The extraction of polymeric collagen from biopsies of human skin

The extraction of polymeric collagen from biopsies of human skin


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M. J. O. F R A N C I S a AND D. C. MAC~V[ILLAN b

aNuffield Department of Orthopaedic Surgery, University of Oxford, Nu~eld Orthopaedic Centre, Oxford OX3 7LD, and bDepartmentof Dermatology, United Oxford Hospitals, Oxford (GreatBritain) (Received J u n e i4th, 1971)


I. Medium sized biopsies (IOO m m 2) of human skin from 14 subjects yielded sufficient polymeric collagen for depolymerisation and ultrastructural investigations. 2. The yields obtained from one skin specimen by the a-amylase, EDTA and lyotropic relaxation (water) methods of extracting polymeric collagen are similar. 3- The responses to depolymerisation treatments of the three polymeric collagen samples extracted by each of the three methods from one skin specimen are crosscorrelated. There are however electron microscopical differences between the three polymeric collagen samples. 4- The results show that it feasible to study the polymeric collagen of normal and diseased human skin from medium sized biopsies.


The study of collagen in normal and diseased skin from adult man has been limited to the small fraction, less than 5%, which can be extracted with dilute salt or acetic acid solutionsl, 2. I t is these soluble collagen fractions that have been most intensively studied by those interested in the relation between disease processes and collagen metabolism 3-5. There are, however, several methods now available for the extraction of the remaining 95% of tissue collagen (insoluble or polymeric collagen) G. In the simplest connective tissue examined to date (bladder or intestinal mucosa) the polymeric collagen can be extracted directly by homogenisation of the salt-extracted tissues in 0.2 M acetic acid solutionsL Other tissues require pretreatment before polymeric collagen can be extracted by acetic acid. Pretreatment with a-amylase or EDTA has proved successful with tendon or skinS, 9 though not with intercostal cartilage or decalcified bone 6. Pretreatment with distilled water alone has also been used successfully with bovine coriuml°, 11. The extracted polymeric collagen can be purified further by salt precipitation 12, Biochim. Biophys. Acta, 251 (i97 I) 236-245



and resulting polymeric collagen fractions then show the low amino sugar content and the amino acid composition found in purified tropocollagene, 13. The polymeric collagen consists of aggregates of collagen molecules held together by non-covalent and covalent bonds14,15. STEVEN14 has described simple indirect methods for measuring the numbers of these bonds in samples of polymeric collagen and showed that the number and type of covalent bonds v a r y both with the age of the animal and with the individual tissue 15-18. The work which we now report shows that it is feasible to extract polymeric collagen from medium sized biopsies of h u m a n skin. We have studied the relative effectiveness of the three methods (a-amylase, E D T A and water) that have been used to extract polymeric collagen from tissues, including skin. We have also compared the ultrastructures and chemical stabilities of the extracted polymeric collagens. In our opinion these results provide a base from which investigations into the relations between the metabolism of polymeric collagen and disease processes can develop. A preliminary account of some of this work has appeared 19. SUBJECTS AND MATERIALS

Skin specimens were taken post-mortem from fourteen patients aged 2o-75 years who had no connective tissue abnormality. Using a template, three side by side diamond shapes of 5o-I5 ° m m 2 were marked on the skin of the lateral thigh, at the level of the greater trochanter. These areas were removed b y dissection and immediately wrapped in aluminium foil to minimise water loss b y evaporation. Taking biopsies of known area allowed us to calculate the collagen contents per m m 2 (ref. 20). a-Amylase (Bacterial Type I I I - A from Bacillus subtilis) and pronase were from Sigma Chemicals. Other reagents were of Analar grade. METHODS

Estimation of collagen The samples were first hydrolysed in 5.5 M HC1 at 123 ° for 6 h which liberates 95% or more of the total hydroxyproline. Then the hydroxyproline content of these samples was measured b y the methods of PROCKOP AND UDENFRIEND 21 a n d BERGMAN AND LOXLEY22 and the collagen content derived from the hydroxyproline concentration 23. These two methods gave identical results for hydroxyproline content of these samples.

