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Biochem. J. (1984) 222, 663-668 663 Printed in Great Britain
Reconstituted collagen fibrils
Fibrillar and molecular stability of the collagen upon maturation in vitro
Carl Christian DANIELSEN Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark
(Received 19 March 1984/Accepted 29 May 1984)
During the maturation in vitro of reconstituted collagen fibrils prepared from rat skin, the mechanical and thermal^ stability^ of collagen increased and the^ pepsin- solubility decreased. At the same time a larger fraction of the pepsin-soluble collagen attained a lower molecular thermal stability that resulted in^ a^ biphasic thermal transition of the soluble collagen. Type-I collagen, with a similar biphasic thermal transition, was isolated from acid-insoluble rat skin collagen.
Reconstituted collagen fibrils attain increasing mechanical and thermal stability during matura- tion in vitro when incubated in air at 37°C (Danielsen, 1981a,b). These changes in stability are similar to those occurring in collagenous tissues during aging in vivo. Usually the 'helix-to-random^ coil' transition upon heating of soluble collagen shows a symmetri- cal transition curve, but some preparations of acid- soluble rat skin collagen have a distinct skewness in the denaturation profile (Danielsen, 1982a). The acid-soluble collagen is supposed to represent the more mature and cross-linked part of fibrillar collagen that is extractable by neutral-salt solutions
and dilute acids^ (Robins, 1980). The^ changed
denaturation profile for this collagen fraction may reflect a^ conformation^ or^ structural^ change^ of^ the
collagen molecule during age-related stabilization
of the fibrils. Therefore an investigation of the
relationship between changes in the denaturation profile of the molecular collagen and the extent of stabilization of reconstituted collagen fibrils^ that were matured in vitro was performed.
Materials and methods Materials
Pepsin (crystallized and^ freeze-dried) was^ pur-
chased from Sigma Chemical Co. The^ DEAE- cellulose (^) ion-exchanger used was Whatman DE-
Reconstitution and maturation of collagen fibrils
Collagen fibrils were^ reconstituted^ and matured
in accordance with a previously described proce-
dure (Danielsen, 1981a). Briefly, a^ stock^ prepara- tion of purified acid-soluble collagen was obtained from the dorsal skin of 60-day-old male Wistar rats. The collagen was reconstituted into fibrils by gradual heating of neutral solutions of the collagen. The collagen fibrils were dried to membranes within 11 days after aggregation. The membranes were then cut into 4mm-wide strips appropriate for mechanical testing. Eight groups of strips were
matured for different time periods (11-150 days
after aggregation) by incubation in air at 37°C. The maturation was stopped by transferring the strips
to liquid N2. Immediately after the completion of
the aggregation, a portion of collagen fibrils was precipitated by centrifugation and stored in liquid N2 until the analyses were performed.
Mechanical testing and determination of thermal
stability of the fibrils
The mechanical (^) strength of the (^) collagen mem- branes that were matured for different time periods after^ aggregation^ was^ determined^ in
accordance with the previously described proce-
dures (Danielsen, 1981a). The thermal stability of
the collagen membranes was determined as the
area shrinkage without tension during heating (AST) and the^ shrinkage^ temperature^ (Ts) was
calculated as the temperature for 50% of this area
shrinkage (Danielsen, 1981b).
