DEFFRACTION STUDIES ON EMMATURE CHECK BONES THOSE: for #56 Deaf“ 6? PH. D.- MICHIGAN‘ STATE UNIVERSITY. Russell J. Kraay, .1955; Jmnmfi Date 0‘169 This is to certify that the thesis entitled Diffraction Studies on Immature Chick Femurs presented in} Russell Kraay has been accepted towards fulfillment of the requirements for _PILD'_degree inmogy l 42: fa/rfifj Major professor Jaw: /7 ”5’3” DIF FRACTION STUDIES ON D'MnTURE CHICK BONES By a}? ‘ 1‘“ Russell J? Kraay AN ABSTRACT albmitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology Year 1955 Approved M fl/M 6474’s” Russell J. Kraay Immature chick femurs were studied using electron and x-ray dif— fraction in an attempt to answer the following questions regarding the process of calcification and the nature of the bone mineral: (1) are crystalline precursors present before the formation of bone apatite. (2) are there detectable changes in the preosseous cartilage prior to ossification, (3) is the first bone mineral actually crystalline or does it become crystalline upon dehydration. (U) is there any change in apatite as bone matures. (5) is bone mineral prepared by ethylene diamine extraction altered in ways which allow conclusions to be drawn with respect to the original bone? Dry. heat ashed. and ethylenediamine extracted embryonic chick femurs were finely powdered. transfered. and examined by electron microscopy and diffraction. In the second experiment. embryonic chick femurs from six to 21 days incubation were studied by x-ray diffraction either in the wet. dry. heat ashed. or ED ashed state. Nickel filtered copper Kpalpha radiation was used with a powder camera having an effective film diameter of 1#.32 cm. By means of electron microscOpy. a thin crystalline species was observed. Its diffraction pattern was always hexagonal. with a cal- culated "an axis of 5.29 X (assmning its innermost spots to be from the 100 plane). Since the crystal was also seen on grids other than those containing bone preparations, it must be regarded as a contaNh inant. No other crystal species ever gave rise to a diffraction pattern. afl xe- r Russell J. Kraay The x-ray diffraction patterns from dry preosseous femurs showed the presence of crystalline sodium and potassium chloride in the ap- proximate ratio of one to three. After bone apatite was detected (from the nine day femurs) no crystalline NaCl could be found and the amount of crystalline KCl was markedly reduced. The possible involve- ment of various binding mechanisms is discussed. No lines were visible on x-ray diffraction pattern from young wet bone. due to the high background. However. in the absence of distinguishable apatite lines. it is possible that the first mineral laid down in normal hydrated bone is non-crystalline. The study of dry embryonic chick bone shows no detectable change in lattice parameters from the time it is first seen. Ash from twenty one day femurs after extraction with ethylene diamine, gave a diffration pattern nearly indistinguishable from that of dry bone. the only difference being the loss of two broad bands. presumably due to the organic portion of dry bone. This indicated that in the process of ED extraction. there had been no detectable shift in lattice parameters. and no induced crystal growth. Heating ED extracted bone produced shifts in lattice parameters as compared with similarly heated dry bone. The ”c" axis retrained unchanged. but the "a" axis was decreased from 9.1423 for heated dry bone to 9.3613 for heated ED ashed bone. The calculated value for heat- °d dry bone corresponds closely to literature values for hydroxyl- apatite. whereas. the value for heated ED ashed bone corresponds more closely to values given for carbonate apatite. DIFFRACTION STUDIES ON IMHATURE CHICK BONES B y as M Russell J? Kraay A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology and Pharmacology Year 1955 Approved 4.22: if 1/424 647— rs’ AC mmmmm‘s The author wishes to express his sincere thanks to Dr. L. F. Wolterink. whose constant inspiration and keen insight have contribut- ed greatly to this work. He is also indebted to Dr. B. V. Alfredson, Head, Department of Physiology and Pharmcology. and to the other members of the depart- ment where this work was carried out in part. Grateful acknowledgment is due to Mr. D. E. Van Farowe. Chief Chemist. and to the office personnel of the Division of Occupational Health. Michigan Department of Health. where the x-ray diffraction experiments were carried out. Also to Mr. H. W. Thomas. Draftsman. for his technical assistance. The author also wishes to thank Dr. H. M. Bendler. Electron HoroscOpist. Physics Department. and Mr. D. A. Libby. Department of Poultry Husbandry. Acknowledgment is also made to the Atomic Energy Commission. Division of Biology and Medicine. for supplying the powder camera used in the x-ray diffraction work. Russell James Kraay candidate for the degree of Ibctor of Philosophy Final examination. June 17. 1955. 