Extraction of polymeric collagen The preliminary extractions removed the soluble collagen and the glycosaminoglycans (which precipitate collagen)24, 25 to leave a residue containing polymeric collagen. First the fat was dissected away, the specimen weighed and a small sample taken for total collagen estimation. Then the three parts of the skin specimen were separately treated as shown in Fig. I. The 'soluble' collagen was estimated from the hydroxyproline content of Supernatant 2 (Fig. I). Residue i was treated with 0.2 M NaH2PO a buffer, p H 5.4, to remove glycosaminoglycans, leaving Residue 2 which contained the remaining collagen. Biochim. Biophys. Acta, 251 (1971) 236-245




SKIN SPECIMEN (3 PARTS) 1) Minceand homogenise for 10 see in 25 ml 5% NaCl at 4 ° (2) Incubate 3 h at 4 ° (3) Centrifuge f_or10 1000 Xg

SUPERNATANT 1 Centrifuge at 40 000 Xg J~ for 60 rain




(I) Extract With 30 ml 0.2 M NaH2PO4 (pH 5.4) (x4)

(2) Extract With water (x2)

5% NaCl-solublecollagen

(Polymeric collagen)

Fig. I. P r e l i m i n a r y e x t r a c t i o n p r o c e d u r e of skin samples.

Extraction of the polymeric collagen from Residue 2 is shown in Fig. 2. Three methods of pretreatment were used. These made the major part of the collagen in the Residue 2 samples dispersable in 0.2 M acetic acid. A different method was applied to each of the three Residue 2 samples derived from one subject. The pretreatments were impure a-amylase 9, EDTA 9 and water ('lyotropic relaxation' method of VEIS et a/)°,11). Fig. 2 gives the details. All mixtures were gently stirred. To prevent bacterial growth the a-amylase mixture contained 2000 units penicillin and 2000 #g streptomycin. The Residue 3 samples from these pretreatments were homogenised in 0.2 M acetic acid (see Fig. 2) and centrifuged to give Supernatant 3, The polymeric SKIN RESIDUE 2 (3 PARTS)



Incubate in 15 ml water at 1 8 - 2 0 ° for 18 h


Incubate in 25 ml 0.1 M NaH2PO4 (pH 5.4) + 1 mg el-amylase at 1 8 - 2 0 ° for 90 h

Incubate in 15 ml 4% (w/v) EDTA at 18-20 ° for 18 h

Wash residues with 30 ml water (x6) SKIN RESIDUE 3 (3 PARTS) I =



omogenise for 8 - 1 0 sec (ultra-turrax disintegrator) in 100 ml 0.20 M acetic acid (x2) ~



10 rain



(contains dispersed polymeric collagen)

(contains undispersed collagen)

Fig. 2, E x t r a c t i o n o f p o l y m e r i c collagen f r o m c o l l a g e n - c o n t a i n i n g skin residues after t h r e e pretreatment methods.

Biochim. Biophys. Acta, 251 (1971) 236-245



collagen dispersed in this supernatant was precipitated b y adding I.O M N a O H until the p H was 6 - 7. The collagen precipitated in two phases. Visible large fibres came down immediately (polymeric collagen fraction i), these were removed on a magnetic stirrer. Over the next 24 h (18-2o °) more gelatinous collagen, without visible fibres, precipitated (polymeric collagen fraction 2). I t was collected by centrifugation at iooo × g for io rain.

