Thermal denaturation of mineralized and demineralized bone collagens

© i970 by Academic Press, Inc. J. ULTRASTRUCTURE RESEARCH

32, 545-551 (1970)

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T h e r m a l D e n a t u r a t i o n of Mineralized and D e m i n e r a l i z e d Bone Collagens ~ LAURENCE C. BONAR2 AND MELVIN J. GLIMCHER3

Department of Orthopedic Surgery, Harvard Medical School, The Massachusetts General Hospital, Boston, Massachusetts 02114 Received August 11, 1969, and in revised form February 25, 1970 Polarization microscopy, X-ray diffraction, and electron microscopy have revealed an extremely intimate relationship between the highly ordered and "crystalline" organic matrix of collagen and the inorganic crystals in bone and dentine (2). In bone it has been shown that a major portion of the mineral phase is deposited within "holes" or compartments formed in the collagen fibrils as a result of the specific way in which macromolecules are aggregated in native type fibrils (2). One of the fundamental properties of collagen is the thermal denaturation and shortening observed when the collagen is heated above a well-defined shrinkage temperature. We have examined, by wide- and low-angle X-ray diffraction, the role of the mineral phase in stabilizing both the short-range (helical) and long-range (the packing of collagen macromolecules in the characteristic staggered arrangement of native collagen fibrils) structure of the collagen against thermal denaturation.

MATERIALS AND METHODS

Chicken bone and tendon. The middle two-thirds of the metatarsal bones of freshly slaughtered chickens 10-14 weeks old were carefully cleaned at 2°C of periosteum and endosteal tissue, split into longitudinal strips, extracted with ether, and thoroughly washed with cold 0.05 M Tris(hydroxymethyl)aminomethane buffer, 0.1 M KC1, pH 7.4. The bone was stored in the Tris buffer solution 4 until used. The tendons were removed from around the metatarsal bones, washed in cold distilled water, and stored in cold Tris buffer until used. Rat bone and tendon. The central two-thirds of the radii and ulnae from 150-g SpragueDawley rats were prepared as described above for chicken bone. Tendons were removed from the proximal end of tails of the rats, washed, and stored in cold Tris buffer until used. 1 This investigation was supported by grants from The John A. Hartford Foundation, Inc. and U.S.P.H.S., National Institutes of Health (AM-06375). Present address: Damon Biomedical SciencesDivision, Needham Heights, Massachusetts 02194. 8 Reprint requests: M. J. Glimcher, Department of Orthopedic Surgery, Massachusetts General Hospital, Boston, Massachusetts 02114. 4 Unless otherwise noted, the Tris buffer solution used was 0.05 M tris(hydroxymethyl)aminomethane, 0.1 M KC1, pH 7.4.

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TABLE I TEMPERATURE TO WHICH BONE AND DEMINERALIZED BONE COLLAGEN WERE HEATED Species

Chicken Rat Bovine Guinea pig

Temperature (°C)

65 60 65 60

82.5 80 82.5 80

100 100 100 100

Bovine bone. Beef shin bone was obtained from freshly slaughtered animals and cleaned of periosteal and endosteal tissue. Longitudinal strips about 2 mm thick were cut from 2-inch lengths of the bone with a band saw. The cut surfaces were then ground off using wet fine-grit silicon carbide abrasive paper. They were then washed with cold Tris buffer solution and stored in buffer until used. Guinea pig bone. The central two-thirds of the tibiae and fibulae of mature animals were prepared as described above for chicken bone. Demineralization. Pieces of bone 10-40 mm long and about 1-5 mm wide were demineralized in cold 0.5 M ethylenediaminetetraacetic acid (EDTA), pH 8.3, then washed thoroughly with Tris buffer solution to remove EDTA. Demineralization was carried out for at least 4 days and at least 1 day beyond the time when X-ray diffraction patterns indicated that no mineral was present. Other samples were ashed and chemical analysis of calcium and phosphorus were carried out confirming the fact that the samples were completely demineralized. Shrinkage temperature measurement. The shrinkage temperature of the tendons and demineralized bone collagen was determined by suspending the material in 0.05 M Tris buffer 0.15 M KC1, pH 7.4, at 40°C, increasing the temperature of the buffer 2°C every 10 minutes, and either measuring the length of the sample under isotonic conditions with an eyepiece micrometer or an electronic length transducer (1) or measuring the force produced under isometric conditions with a strain-gauge transducer. Thermal denaturation. Separate samples of mineralized bone and EDTA-demineralized bone collagen about 10 m m long and 2 m m wide were heated in Tris buffer for 10 minutes and for 30 minutes at each of the three temperatures listed in Table I. The lowest of these temperatures is about 5°C above the temperature at which the material was completely shrunk, the next is halfway between this temperature and 100°C. The samples were placed in the solution maintained at the desired temperature in a thermostat and stirred gently during heating. Precautions were taken to minimize evaporation of water from the buffer. At the end of the 10-minute or 30-minute period of heating, the samples were transferred to cold Tris buffer for 24-48 hours. All samples, regardless of whether they had been previously demineralized or not, were then treated with EDTA as described above, washed with Tris buffer, and stored in the buffer until used for X-ray diffraction studies. This sequence is shown in Fig. 1. X-ray diffraction. Low angle X-ray diffraction patterns were obtained with a RigakuDenki rotating-anode X-ray generator operated at 40 kV and 100 mA. Specimen to film distance was 110 ram, and collimation conditions were selected to give adequate resolution of the collagen low-angle diffraction pattern. Wide-angle patterns were obtained with a