10:00 A. M.. Room 213A. Giltner Hall Dissertation: Diffraction Studies on Immature Chick Bones Outline of Studies Major subject: Physiology Minor subjects: Endocrinology, Biochemistry Biographical Items Born. April 2h. 1926. Munster. Indiana Undergraduate Studies, Hope College. 1943-#, Cont. l9U6-9 Graduate Studies, Michigan State University. 1950-1955 Experience: U. S. Navy. 194h-1946. Control Chemist. 1950. DePree Chemical Company. Holland. Michigan. Radiochemical Technician. Michigan State University. 1950-1951. Instructor in Physiology, Michigan State University. 1951-1952. Graduate Assistant. Michigan State Uni— versity, 1952-195U. Industrial Hygiene Chemist. Michigan Department of Health. 1954-1955. iii TABLE OF CONTENT ACICnOW-ledg'rlents o e o o o o o o e e s e 0 List Of Tables 0 0 o o a C I O o o o o O List of Figures C O U O O I O I D O D O O I. II. III. Introduction and Literature Survey The Nature of Apatites . . . Relationship of Home to Funeral Tissue Upon Heating . . . . . Size and Surface . . . . :30 ’11 EHUOUJZD Experimental Methods . . . . . . . A. Electron Diffraction Studies . 1. Preparation of Materials . 2. Instrumentation S 3. Mathematical Considerations . B. X—Ray Diffraction Studies . . . 1. Preparation of Materials . 2. Technical Aspects . . . . . Apatites Views Concerning the Nature of Bone Mineral . Techniques of Bone Preparation. . . . . . Information Obtained from Behavior of Calcified Chemical Properties of Bone Crystals Related to C. General Conventions Regarding Diffraction Presentation of Beta . . . . . . . . . . . . A. Electron Diffraction Studies . . . . . . B. X-Ray Diffraction Studies . . . . . . . . 1. Data from Dry Ehbryonic Femurs of Six Days Incubation iv Chemistry of the Process of Calcification . . Objectives of Thesis . . . . . . . . . . to Ten Page ii vi viii H \OO\$'l—‘ 13 15 19 19 19 20 21 21 23 25 26 26 3o 30 i——m - V7 ‘wq‘ 2. X-Ray Diffraction by "Wet" Fen'urs . . . . . . . 3. Comparison of Values from Dry and Ethylene- diamine Ashed Twenty-One Day Fezmrs Ignited at High Temperatures . . . . . . . . . it. Values Obtained from Ashing Young Embryonic Femer................... IV. DiscussionofResults..... . . ... .. .. .. . A. Electron Diffraction Studies . . . . . . . . . . . B. X-ray Diffraction Studies . . . . . . . . . . . . . 1. Discussion of Data Obtained from Dry Enbryonic Femurs After Six to Ten Days Incubation . . . 2. Studies on the Nature of Bone Mineral . . . . . V. Summary and Conclusions . ... . . . . . . . . . . . . . AppendixA. Tables .................... AppendixB. Figures .. Literatllre Cited . O I O O O O O O I O O O I l O O O I C C U 51 58 62 65 89 II III IV VII VIII XII XIII LIST OF TABLES Some Substituted Sodium and Potassium Phosphates. . . Conceptions of the Constitution of the fiineral Phase of calCfliEdTissueooooooooooooeoooo Calculation of Errors Involved in Estimation of Spot Position on an Electron Diffraction Pattern . . . . Calculation of Errors Involved in Estitation of Line Pbsition on an X-Ray Efiffraction Pattern . . . . . Average values for Six Largest Spacings of Crystals Observed Crystalline Components of Dry Early Embryonic Chick Femurs Average Values Obtained from Dry Bones at Different Ages Chemical Analyses on Embryonic Femurs . . . . . . . . Observed values for Bone Apatite Compared to Values Calculated for Bone Apatite Based on Lattice Parameters Observed Values for Whitlockite Compared to Values in literature Calculated Amounts of Sodium, Potassium. and Chloride Associated with Several Possible Binding Mechanisms in Cartilage Example of Individual keasurements from an Electron mffraCtion Pattern O O O O O O O O O O O O O O O 0 Summary of Electron Diffraction Data Comparison of values Obtained from a 1:1 Rfixture of NaCl and KClywith Those Found in the.ASTM.File . . Values Obtained From Six Day Dry Embryonic Femurs . . vi 26 33 34 36 42 55 67 68 69 XVI Values Obtained From Seven Day Dry Embryonic Femurs . . XVII Values Obtained From Eight Day Dry Embryonic Femurs . . XVIII Values Obtained From Nine Day Dry Embryonic Femurs . . XIX Values Obtained From Ten Day Dry Embryonic Femurs . . . XX Values Obtained From Eleven and Twenty—One Day Dry FemurS 0 O... D on. I o 0 II s on o oo‘O-U XXI Values Obtained From Dry 21 Day Femurs with Increasing HeatingandTemperature o o a o o o a o o o a a o o O XXII Values Obtained From Ethylemdiamine Ashed Femurs With Increasing Heating. Time and Temperature . . . . . . XXIII Comparison of Values From High Temperature Incinflerated DryBoneandEDAshedBone............. XXIV Comparison of Values from Heat Ashed Eight to Ten Day Femurs with Heated Mixture of NaCl and KCl . . . . . DIV List of Crystallin Materials with an "3." Axis Near 5029101'10058 oooooooooooooo-oeo vii a’e 70 72 73 71b 75 78 79 fim’J L. ' _——_ 2-339 E-‘M’b‘l: «I—W'A‘- - ll}. 