Depolymerisation treatments of polymeric collagen fraction I Polymeric collagen fraction I samples were tested for their stability to depolymerisation treatments by methods adapted from STEVEN14. Polymeric collagen fraction i samples were divided into four aliquots (each of o.2-3.5 rag) and one aliquot tested by each of the following treatments: (z) Thermal denaturation. After suspension in 5 ml water the polymeric collagen fraction I aliquot was boiled at ioo ° for I h. The soluble gelatin fraction was filtered off. The hydroxyproline contents of the soluble and insoluble were measured. (2) Cold alkali treatment. The polymeric collagen fraction I aliquot in I ml water was cooled to 4 ° and i ml 4 M N a O H added; the mixture was incubated at 4 ° for 6 days with occasional shaking. Then the p H was raised to 5-7 with 1.5 M acetic acid and the supernatant removed. To ensure t h a t the reaction mixture reached a neutral p H a further 2 ml of 15 mM acetic acid was added and the mixture allowed to stand for at least 2-3 h. The supernatant was removed after centrifugation of the mixture at iooo × g for IO min. The supernates were combined and filtered and the hydroxyproline content measured (cold alkali-soluble collagen). Then the residue was denatured by boiling as above (thermal denaturation). The p H was checked after boiling to ensure that depolymerisation of the collagen was not due to any residual alkali. (3) Hydroxylamine treatment at 4 o°. I ml 2.0 M hydroxylamine, p H 9.0, was added to an aliquot of polymeric collagen fraction I in I ml of water. The mixture was incubated for 2 h at 4 °0 and the supernatant removed. The residues were washed 2 times with 4 ml water to remove traces of hydroxylamine. The supernates were combined, filtered and the hydroxyproline content measured (hydroxylamine-soluble collagen). The residue was denatured by boiling as above (thermal denaturation). (4) Pronase treatment. 5 ml o.oi M CaCI~, IO mM Tris buffer solution, p H 7.0, containing 0.05 mg pronase was added to an aliquot of polymeric collagen fraction I and incubated for 20 h at 20-22 °. The supernatant was removed, filtered and the hydroxyproline content measured (pronase-soluble collagen). The residue was denatured by boiling as above (thermal denaturation).

Electron microscopy Polymeric collagen samples were fixed in 3% glutaraldehyde in o.I M cacodylate buffer, p H 7.0. Samples were part fixed in 1% osmic acid, stained in I°/o phosphotungstic acid and then embedded in araldite and sectioned. The sections were examined in a Phillips ioo electron microscope.

Biochim. Biophys. Acta, 251 (1971) 236-245

24 ° TABLE

.xl. j . o . F R A N C I S , D. C. M A C M I L L A N I


Extraction method

Total collagen

5% NaCl-soluble collagen

Polymeric collagen

Final residue

Water a-Amylase EDTA

39 39 39.5

0.37 0.32 0.39

26.5 23-5 3°.o

12 I5 9

(B) COLLAGEN" (°' o O F T O T A L C O L L A G E N ) I N T H E D I F F E R E N T


S a m e s p e c i m e n a s i n (A)

Extraction method Water a-Amylase EDTA

5°o NaCl-soluble collagen i 0.8 i

Polymeric collagen Fraction •

Fraction 2

68 60 76

i 3 i

insoluble (residue) 3° 36 22


Extraction of collagen from human skin The total skin collagen of the fourteen specimens was 15o-6oo #g/mm 2 and the extraction procedures removed lOO-35o #g/ram 2 (45-90%) of the total skin collagen. The three types of pretreatment (water, a-amylase and EDTA) gave similar results on the same skin specimen. Table I, A and B, shows typical figures for the three methods on the same skin sample; the results are given as percentages of the dry weight. Collagen accounted for nearly 40% of the dry weight. The three methods (Table IA) gave comparable yields of polymeric collagen. The distribution of collagen into the different fractions, expressed as percentages of the total collagen, are given in Table IB (same skin specimen). 1% or less of the collagen was soluble in 5% NaC1, 61-77 % of collagen was extracted (the polymeric collagen) and the remaining collagen was in the residue. In this table the two polymeric collagen fractions, of large visible aggregates (polymeric collagen fraction I) and small gelatinous aggregates (polymeric collagen fraction 2), are shown separately. Nearly all the extracted collagen was in the polymeric collagen fraction I. The polymeric collagen fraction 2 was never greater than lO%. The amount of polymeric collagen fraction 2 can be increased either by ultraturraxing Residue 3 (Fig. I) in acetic acid for longer periods, e.g. I5 rain, or by centrifuging the acetic acid dispersion of collagen at higher g (see VEIS et a1.1°,11). However, these treatments decrease the overall yield of polymeric collagen. The least effective pretreatment method for the specimen in Table I was aamylase, and a-amylase pretreatment was the least effective method in 7 of the i i specimens to which all three pretreatments were applied. But the reduced extraction after a-amylase pretreatment was not significant at the 5% level. There was no significant difference between the water and EDTA methods. The percentage extraction of polymeric collagen from skin did not correlate with the size or weight of the biopsy nor, on this mature skin, with the age of the subjects. Biochim. Biophys. Acta, 251 ( i 9 7 1 ) 2 3 6 - 2 4 5