Solubilization and isolation of collagen Samples of the stock preparation of acid-soluble
collagen and of the^ reconstituted^ collagen fibrils
that were matured for 0-150 days were incubated
with stirring in 0.5M-acetic acid at 1 :10 pepsin/col-
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C. C. Danielsen
lagen weight ratio at 4°C for I week. After the incubation the (^) suspensions were centrifuged
(50000g for Ah). The resulting supernatants were
dialysed against 0.15M-CaCl2/0.05M-Tris/HCl buffer, pH 8, and re-centrifuged (1 h), and NaCl was added to give 4M. The precipitated collagen was dissolved with (^) 5mM-acetic acid and centri-
fuged (50000g for 1 h). The solubility of a sample
was defined by the amount of hydroxyproline in the (^) resulting supernatant divided by the total amount of hydroxyproline in the supernatant and the pooled precipitates. A collagen membrane that was prepared and matured for 85 days, as described above, was subjected to more extensive pepsin digestion in 0.5M-acetic acid at 4°C for 4 days. Pepsin was added to give a pepsin/collagen weight ratio of 1: 5 at the start of the incubation and added again after 2 days' incubation to give the same weight ratio. The insoluble residue resulting from the extrac-
tion of 40g (wet wt.) of rat skin with 0.5M-acetic
acid from the 60-day-old rats (Danielsen, 1981a)
was (^) re-homogenized in 100 ml of 0.5M-acetic acid,
combined with 100mg of pepsin and incubated
with (^) stirring at 4°C for 1 week. The incubation
mixture was then centrifuged (500OOg for 1 h) and
the resulting supernatant dialysed against 0.15M-
CaCl2/0.05M-Tris/HCl buffer, pH7.5. After^ re-
centrifugation (50000g for 1 h), the collagen in the supernatant was (^) precipitated by the addition of NaCl to give 4M and dissolved in 5mM-acetic acid. Thereafter the collagen solution was diluted 1:
with a 0.4M-NaCl/O.lM-Tris/HCl buffer, pH7.4,
and chromatographed on a DEAE-cellulose col- umn by the procedure of Miller (1971). The break- through fractions were pooled, dialysed against 5mM-acetic acid and diluted 1:1 with 2M-
NaCl/0. 1 M-Tris/HCl buffer, pH 7.4, and, after
adjustment of pH to 7.4 with (^) 1M-NaOH, the collagen was fractionated by the method based on
that of Chung & Miller (1974) by sequential
addition of NaCl to give 1.7M, 2.5M and 4M. The
4M-NaCl-precipitated fraction^ was subjected to DEAE-cellulose chromatography by the procedure
of Bentz et al. (1978). The collagen was dissolved
in 2M-urea/20mM-NaCl/0.05M-Tris/HCl buffer,
pH 8.6, and applied to a column that was (^) equili-
brated with the same buffer at 15°C. The absorbed
collagen was eluted with 100mM-NaCl.
Sodium dodecyl sulphate / polyacrylamide -^ gel electrophoresis Polyacrylamide-gel electrophoresis was carried
out in 5% (w/v) acrylamide gels in the presence of
sodium (^) dodecyl sulphate at (^) 6mA/tube for 6 h at
room temperature according to a previously de-
scribed procedure (Danielsen, 1982b) based on that
of Furthmayr & Timpl (1971).
Absorbance temperature transitions Duplicate determinations of the thermal stabil- ity of molecular collagen and production of smoothed denaturation profiles were performed by the procedures described in detail previously (Danielsen, 1982a). Briefly, the 'melting' of colla- gen was measured by recording the absorption difference at 227 nm between identical sample and reference collagen solutions (0.10-0.25mg/ml in 5mM-acetic acid) during gradual heating of the sample (0.24°C/min). For comparative purposes,
the reproduced denaturation profiles (the first
derivative of the absorbance versus temperature) were normalized to an area of one unit by dividing the first derivative by the total transition (^) absorp- tion change. The temperature for each successive 5% absorption change in the total transition absorption change was calculated. The fraction of collagen that (^) 'melted' below a certain temperature was calculated from these data by interpolation. The 'melting' temperature (Tm) was defined as the temperature at which 50% of the transition absorption change had occurred.
Results and discussion The stability of the reconstituted collagen fibrils increased during the maturation (Fig. 1). The mechanical stiffness (and strength) of the collagen membranes increased 3-fold from the 11th to the 150th day of maturation. The area (^) shrinkage without tension during heating and the fraction of
80 r (^0) to E 2= 60 2-. (^20) r__^ 0.- (^0) Z r: - ua E (^20) ._ x ce 7-
0
i4,
I
50 100 Maturation time (^) (days)
150
100
75 wD 0
(^5 0) as A: C (^2) S. 25 1--
To
Fig. 1.^ Stabilization of reconstituted collagen fibrils upon maturation The (^) collagen fibrils were incubated at 37°C in air and after various times removed for determination of mechanical strength (maximum stiffness, 0), percentage area shrinkage during (^) heating (AST, A) and (^) solubility by limited (^) peptic digestion (%, 0). (Vertical bars indicate + S.E.M.)