15. LIST OF FIGURES Electron Micrographs of Powdered Bone . . . . . . . . Electron Diffraction Pattern from Thin Crystals in Fig. l X-Ray Diffraction Pattern fromPreosseous Femurs Compared withSaltMixture................... X-Ray Diffraction Patterns from Ez-bryonic Femurs . . Comparison Of my and ED A~3hed Bone o o o o o o o o 0 Effect of Heating Dry and ED Ashed Bone . . . . . . . Conversion of Dry and ED fished Bone to Whitlockite . X-Ray Diffraction Patterns of Dry and ED Ashed Bone Con- vertedtOWhitlocki‘be........o......o LRay Diffraction Patterns of 6 Day Embryonic Femurs X-Ray Diffraction Patterns of 7 Day Dubryonic Femurs X—Ray Diffraction Patterns of 8 Day Enbryonic Femurs X—Ray Diffraction Patterns of 9 Day Embryonic Femurs Comparison of Diffraction Patterns from Embryonic Femurs oflOtOZIDayS......o............ Comparison of Diffraction Patterns of Various Parts of 21 my Femur O O O C O O O O C O O O O O O O O O O 0 Comparison of Diffraction Patterns of Heat Ashed Pre- osseous Femurs with Heated Salt Mixture . . . . . . . . viii 1L1 82 83 85 ,__._.——..— a‘. I’ ‘Vv I. INTRODUCTION AND LITERATURE SURVEY A. We Crystallographic investigation into the nature of the bone mineral began with the x-ray diffraction work of DeJong (1926) in which he recognized the similarity between the diffraction patterns from bone and those from the apatite minerals. Earlier. however. Berzelius (184-5), Heppe - Seyler (1862) and Werner (1907). in attempt- ing to combine analytical data for bone into the chemical formula for a single molecule. had called attention to the apatite nature of the bone salt. lflneralogically. the apatites are a large group of naturally occurring hexagonal crystals belonging to the tripyramidal class (Dana. 1932). The apatite most thoroughly studied is fluorapatite, whose unit cell consists of lo-Ca. 6-P. zit-O. and 2-F. Its formula is 3[Ca3(POu)2] - Can. and the structural arrangement of the atoms in its lattice has been worked out by hhmel (1931). The apatites form ionic. rather than molecular or metallic crystals. despite their generally low solubility. Within the same general lattice. considerable substitution can occur. with only slight shifts in lattice parameters. Replacement of the F; with 01; or (OH)§' gives the corresponding chlorapatite or hydrmhpatite. with no significant detectable change in lattice Parameters as determined by x-ray diffraction methods (Altschuler. Cisney. and Barlow. 1953)- ——.. ‘— '-.. "J - .3- Carbonate apatites are also known having the general formula Ca003 - {933(P0u)2]n. Xpray diffraction reveals slight but significant differ- ences in lattice parameters as compared with fluoro- and hydroxylapatite. fltschuler. Disney and Barlow give the lattice parameters of OH apatite as a = 9.1+13 K. c = 6.875 3 whereas carbonate apatite parameters are given as a = 9.3144 R. c = 6.881 K. Roseberry. Hastings and Horse (1931). studying bone and teeth by x~ray analysis. concluded that the diffraction patterns of bone and teeth are essentially those of dahlite. a mineral carbonate apatite. The problem of the position of carbonate in the apatite has been particularly studied by Gruner and McConnell (1937). It (was recognized that the carbonate of calcified tissue might very well occupy the same position as that of the carbonates in the mineral apatites. From a study of francolite. they originally proposed a 1:1 substitution of certain 00;. groups to r splace P014. groups. Hendricks and Hill (1942) regard the replacement of 3 P01, groups by ll- CO3 groups as much more likely. Investigators using. in addition to x-ray diffrac- tion. crystal Optics and mole refractivity. have not been able to rigorously prove the exact position of carbonate in large mineral crystals. since a number of equally plausible alternatives exist. Agreement is qualitative or semi-quantitative at best. Additional substitutions which have been postulated are discussed by Neuman (1953). Heteroionic exchange of a sodium (Hodge. et 31.. 1943). strontium (Hodge. 1945) and uranyl ion (Neuman. et a1.. 1949) have been studied in bone. However. no careful crystallographic stucbr has ever established the actual presence of any of these ions in the apatite lattice. Simply on the basis of ionic radii. some ions are denied a position in the lattice. Potassium ion. for example. with an ionic radius of 1.33 K could not replace calcium in apatite with an ionic radius of 0.99 K‘without seriously disrupting the lattice structure. Although this statement is commonly repeated by such authorities as McConnell. Hendricks. Armstrong. and Hodge. there are numerous examples in mineralogy where K is assumed to replace Ca in a hexagonal phosphate lattice. Thus: TABLE I"I SO 30 AND A SIUM 31331' D PHD ATES Mineral a c Author NaSrPOu 10.65 5. 81 flement KSrPOQ, 10. 