Ultrastructure of polymeric collagen None of the polymeric collagen samples contained any adhering electron dense material. This suggests that all three pretreatments have effectively removed the maior proportion of the interfibrillar matrix present in vivo. All the fibres showed the characteristic cross-banding and staining of collagen. The fibres in the polymeric collagen fraction i isolated after a-amylase pretreatment were compact and rod-like. But the fibres in the polymeric collagen fraction i isolated after either water or EDTA pretreatments were swollen and less compact. The ends of some of these fibres were shredded and showed protofibrils partly unwound from the larger fibrils. Some of the fibrils consisted of loosely aggregated collections of protofibrils which had unwound. The polymeric collagen fraction 2 contained only fine fibres with no discernible protofibrils. The fine fibres had not formed into the thick fibres seen in polymeric collagen fraction I. The length of the fibres was the same in both types of polymeric collagen.

Depolymerisation of polymeric collagenfraction I The polymeric collagen fraction I extracted by the water, EDTA and a-amylase methods were each divided into four. One was denatured by boiling, the others were pretreated with cold alkali or hydroxylamine or pronase before boiling. These treatments break some of the bonds which hold together the aggregates of collagen molecules 14,15. The extent of the resulting depolymerisation is measured from the amount of hydroxyproline in the supernatant fractions. Table I I gives the results from a single skin specimen and these are representative for the 14 specimens studied. Boiling 'solubilised' up to 8% of tile collagen. Pretreatment with hydroxylamine and pronase solubilised only a further 2-3%. The cold alkali treatment was much more effective with up to 45.5% of the collagen entering the supernatants. TABLE II STABILITY OF POLYMERIC COLLAGEN FRACTION I EXTRACTED BY THE ~VATER, (I-AMYLASE AND E D T A METHODS FROM A SINGLE SAMPLE OF HUMAN SKIN Treatment methods

Collagen ' solubilised' ( % ) Extraction method : Water a-A mylase


(I) Boiling alone


7 .8


(2) P r e t r e a t m e n t + boiling (a) 4°°hydr°xyamineBoiling

7.5 2 } 9.5

63 } 9


(b) Pronase Boiling (c) Cold alkali Boiling

o / 6.7 6. 7 J" 7.5 ~ 39.5 32 J"

o / 9.2 9.2 8.5 ~45.5 37 f


o } 8.0 8.o 13"5 } 28 41.5

In this specimen (Table II), the polymeric collagen fraction I obtained after a-amylase pretreatment was less stable than the other two polymeric collagen fractions i in three of the four treatments. In the 43 depolymerisation treatments perBiochim. Biophys. Aeta, 251 (1971) 236-245



formed the polymeric collagen fraction i extract after a-amylase pretreatment was the least stable in 25, that after E D T A in I I , and that after water in 7. The average differences in stability were small (0-5%) . The stabilities of the polymeric collagen fractions I obtained after a-amylase pretreatment were significantly less than those obtained after water pretreatment (P < 0.02) and nearly significantly less than those obtained after the E D T A pretreatment (P < o.Io) (Student t test26). The degree of solubilisation differed in the fourteen specimens of polymeric collagen fraction I. The polymeric collagen fractions I from the two less mature subjects (aged less than 40 years) were distinctly less stable than those of the nine from more mature subjects (aged more than 60 years) as has been found by STEVEN for the polymeric collagen extracted from tendon 14,~5. In these samples the stability of a polymeric collagen fraction I sample was not correlated with the original weight or size of the sample nor with the percent extraction of the total collagen into the polymeric collagen fraction I (and so not with the size of the residue). It was unrelated also to the relative size of the polymeric collagen fraction 2 (small fragment) fraction. To see if the stability of the polymeric collagen fraction I in the individual subjects was varying due to the method of pretreatment the results were analysed for cross-correlations by Friedman's test 27. The stabilities to boiling of the three samples of polymeric collagen fraction I extracted from the same specimen of skin were highly significantly correlated (P < o.oi). The stabilities of the samples to cold alkali treatment and also to the subsequent boiling were correlated similarly (P o.oi). The pronase and hydroxylamine results were less markedly correlated (o.oi < P < 0.05. This analysis suggests that the covalent bonds within and between the collagen molecules of the polymeric collagen fraction I aggregates were little altered during the extraction procedures. The interrelationships between the effects of the different depolymerisation treatments on the polymeric collagen fractions I isolated from skin samples of the 14 subjects were further analysed by Spearman's correlation test 27. A positive correlation would imply that the treatments could be acting on similar sites in the collagen aggregates; absence of a correlation that the sites of action were different. Boiling alone non-specifically denatures and hydrolyses collagen as opposed to the more specific degradation produced by hydroxylamine, pronase and alkali 15. However, the effects of boiling alone and hydroxylamine were correlated (P < o.oi). This result suggests that hydroxylamine does not specifically degrade these collagen (polymeric collagen fraction I) samples. The effect of the other treatments were not significantly cross-correlated and this might be expected from their different modes of action 1~. DISCUSSION Ways of extracting polymeric collagen from mature tissues have only recently been developed. STEVEN6,8,9,17 has made empirical use of a-amylase and chelating agents, particularly EDTA, and obtained high yields of polymeric collagen from several tissues including skin. VEIS et al.l°, n obtained much lower yields with these methods and with their own method of water alone (lyotropic relaxation). However, they homogenised more thoroughly than STEVEN and also centrifuged their extracts at higher speeds1°, n. Both these differences might reduce the final yield of polymeric collagen and we found this to be so. These considerations suggested that where the Biochim. Biophys. Acta, 251 (1971) 236-245



