pont6n and L. Philipson, The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro, Exp. Cell Res., 42 (1966) 673-684. 18 A. Macieira-Coelho, J. Pont6n and L. Philipson, Inhibition of the division cycle in confluent cultures of human fibrobalsts in vitro, Exp. Cell Res., 43 (1966) 20-29. 19 P. Holm-Pedersen, Studies on Healing Capacity in Young and Old Individuals, Munksgaard, Copenhagen, 1973. 20 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
185 21 E. Volkin and W. E. Cohn, Estimation of nucleic acids, Methods Biochem. Anal., 1 (1954) 287-305. 22 A. M. Fenstad, The influence of female sex hormones on the oral mucous membrane, Ph. D. Thesis, University of Minnesota, 1973. 23 K. W. Giles and A. Meyers, An improved diphenylamine method for the estimation of deoxyribonucleic acid, Nature, 206 (1965) 93. 24 R. J. Boucek, N. L. Noble, K.-Y. T. Kao and H. R. Elden, The effects of age, sex, and race upon the acetic acid fractions of collagen (human biopsy-connective tissue), J. Gerontol., 13 (1958)2-9. 25 W. Doberauer, Beeinflussung von Wundheilungsvorgangen durch das I.ebensalter, Gerontol. Clin., 4 (1962) 112-127. 26 J. C. Houck and R. A. Jacob, The effect of age on the chemistry of inflammation, J. Invest. Dermatol., 42 (1964) 377-371. 27 G. Junge-H/Jlsing and H. Wagner, Alterations in mucopolysaccharide (glycosaminoglycan) metabolism with age. In A. Engel and T. Larsson (Eds.), Aging of Connective and Skeletal Tissue, Nordiska Bokhandelns F6rlag, Stockholm, 1969, pp. 213-228. 28 P. Holm-Pedersen and B. Zederfeldt, Granulation tissue formation in subcutaneously implanted cellulose sponges in young and old rats, Scand. J. Plast. Reconstr. Surg., 5 (1971) 13-16. 29 P. Holm-Pedersen and B. Zederfeldt, Respiratory gas tensions and blood flow in wounded areas in young and old rats, Scand. J. Plast. Reconstr. Surg., 7 (1973) 91 96. 30 P. Holm-Pedersen and A. Viidik, Tensile properties and morphology of healing wounds in young and old rats, Scand. J. Plast. Reconstr. Surg., 6 (1972) 24-35. 31 P. Holm-Pedersen, K. Nilsson and P. I. Br~memark, The microvascular system of healing wounds in young and old rats, Advan. Microcirc., 5 (1973) 80-106. 32 T. Gillman and J. Penn, Studies on the repair of cutaneous wounds, Med. Proc., 2 (1956) 121-186. 33 R. A. MacDonald, Origin of fibroblasts in experimental healing wounds: autoradiographic studies using tritiated thymidine, Surgery, 46 (1959) 376-382. 34 H. C. Grillo, Origin of fibroblasts in wound healing: an autoradiographic study of inhibition of cellular proliferation by local X-irradiation, Ann. Surg., 157 (1963) 453-467. 35 R. Ross, N. B. Everett and R. Tyler, Wound healing and collagen formation. VI. The origin of the wound fibroblast studied in parabiosis, J. Cell Biol., 44 (1970) 645-654. 36 B. L. Strehler, Aging at the cellular level. In I. Rossman (Ed.), Clinical Geriatrics, Lippincroft, Philadelphia, 1971, pp. 49-81. 37 B. L. Strehler, Aging and the utilization of informational patterns, Proceedings 4th European Symposium on Basic Research in Gerontology, Varberg, 1973. 38 R. Johnson, C. Chrisp and B. Strehler, Selective loss of ribosomal RNA genes during the aging of post-mitotic tissues, Mech. Age. Dev., 1 (1972) 183-198. 39 H. P. von Hahn, Primary causes of ageing: A brief review of some modern theories and concepts, Mech. Age. Dev., 2 (1973) 245-250.