70 5- 87 " NaCaPOu 10.53 5.76 " moron 10.60 5.81; n CaNaPOz‘ 5.23 7.13 Bredig CaKl’Ou 5.58 7.60 - "‘ All values found in this Table were taken from Denny and Nemacki (1951*). Numerous examples may also be found in the silicates. In view of the examples cited. the statement that potassium is denied a position in the lattice must be regarded as questionable. It should be pointed out that the substitutions of sodium and potassium reduce the Ca/P ratio and that they represent approaches toward secondary phosphates 01+ substituted by monovalent cation). On the other hand. strontium. with a radius of 1.13 X is apparently able to replace calcium. If monovalent sodium (ionic radius 0.95 K) replaces calcium. as it might well do on the basis of size. the question of the unsatisfied valence must be answered. As yet. biological studies regarding heteroionic eXChange must wait for mineralogic evidence that am of these ions are actually ever part of the lattice. Returning to the problem of bone. Armstrong (1950) reviews ten preposed formulations for the nature of the bone salt (See Table II). All start with what is regarded as the best analytical data. To this they have added a series of secondary observations: solubility. in- congruent solubility. temperature curves. and diffraction. The present status of bone is subject to the following limitations. First. since the composition of adult bone is not uniform. the problem has been to explain the variations within the framework of what is generally lmown about apatite structure. Second. the origin of the apatite lattice in calcified tissues is far from clear since it goes down only in selected biological situations. and under conditions such that it does not have a true solubility constant. In view of the widespread occurrence and uses of the apatite phosphates. as fertilizers. there is a surprising lack of information regardirg the mechanisms of precipitation so as to give the various ionic ratios encountered. The chief reason for this is that there are primary. secondary and tertiary orthOphosphates. all of which form various types of mixed complexes depending on pH and the presence or absence of a variety of both cations and anions. The presence or absence of water in the final crystal lattice is also critical. .- .4.— TABLE II“ CONCEPTION OF THE CONSPITUTION OF THE MNERAL PHASE 0!“ CALCIFIED TISSUE Formulation Proponents Year 8: Ref. Ca/POu, 1. Mixture)of Gag“): (P01; . CaO . Cagz. etg. 3 2. 0.151120%“)2 Berzelius 1845 (2) 2.5 3. Ca[(0.P030a)ZCa]3CO3 Hoppe-Seyler; 1862 (3) 1.67 Werner; Cnssman 1907 (a) Gassman 1937 (5) 1.. Ca(OH) [c.( (you) 3 Klement 1929 (6) 1.67 + 31 e c bone as and bicarbonates 5. Cacog[ca3(ro.,)z]n Bogert and Hastings 1931 (15) 1.67- n - or 3 1-75 6. >26. - Gruner. McConnell. 1937 (13) [(P.C)8L,J6(Ca.0)l&] and Armstrong 7. Cam) - Ca (90 )] Hodge. et al.; 1938 (20) 1.67 2 l: 3 a 2 3 Bale: 1936 (21) Thewlis. Glock. and 1939 (22) 151er 8. C30 - Ca( )] Sobel. Rockenmacker.19’*5 (23) .BE- 3 P0” 2 and Kramer m Camu'n £33603); 9. (CaS ()ng Na)9- (P0 . Hendricks and Hill 1947 (2a) 320 (s betitutes hydrated tri- calcium phosphate) 10. c. on (on)2 Dallemagne 19117 (25.31) +3agou %3%603. ate. * This Table appears as Table 1. page 12. Armstrong (1950). slightly modified. 4”: .— Although the apatites are Classically described as anhydrous crystals. McConnell is convinced that water occupies a lattice position and Neuman and Neuman (1953) suggest the presence of hydronium ions. Both agree that water is lost betwoen BOO—500° C. as reported by Dallemagne (1952). It is clear that in the case of bone. the problem is that of a true heterogeneous system. in the physical chemical sense. with numer- ous "impurities". and in which exchange and adsorption play highly significant roles. Co-precipitation has often been suggested but never proved. Therefore. the biologic situation cannot be described only in terms of simple analytical values for calcium and phosphorus. The significant components of this system are still. not completely established. As a result. in a recent review (Neuman and ileum. 1953) the following statement is made. "It is generally agreed that the apatite lattice of bone mineral approximates the structure of hydroxylapatite." This is a very small advance in the years since the observations of DeJong. Obviously. proof for the many hypotheses currently in print requires more rigorous and sustained investigation than is presently being pursued. 0- W In addition to its major constituent. basic calcium phosphate which is present to the extent of 80 - 85%. bone mineral contains 3 - 5% carbonate (Neuman and Neuman. 1953). with small amounts of citrate - 27%. sodium - 180 mEq/Kg. potassium - l9 mEq/Kg. magnesium - 1% and a trace of fluorine (Gabriel. 1891). Bergstrom. 1952. Huggins. 1937. PbLean and Urist. 1955). The Ca:P04 ratio does not vary much in normal adult bone and is about 1.92-1.99 (Huggins. 1937). Leulier. Policard. axui Revol (1991) reported a calcium to phosphate ratio of 1.8 in the diaqfluyseal bone of chick embryos. Low calcium. high phosphate diet ‘has been shown to decrease the CazPOu nmle ratio (Sobel. Rockenmacher. Kramer. 1945). In spite of the variations encountered in bone compo— sition. x-ray diffraction has shown only the presence of an apatite structure (Neuman and Neuman. 