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C. C. Danielsen
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0 50 100 150 Maturation time (days) Fig. 3. Fraction ofpepsin-solubilized collagen that 'melted' below 37°C The fractions represent the transition absorption change at temperatures below 37°C relative to the total transition absorption change during thermal denaturation for the pepsin-soluble part of the reconstituted collagen fibrils that were incubated at 37°C in atmospheric air for 0-150 days. (The standard deviation of the difference of duplicate determinations is 0.0063.)
resulted in a pronounced transition between 34 and 36°C (Fig. 4). The fraction of soluble collagen from this experiment that 'melted' below (^) 37°C was approximately 3-fold higher than the correspond- ing fraction^ of the collagen from which the fibrils were prepared. Therefore the change in the denaturation (^) profiles that (^) was observed after maturation of the collagen in fibrillar form represents a (^) molecular destabilization of a fraction of the collagen during the maturation. The de- stabilization revealed as diminished molecular thermal stability of the matured collagen may either be a molecular change occurring during the maturation or reflect an alteration of the (^) collagen that was induced by the subsequently performed peptic digestion. If (^) this possible alteration is induced by the peptic digestion, then the collagen matured (^) in vitro must be more prone for such an alteration, since the thermal (^) stability of acid- soluble collagen was unaffected by the digestion procedures that were applied. The electrophoresis of the pepsin-solubilized collagen fraction of the fibrils indicated that degradation of the collagen chains could not account for the changed denaturation pattern (Fig. 5ii).
4 0.
0.1I
34 38 Temperature (0C)
46
Fig. 4. Denaturation profiles of soluble collagens isolated from reconstituted and native (^) fibrils Pepsin-solubilized collagen from^ reconstituted collagen fibrils that were incubated at 37°C in atmospheric air for 85 days ( ), pepsin-solubilized acetic acid-insoluble collagen from skin that was precipitated by 4M-NaCl and un- adsorbed on the ion-exchanger during the DEAE-cellulose (^) chromatography by the (^) procedure of Bentz et al. (^) (1978) (. (^) ), and the stock (^) preparation of acid-soluble (^) collagen not subjected to heat aggregation (------).
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t- 4.) 0 0.
C on 'd (^) 0. (^0) c) to.- 0 r. CIO
Stability of collagen on maturation in vitro
(ii)
2 4 6 I^0 Migration distance^ (cm)
(^2 4 )
Fig. 5.^ Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis ofsoluble collagens isolatedfrom reconstituted and^ native fibrils (i) Acetic acid-soluble collagen. (ii) Pepsin-solubilized collagen from reconstituted collagen fibrils incubated at 370C in (^) atmospheric air for 85 (^) days. (iii)-(v) Pepsin-solubilized acetic acid-insoluble (^) collagen from skin that was precipitated by 4M-NaCl (iii) and that on subjection to DEAE-cellulose chromatography by the procedure of Bentz et al. (^) (1978) was (^) separated in (^) collagen unadsorbed (^) (iv) and adsorbed (^) (v) on the (^) ion-exchanger.
The pepsin-solubilized acetic acid-insoluble skin collagen that^ was^ precipitated by 4M-NaCl during the sequential precipitation by 1.7M-, 2.5M- and
4 M-NaCl constituted 7% of the solubilized collagen
and contained in addition to type-I collagen an electrophoretic band with mobility similar to that of a (^) type-V collagen chain (Brown & Weiss, 1979) (Fig. 5iii). The latter collagen type was adsorbed on the ion-exchanger during the DEAE-cellulose chromatography by the procedure of Bentz et al.
(1978) (Fig. 5v). The denaturation profile of the resulting type-I collagen fraction (Fig. 5iv) was similar to that of the (^) collagen isolated from the collagen fibrils matured in vitro (Fig. 4). This similarity suggests that a modification of collagen is (^) occurring both in vitro and in vivo. Attempts to isolate collagen with a biphasic thermal transition from skin gave the highest yield for (^) peptic digests of the acetic acid-insoluble fraction. They have been unsuccessful for neutral-
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