water method was used in conjunction with the shorter mechanical disruption times of STEVEN'S procedures, it would give a high yield and our results confirm this. As performed in this study, the waterl°, 11 a-amylase s and E D T A 9 pre-extraction procedures dispersed equivalent amounts of polymeric collagen into acetic acid. Although the yields were similar, ultrastructural differences between the polymeric collagen fractions were found. The a-amylase pretreated fraction contained mostly compact, normal collagen fibres TM, whereas the fibres in the water and E D T A pretreated fractions were more swollen and loose with m a n y of the smaller protofibrils visible. The studies of FINLAY et al. 2s have shown that a-amylase pretreatment applied directly to 6o-#m sections of adult human skin does not damage the collagen fibrils. Hence the fibril disintegration seen in our studies could be due to the general homogenization steps used in our extraction methods. However, similar homogenization times were used in all three methods. Our findings suggest therefore that the pretreatments must be having some additional effect on the polymeric collagen which is seen on reaggregation of the extracted polymeric collagen fibrils. The looseness of the polymeric collagen fraction I fibrils extracted after water or E D T A pretreatment is not associated with a reduced resistance to depolymerisation. Rather the reverse, for the a-amylase pretreated polymeric collagen fraction I was slightly, but significantly (P - o.0i), the least stable of the three fractions. The mechanism of this reduced stability after the a-amylase pretreatment is not clear. The studies of FINLAY et al. ~s support earlier suggestionsS, ~9 that the impure aamylase preparations remove the glycoprotein component of the fibres without any proteolysis. Limited proteolytic activity on tropocollagen has been detected in such preparations of impure bacterial a-amylaseS°, sl but this activity is negligible at the low pHs and enzyme concentrations used in our extractions. The facts that collagenase is stabilised by its substrate collagen 32 and that it m a y be secreted as a pro-enzyme 32 suggest alternative explanations for our findings. In contrast to the conditions that are most frequently used in other studies, our collagen samples have, at no stage, been extracted w i t h lipid solvents which might inactivate collagenase or its proenzyme. Collagenase digestion of polymeric collagen could therefore take place during the pretreatment, most particularly during the a-amylase pretreatment which is the most prolonged. Furthermore the proteases present in the a-amylase preparation could also convert the inactive pro-enzyme of collagenase to its active form and so increase further the digestion of polymeric collagen. The lack of correlation between looseness of the polymeric collagen fraction I fibres on electron micrographs and decreased stability to depolymerisation in this skin study, is paralleled by a similar finding for the collagen derived from the intestine. Intestinal collagen has a very disordered ultrastructural appearance, yet is remarkably resistant to depolymerisation treatment ~. A cross-correlation was present between the stabilities of the three polymeric collagen fraction I fractions derived from the same specimen of skin. This was reassuring for it indicates that the pretreatments had little effect on the stability of the collagen molecules and so the polymeric collagen fractions are probably representative of tissue collagen. These results thus support STEVEN'S6 claim that he is achieving dispersion of mature collagen without producing significant denaturation. The presence of an association between resistance to depolymerisation and the presence of covalent bonds has been mentioned above. The chemical structures of Biochim. Biophys. Acta, 251 (1971) 236-245