1953). However. it is impossible to rigorously prove the presence of many plausible minor crystal species in the presence of strong apatite patterns unless their concentration is considerably greater than might be eXpected. The "formulation M from Armstrong's Table 64092 [Ca3(P0u)2]3 proposed by Klement (1929). indicates that the basic calcium phos- phate. hydroxylapatite.is the principal constituent of bone. This formula tacitly assumes that either carbonates or bicarbonates of Na. K and Ca are mixed in the bone mineral as separate species along with hydroxylapatite or that non-crystalline components exist. The problem of carbonate in bone has been extensively discussed by (banner and leonnell (1937). McConnell (1952). Hendricks and Hill (1912 and 1951). and Hendricks (1952). Both groups studied mineral carbonate apatite and attempted to relate their findings to bone. Their formulations (numbers 6 and 9 in Table II) again may be viewed as an effort to relate the analytical data to a molecular structure. Even though both groups agree that carbonate can be and probably is present in the apatite lattice. the exact position and total amount so incorporated is vigorously argued. McConnell. as a professional crystallographer. has not yet felt it necessary to abandon the attempt to fit analytical components into a unified crystal structure. Hendricks. on the other hand. with crystallographic experience also dating back to the early twenties. is frankly willing to admit various types of association short of incorporation into an apatite lattice. Since they are in possession of the same experimental data. but draw divergent conclusions. it is obvious that present data are not suffi- cient for the solution of the problem. There is a great deal of evidence in favor of hydroxylapatite as such. Among the foremost proponents of this concept are Thewlis. Glock and Murray (1939). Hodge and co-workers (French. at al.. 1938) and Bale (1940). These workers have demonstrated that x-ray diffrac- tion patterns obtained from "calcified tissue" and I'synthetic hydroxyl- apatite“ are indistinguishable. Unfortunately. doubt may be expressed both as to whether their "bone" was in the "normal" state and whether their synthetic hydroxylapatite was 'pure'. in the sense of being car- bonate free. The general feeling amorg these investigators is that carbonates and other salts are occluded. adsorbed or interstitially crystallized. Hendricks and Hill (1951) quoted Brasseur and mllemagne as saying that crystalline calcium carbonate of bone could not be de- tected in the amounts present. about 1» to 5 percent. However. Hendricks concluded that the methods used would detect even 1 percent carbonate (as calcite). In a very recent review. Posner (1955) claims to have shown the presence of calcite mixed with the phosphates of the in- organic portion of bone and teeth. and also in the mineral francolite by use of infrared spectroscopy. Dallemagne in 1952. primarily on the basis of dehydration studies. concluded that alpha (or hydrated) tri- calcium phosphate is the principal bone salt. This may be represented by the formula [033(P0h)2]3 . H2(0H)2 to show its similarity to [Ca3 , (I‘Mg)é]3 Ca(OH)2. givegrhydroxylapatite. However. it is evident that <>L tricalcium phosphate has a lower CazPOu ratio than hydroxylapatite. D. Tghnigges of Bong fleggation A good deal of the confusion which exists regarding the nature of mineral phase of calcified tissue arises from the fact that untreated bone gives very poor diffraction patterns. Since the lines from un» treated bone are broad and poorly defined (DeJong. 1926. Taylor and Sheard. 1929) a valuable clue is given regarding crystal size. At the same time. the poor resolution of the broad lines makes the pattern from.untreated bone indistinguishable from those arising from a wide variety of mineral apatites (Roseberry. Hastings and Horse. 1931. and Bredig. 1933). Therefore. a number of techniques of bone preparation have been used in an effort to more rigorously establish the nature of the bone salt (Neuman and Neuman. 1953). Any-manipulation may be criticized since even after simple removal from the intact organism and drying. bone can no longer be considered completely normal. Dny heat ashing. one of the most drastic techniques. produces an ash which gives a much sharper diffraction pattern than fresh bone (Klement & rrdmel. 1932). The procedure employed by Gabriel (1894) (boiling glycerol and KOH extraction of the organic matter) has been used ex- tensively in bone preparation and has the advantage of a rather low temperature in an.anhydrous medium. Even though some structural modi- fications in the bone salt undoubtedly occur. the diffraction pattern does not change (Dallemagne. 1952). A modification using ethylene 10 glqvcol.and KOH has been used by Crowell. Hodge and Line (1934). Boiling bone for 21+ hours followed by t‘yptic digestion was em. ployed by Bell. Chamber and Dawson (l9h7). This method causes structural modifications and loss of mineral elements (Dallemagne. 