547

T H E R M A L D E N A T U R A T I O N OF B O N E C O L L A G E N INTACT, MINERALIZED BONE

L

OEMINERALIZE WITH 0.SM EDTA

1

I WIDE-ANGLE AND LOW-ANGLE XRAY DIFFRACTION

HEAT TO Ts 4- 5°, 80-82.5 °C or 100°C

HEAT T O T s + 5 °, 80-82.5°C or 100°C

1 1

COOL 24 HOURS

COOL 24 HOURS

DEMINERALIZE

TREAT WITH 0.5 M EDTA

WITH 0.5M EDTA

1

WIDE-ANGLE & LOW-ANGLE XRAY DIFFRACTION

WIDE-ANGLE & LOW-ANGLE XRAY DIFFRACTION

HEATED IN THE DEMINERALIZED STATE

HEATED IN THE FULLY MINERALIZEDSTATE

Fro. 1. Preparation of bone for X-ray diffraction studies.

Phillips microcamera, specimen to film distance 15 ram, equipped with a 100 #m bore glass capillary collimator, or a flat-film camera, specimen to film distance 57 mm with a beam of about 1 mm diameter of the film. Specimens for X-ray diffraction were washed briefly in distilled water to remove buffer salts, then dried in air with a small weight (5-10 g) attached so they remained straight and grossly oriented during drying. Reflections of all X-ray diffraction patterns were measured, as were their relative intensities. RESULTS The mean shrinkage temperatures (the temperature at which 50 % of the decrease in length had occurred) for the collagen samples studied are listed in Table II. Some of these values are several degrees lower than previously published values (7). The observed shrinkage temperature is k n o w n to be influenced by both the rate of heating and the force against which the sample must shorten. Slow rates of heating, and

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TABLE II SHRINKAGE TEMPERATURES FOR TENDON AND DEMINERALIZED BONE COLLAGEN c~

Sample

Chicken Demineralized bone collagen Tendon Rat Demineralized bone collagen Tail tendon Bovine Demineralized bone collagen Achilles tendon Guinea pig Demineralized bone collagen

Temperature (C °)

57-59° 57° 55-56° 54° 53 55° 59-62° 55-56°

a Temperature for 50 % of maximum shortening.

small applied loads, which would tend to yield the lowest values, were used in the studies reported here. Figs. 2 and 3 show wide-angle and low angle X-ray diffraction patterns obtained from unheated samples of demineralized chicken bone and rat bone collagen, respectively, with comparison diffraction patterns obtained from chicken and rat tendon collagen. Fig. 4 shows the wide-angle pattern obtained from both bone collagens kept at 65°C, i.e., above the shrinkage temperature; it shows only a diffuse halo at 3.9 A, typical of denatured (hot) gelatin (4). Figs. 5, 6, and 7 show X-ray diffraction patterns obtained from samples of chicken bone which had been heated to 65°C, 82.5°C, and 100°C, respectively without prior demineralization (i.e., heated in the fully mineralized state), then cooled and demineralized. Comparison X-ray diffraction patterns obtained from samples of chicken bone which had been demineralized prior to being heated (i.e., heated in the demineralized state), then cooled and treated with E D T A are also shown. Fig. 5 shows that after heating to 65°C (5 ° above the temperature at which the demineralized bone collagen is completely shrunk) for 30 minutes followed by cooling and treatment with EDTA, samples of chick bone which had been demineralized prior to heating, and samples which were heated in the mineralized state both gave wide-angle and low-angle X-ray diffraction patterns that are virtually indistinguishable from patterns obtained from unheated samples of demineralized bone collagen. From Fig. 7 it can be seen that, following heating to 100°C for 30 minutes and subsequent cooling, the basic features of the collagen wide-angle diffraction p a t t e r n - -

THERMAL DENATURATION OF BONE COLLAGEN

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FIG. 2. X-ray diffraction patterns of chicken collagen. (a) Wide-angle X-ray diffraction pattern of dry demineralized bone collagen. (b) Low-angle X-ray diffraction pattern of dry demineralized bone collagen. (c) Wide-angle X-ray -diffraction pattern of dry tendon. (d) Low-angle X-ray diffraction pattern of dry tendon. (a) and (c): Specimen-tofilm distance 57 ram, no enlargement; (b) and (d): specimen-to-film distance 110 ram, enlarged 2 x.