these bonds are being extensively studied and several different types have already been detectedla, aa-37. GTEVEN'S14,15 use of cold alkali, pronase and hydroxylamine as depolymerisation treatments has already shown that the collagen from different tissues probably contains different types of covalent bonds. STEVEN6,17found that the stability of polymeric collagen derived from skin was markedly reduced by cold alkali but little affected by hydroxylamine or pronase and we have confirmed this pattern of response. Another important source of variation in the covalent bonds is that their number increases with age2,16-18. This increase does not cease when an animal or man reaches adult age, for increases in stability of polymeric collagen continue throughout an animal's life2,8,15,17,is. Even in our small series the stability of the polymeric collagen increased from the under 40 years of age group to the over 60 years of age group. It will therefore be important, when selecting the control subjects for studies of diseased connective tissue, to match the ages of the subjects. From the view point of studies on human skin, our results on fourteen subjects show that sufficient polymeric collagen can be extracted from medium sized biopsies for detailed studies. The yields of polymeric collagen from the fourteen subjects varied widely as did the stabilities of the polymeric collagen fractions I to depolymerisation treatments. However, the three extraction procedures gave similar yields of polymeric collagen from the skin of any one individual. The polymeric collagen fractions I so obtained also behaved similarly in the depolymerisation treatments. However, the water method is the simplest to use. We suggest therefore that it is the method of choice for comparative studies in man. These techniques are now being applied to skin biopsies from normal subjects and to those with various connective tissue disorders. ACKNOWLEDGEMENTS

We are grateful to Mrs. J. Farmer for carrying out the electron microscopy and to Mrs. J. Marsh for technical assistance. We thank Drs. H. R. Vickers and R. Smith for advice and encouragement during the course of this work. This work was supported by a Medical Research Council Grant to M.J.O.F. REFERENCES i A. VRIS, in G. N. I{AMACItANDRAN, Treatise on Collagen, Vol. I, A c a d e m i c Press, N e w Y ork, I967, p. 367 • 2 S. BAKERMAN, Biochim. Biophys. Acta, 90 (1964) 621. 3 E. D. HARRIS AND A. SJOERDSMA, Lancet, ii (1966) 707 . 4 J- UITTO, O. LAITINEN, ~3,1. HANNUKSELA AND I(. K. MUSTAKALLIO, Scand. J. Clin. Lab. Invest., 19, Suppl. 95 (1967) 415 0 . LAITINEN, J. UITTO, M. HANNUKSI~LA AND K. I. KIVIRIKKO, Clin. Chim. Acta, 21 (1968) 321. 6 F. S. SXEVEN, in E. A. BALAZS, Chemistry and Molecular Biology of the Intercellular IVIatrix, Vol. i, A c a d e m i c Press, N e w York, 197 o, p. 43. 7 F. S. STEVEN, D. S. JACKSON, J. D. SCHOFIEI.D AND J. B. L. t~ARD, Gut, IO (1969) 484 . 8 F. S. STEVEN, Ann. Rheum. Dis., 23 (1964) 300. 9 F. S. STEVEN, Biochim. Biophys. Acta, 14o (1967) 522. IO A. VEIS AND R. S. BHATNAGAR, in E. A. BALAZS, Chemistry and Molecular Biology of the Intercellular ,'~1atrix, Vol. i, A c a d e m i c Press, New Yor k, 197o, p. 279. I I A. VEIS, R. S. BHATNAGAR, C. A. SHUTTLEV,'ORTH AI'qD S. MUSSELL, Biochim. Biophys. Aeta, 200 (197 o) 97.

Biochim. Biophys. Acta, 251 (1971) 236-245

POLYMERIC COLLAGEN FROM HUMAN SKIN 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37


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Biochim. Biophys. Acta, 251 (1971) 236-245