1952). For preparation of bone and teeth for electron microscopy. Robinson and Bishop (1950) autoclaved fresh bone at 27 pounds for 2 hours and blended it in distilled water. Later. Robinson and Watson (1952) obtained an electron diffraction pattern from n-butyl-methacry- late embedded bone. which showed good agreement with x-ray diffraction data on bone treated by various other methods. Again the rigors of the embedding process may have altered the bone mineral considerably. Recently ethylenediamine (ED) has been used by Arnold (1952) to study the amount of Can5 combined with the organic matrix of bone at various times after deposition. This report did not give a chemical or x-ray diffraction analysis of the ED ashed material. so it is not possible to estimate an effect on the crystal lattice from the data rewrtedo E. f a a F hav 0 C T U Hea Taylor and Sheard (1929) showed that incinerated bones produced much sharper lines on a diffraction pattern. and attributed this to growth of the crystals of bone mineral. 0n the basis of the diffuse lines obtained from unheated bone they concluded that the bone crystals probably contained only a few hundred molecules. Klement and Trbmmel (1932) also showed this sharpening of lines upon heating bone at 600° C. They also prepared a synthetic apatite with the material precipitating at pH 7.15, which when heated showed some fl—Ca3(POL,)2 lines. later, Hodge. LeFevre, and Bale (1938) conducted a thorough study of diffraction patterns of several calcium phOSphates. They showed that precipitates containing a Ca:P mole ratio of 1.93 to 2.1.2 all ex- hibited a hydroxylapatite diffraction pattern before ignition, but after ignition at 900°C. for one hour. precipitates with CazP ratios of 1.93 and 1.98 gave a pattern of fl-Ca3(POu)2, whereas. those with Ca:P ratios of 2.10 and 2.12 still gave a pattern of hydroxylapatite. They concluded that a reaction took place between the hydroxylapatite and excess ad- sorbed phosphate ions to give ,8-0a3 (P0202 in the cases of Ca:P ratios of 1.93 and 1.98. Sobel and co—workers (Hirschman. at 8.1.. 1947 and Sobel. et a1.. 1945 and 1949) were able to lower the Ca :POz, mole ratio of bone below 1.93 by diet and in each case verified the findings of Hodge. IeFevre and Bale on precipitated calcium phosphates. The pattern of ignited bone then can be used as a rough index of the Ca:P ratio. Neuman (1951). in discussing the work or Kunin. viewed the high phosphate bones of Babel as a substitution of some of the calcium by hydronium ions which will dehydrate at about “00°C. When this occurs. the lattice con- figuration of apatite presumably collapses, giving lB-tricflcium phos- phate. F. WWWLOW Since the work of Taylor and Sheard (1929). in which they concluded that bone crystals probably contain only a few hundred molecules. much % . 12 work has been done on the estimation of crystal size. The earlier workers based their conclusions on the line broadening of the diffraction pattern. and estimated the crystals of bone salt at 10"5 to 10"6 cm (Iogan. 1940). Determination of surface area by the gas adsorption technique (Woods. 19“? and Neuman. 1950) tend to confirm that the cry- stale are of this order of magnitude. Confirmation of the size of bone crystals has come from the eac- cellent work of Robinson (1951) and of Robinson and Watson (1952, 1953). By using the electron microscope they were able to see and measure bone crystals. and obtain the typical apatite pattern from them. They report the average dimensions for bone crystals as 500 x 250 x 100 3, although in sectioned material from the outer cortex of the human rib. the aver- age crystal is about half this size. This would give between 100 and 250 square meters of exchangeable surface area per gram of bone crystals. In terms of the entire skeleton of a 70 kg. man. this would exceed 100 acres (McLean and Urist. 1955). It is easy to see then why Neuman (1950) comented that "increasing numbers of investigations have been forced to use surface chemistry to explain results obtained in bone studies. " This approach has strengthened the effort to explain the Variation in the euro“ ratio in bone and synthetic precipitates. (and indeed the presence of carbonate. citrate. Na. and other ions "foreign” to the apatite lattice) by assuming "adsorption" rather than incorporation into the lattice. The fact that synthetic precipitates of "tricalcium phosphate hy- drate" possess a Gamma mole ratio of 1.50 rather than 1.67 (theoretical fOI‘ hydroaquapatite) has caused two groups of investigators to invoke , —.v—-_'.yw L“ ..s 35-1: , _ _—'—V 3.49' no “W l ...- T‘P“ ‘- an adsorption hypothesis. but each with a different interpretation. Dallemagne (1952) summarizes one position by pointing out that'tri- calcium phOSphate hydrate with a CazPOu, mole ratio of 1.50 may be thought of as a calcium deficient hydroxylapatite. The ratio could be increased by adsorption of calcium hydroxide. which might not be fixed in a crystal lattice unless brought to a high temperature. Hodge (1938) and co-workers have supported the alternative opinion that tricalcium phosphate is simply hydroxylapatite with sufficient Pqu ions adsorbed to lower the Ca:PQu ratio to 1.50. Posner and Stephenson (1952) also support this view. Neuman and Neuman (1953) point out the limitations of the adsorption theory when considered in the light of surface area. It would require adsorption of one P9“ for each # th ions in the crystal to give a Ca:PQu mole ratio of 1.33. Precipitates with a Ca:PQu ratio above 1.33 give only an apatite diffraction pattern (Arnold. 