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the 2.86 N meridional and 12 ,~ equatorial reflections--are present both in bone demineralized prior to heating and bone demineralized after heating. Furthermore, samples of bone which had been heated to 100°C in the mineralized state, then cooled and demineralized, also gave a low-angle X-ray diffraction pattern similar to that of unheated demineralized bone collagen. In contrast, bone collagen which had been treated after demineralization and then cooled and treated with EDTA, did not give a collagen low-angle pattern. After heating to 82.5°C followed by cooling and EDTA treatment, bone collagen demineralized before heating gave an essentially normal wide-angle pattern, but only very faint traces of a low-angle pattern, as shown in Fig. 6. Wide-angle and lowangle patterns obtained from fully mineralized bone heated to the same temperature, then cooled and demineralized are identical with the control patterns. The above results can be summarized as follows: Chicken bone heated to various temperatures in the mineralized state, then cooled and demineralized gave wideand low-angle X-ray diffraction patterns identical to patterns obtained from unheated demineralized bone collagen; bone which had been demineralized, then heated to 65°, cooled, and further treated with EDTA also gave both wide-angle and low-angle collagen X-ray diffraction patterns. However, demineralized bone heated to 100°C, then cooled and treated with EDTA, gave essentially normal wide-angle collagen X-ray diffraction patterns, but no low-angle pattern. Previously demineralized bone heated to 82.5°C then cooled and treated with EDTA gave essentially normal wideangle collagen X-ray diffraction patterns, but only faint traces of the low-angle pattern. Results obtained with rat, guinea pig, and bovine bones were similar to those obtained with chick bone, except that samples of previously demineralized bone heated to 80°C, followed by cooling and EDTA treatment did not give even traces of lowangle collagen X-ray diffraction patterns. Samples of bovine Achilles tendon and chicken tendon heated to 65°C for 10 or 30 minutes, then cooled and treated with EDTA, gave characteristic collagen wideangle X-ray diffraction patterns, but no low-angle X-ray diffraction patterns. DISCUSSION The similarity between the wide- and low-angle X-ray diffraction patterns of demineralized bone collagen and tendon collagen from the same species indicates that bone collagen has the same basic molecular architecture as soft tissue collagen. That is, both the short-range order (the helical backbone structure and the lateral intermolecular packing) and the long-range order (the macromolecular structure and aggregation state) are essentially identical in bone and soft-tissue collagen. There is

THERMAL DENATURATION OF BONE COLLAGEN

551

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Fr~. 3. X-ray diffraction patterns of rat collagen. (a) Wide-angle X-ray diffraction pattern of dry demineralized bone collagen. (b) Low-angle X-ray diffraction pattern of dry demineralized bone collagen. (c) Wide-angle X-ray diffraction pattern of dry tail tendon. (d) Low-angle X-ray diffraction pattern of dry tail tendon. (a) and (c): Specimento-film distance 57 mm, no enlargement; (b) and (d): specimen-to4ilm distance 110 mm, enlarged 2 ×. 3 6 - 701829 J . Ultrastrueture Resectreh

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BONAR AND GLIMCHER

FIG. 4. X-ray diffraction patterns of melted wet bone collagen, with the specimens held at 65°C during diffraction. (a) Demineralized chicken bone collagen. (b) Demineralized rat bone collagen. Specimen-to-film distance, 15 mm, enlarged 3.3 x.

THERMAL DENATURATION OF BONE COLLAGEN

5a

553

5c

FI6. 5. Wide- and low-angle X-ray diffraction patterns of demineralized chicken bone collagen after heating 30 minutes at 65°C, then cooling. (a) and (b). Bone heated, then cooled and demineralized. (c) and (d). Bone demineralized, then heated and cooled. (a) and (c): Specimen-to-film distance 15 ram, enlarged 3.3 ×; (b) and (d): specimen-to-film distance 110 mm, enlarged 2 x.