1950). Neuman and Neuman (1953) point out that because of the extremely small size of the crystals. one half to two-thirds of the unit cells are located in the surface. Postulation of an isomorphic substitution of hydronium ions for surface calciums would explain the existence of precipitates with CazPQu mole ratios of 1.9 to 1.8. Such a system would still retain a characteristic apatite lattice structure. This hypothesis approaches that of Dallemagne. G. thgistgx of the Procggs of Calcification "Knowledge of the composition of primary calcification. i.e.. of the composition of bone salts immediately after’deposition. would Probably throw considerable light on the mechanism of calcification" , i W on» ’ ..-——.-—-- {is 11+ (Shear and Kramer. 1928a.) . The simplest structural unit of apatite contains 18 ions. It is inconceivable that this large a number of ions could spontaneously aggregate in the prOper spacial configuration of the lattice. This has led investigators to postulate some inorganic intermediate in the process. which has gone undetected. or some organic complex or template which would serve as a nucleation center (Roseberry. Hastings and Horse. 1931). These two approaches have been reviewed in a number of papers, (Huggins. 1937. Iogan, 191m. and Neuman and Neuman. 1953). An extensive stuck of calcium phosphate precipitates was carried out by Holt, Lafler. and Chown (1925) in an attempt to better understand the process of calcification. Shear and Kramer (1928a) develOped the idea that CaHPOu was an intermediate in the formation of bone salt. even though it is not stable at the pH of body fluids. No Cal-I130” has been detected in bone by x-ray diffraction techniques (Taylor and Sheard. 1929. and Roseberry. Hastings and Horse. 1931) even in ‘young" bone. Nor did Hirschman. Sobel. and Fankuchen (1953) find it in in m calcification studies of epiphyseal cartilage. However. CaHP04 could not be excluded from consideration on this basis alone since CaHl’O,+ was converted to apatite when placed in the solutions employed 1‘ or calcification in mm. A number of points of evidence for and a‘Sainst the importance of CaHPOn in the process of calcification has been presented by Hodge (1950). Il'he recent work of Robinson and Watson (1953) showing the inti- mate relationship between the collagen fibers and bone crystals has given considerable impetus to the concept of some organic nucleation 15 center. This center would initiate a stepwise binding of the prOper ions into the apprOpriate configuration for the complete crystal (Neuman and Neuman. 1953). The close association between the periodi- city of the collagen fibers and the presence of bone crystals is suggestive. Whether periodic collagen side groups initiate the crystal lattice or merely prevent the growth of the crystal beyond a certain size is entirely within the realm of conjecture. After some twenty-five years of concentrated effort we still read. ”The calcification process is so poorly understood that no comprehensive hypothesis can be given" (Neuman and Neuman. 1953). Ho MW From the foregoing. it is evident that we do not really know the structure of bone. other than that finally some apatite lattice is in- volved. The biochemical relation of this general lattice to specific ions is obscure and the role. either active or passive. of the organic material is conjectural. How then. can one understand the initiation and development of this tissue when the significance and inter-relations 01‘ its component parts are unknown? The exact roles of vitamin D and 0f the parathyroid hormone and other agents in regulating exchange of radioactive calcium into and out of the system cannot finally be under- stood except in terms of the ionic anatomy which has been sketched above. There is yet no adequate explanation for the direct effects either of vitamin D or of parathormone on bone. although the "gross" histological picture is reasonably clear. It is surprising that no work has been done on very young bone 7 If if V‘— T““-_ I . I ‘— w fiflm_.