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FIG. 6. Wide- and low-angle X-ray diffraction patterns of demineralized chicken bone collagen after heating for 3IN minutes at 82.5°C and cooling. (a) and (b). Bone heated, then cooled and demineralized. (c) and (d). Bone demineralized, then heated and cooled. (a) and (c): specimen-to-film distance 15 mm, enlarged 3.3 x ; (b) and (d): specimen-to-film distance 110 mm, enlarged 2 ×.

THERMAL DENATURATION OF BONE COLLAGEN

Ta

555

7c

FIG. 7. Wide- and low-angle X-ray diffraction patterns of demineralized chicken bone collagen after heating 30 minutes at 100°C and cooling. (a) and (b). Bone heated, then cooled and demineralized. (c) and (d). Bone demineralized, then heated and cooled. (a) and (c): Specimen-to-film distance 15 mm, enlarged 3.3 x ; (b) and (d): specimen-to-film distance 110 mm, enlarged 2 x.

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BONAR AND GLIMCHER

no evidence that demineralization with EDTA adversely affects the integrity of the collagen structure. The results described in the preceding section indicate that demineralized bone collagen will thermally denature and shrink when heated to the appropriate temperature. This is accompanied by a loss of both the short-range order indicated by the loss of the wide-angle X-ray diffraction pattern, and long-range order indicated by the loss of the low-angle X-ray diffraction pattern. The short-range order is regained following several hours of cooling, even when heated for 30 minutes at 100°C. The long-range order is at least partially regained if the bone collagen is heated to only 5° above the shrinkage temperature, but is irreversibly lost on heating to higher temperatures. (An exception is chick bone, which partially regains its long-range order after heating at 82.5°C.) Fully mineralized bone does not shrink, even when heated at 100°C for 30 minutes. Furthermore, after cooling and demineralization, the collagen retains both its shortrange and long-range structure, and appears unchanged, as far as X-ray diffraction properties are concerned, by the heating. The powerful protective effect of the mineral phase in preventing the thermal denaturation of the collagen phase suggests a close, intimate relation between the mineral and the collagen fibrils. Available electron microscope evidence indicates that a major portion of the bone mineral is, in fact, located in "holes" or "channels" approximately 20 • or so across, resulting from the specific intermolecular packing arrangement of collagen (2). Such an arrangement readily explains the absence of thermal denaturation upon heating reported here. The stabilizing effect of the mineral may be a purely mechanical phenomenon, with the mineral, by its presence within the collagen fibrils, physically restraining the collagen from uncoiling and shortening. Chemical interactions between the Ca 2+ and PO48- ions and the side chain and backbone groups may also play a role. It has been reported that the shrinkage temperature of collagen is increased by high concentrations of phosphate, and that in some cases the collagen did not shrink even at 90°95°C (6). Fully mineralized bone, specific gravity 2.1, contains about 67 % mineral and 12.5 % PO4 corresponding to an effective concentration of 2.75 moles PO4 per liter. Krane and Glimcher (5) have reported that the nucleotidase activity of bone collagen is destroyed by heating the demineralized bone collagen above its shrinkage temperature. However, if the bone is heated to the same temperature in the fully mineralized condition, full nucleotidase activity is retained following demineralization. This observation of the protective effect of mineral on the catalytic potential of bone collagen is further evidence of the intimate relationship between mineral and protein in bone.

THERMAL DENATURATION OF BONE COLLAGEN

557

It is interesting to note that demineralized bone collagen, when heated 5°C above its shrinkage temperature, regains both its wide-angle and low-angle X-ray diffraction patterns, while tendon collagen from the same animals recovers only the wideangle pattern. This may be a manifestation of differences in the number of intermolecular bonds present, or their nature and distribution, as has been suggested from solubility studies (3). This phenomenon is being investigated more fully and will be described in a separate report. REFERENCES 1. BONAR,L. C., in preparation. 2. GLIMCHER,M. J. and KRANE,S. M., in RAMACHANDRAN,G. N. and GOULD,B. S. (Eds.), A Treatise on Collagen, Vol. II B: Biology of Collagen, p 67. Academic Press, New York, 1968. 3. GLIMCHER,M. J. and KATZ,E. P., 3. Ultrastruct. Res. 12, 705 (1965). 4. KATZ, J. R., DERKSEN,J. C. and BON, W. F., Rec. Tray. Chim. Pays-Bas 50, 725 (1931). 5. KRANE, S. M. and GLIMCHER, M. J., in LACRoIX, P. and BuDY, A. M. (Eds.), Radioisotopes in Bone, p. 419. Blackwell, Oxford, England, 1962. 6. VILLARICO,E. A., M. S. Thesis, Florida State University, Tallahassee, 1967. 7. WEIR, C. E., J. Res. Natl Bur. Std. 41, 279 (1949).