‘m.v“. ‘ , ‘7' g, ’ ..i, 16 which is just beginning to calcify. Nor has any serious attempt been made to study calcification of arteries by the physical methods initiated by DeJong. The study of forming bone has been done almost entirely on rachitic cartilage placed in various artificial calcifying media. There is no good chemical infornation other than total 0810111111 and phosphorus. for any of these situations. or for the preosseous cartilage in which bone is about to form. In fact. for cartilages in general. Eichelberger (1952) was able to cite only two references to its ionic composition. And yet. would one expect to find a crystalline precursor for apatite. or an organic template. in mature bone in which growth has ceased and only the slow remodeling is going on? No one has yet looked at the diffraction pattern of may formed bone with the object of comparing it with older bone. Would one choose for such a study the growing bone of a weanling rat in which the actual new bone laid down in a day represents less than 1% of that already present? What about the preosseous cartilage? Does it show any evidences at all of impending change? It was these questions which led to the selection of the embryonic chick femurs as the biologic material for this investigation. Although general pessimism has been expressed concerning the adequacy of the various diffraction techniques. as currently employed. the data they provide are nevertheless clearly more sensitive than those resulting from routine chemical analyses. This will be demon- i strated again in the data which follows. Further. electron diffract on ' d With Selected Area Aperture. a technique which has scarcely been trle 17 in biology is an ideal method for circumventing the "averaging”or “weighting" effect inherent in standard x-ray diffraction. This thesis. then. reports the data resulting from study. by diffraction methods. of the initial stage of bone formation. Search was made for crystalline "precursors" of apatite in a biologic situ— ation where they should be relatively unobscured (if present at all) and using techniques to minimize interference by heavy apatite patterns. Further. specific study of the 'apatite" patterns themselves was made to determine whether shifts could be detected between the time of initial lay down and later times when the lattice might be regarded as being more'mature'. The heat conversion of apatite to whitlockite (3033(P0g)2) was also studied both on dried and on ethylene diamine extracted bone mineral in an effort to find lattice differences associated with removal of organic matter in the extraction procedure. Thus a serious effort was made to obtain direct crystallographic evi- dence for the presence of constituents of types which have often been regarded as necessary iniheory but which existing data could neither prove nor disprove. This search was limited only by the instrumen- tation available. Alternative approaches are conceivable but in general. they are less than satisfactory for the objectives of this study on two counts. First. many(very useful) animal experiments can be devised to show the importance of a variety of variables on such things as the rate of lay down of the "apatite'I lattice. for example. Such experiments would prove only how fast ‘something" goes down but could not tell what that ”something" was. In this investigation. we are concerned 18 with exactly what happens when ossification begins. Second. alternate physical or chemical methods which would be use- ful in gross studies. in general. lack sufficient sensitivity for work on the micro scale required in this problem. Thus. even chemical analysis by diffraction often has a much greater sensitivity than con- ventional analytical methods.particularly when used on mixtures with H“ interfering' substances. The crystals being investigated are extremely small (less than the distance between the 640 X spacing on collagen fibres. for example) and are surrounded by a formidable mixture. con- taining both electrolytes and non-electrolytes. This situation also rules out the usual techniques of crystal Optics and molecular refrac— tivity which have been used in studies of the large macro crystals of classical minerology. The only logical alternative for the intimate study of the inti- mate study of the physiology of bone mineral formation involves the construction of more highly refined diffraction equipment. Although such work will undoubtedly be done in the next decade. it is obviously beyond the scope of this thesis. II. EXPERIMENTAL METHODS A. Electronufligfraction Studies 1. Preparation of Materials Femurs were harvested from chick embryos and placed in two groups for further treatment. One group of bones was dried at 100° C. for 2“ hours. A second group was ashed at 600° C. for 5 hours. Another group of femurs was taken from two-week old chicks. These were wet ashed with ethylenediamine for several hours (until the ash appeared nearly white. and the SOIVent in contact with the bone was colorless). Ethylenediamine ashing was carried out by placing dry bones in the thimble of a continuous extraction apparatus with boiling ethylenediamine (ED) as the solvent. (B.P. 117° C.) In such a procedure. the temper- ature of the bone never exceeded the boiling point of ED. Extraction was usually complete in four hours. after which excess ED was removed by washing the ash with distilled water. The residue was air dried at 38° C. and the fragile white ash ground to pass a 200 mesh sieve. For electron microscopy all preparations were further reduced in size by Placing the mortar ground material between two ground glass disks. one Of which was rotated at about 500 revolutions per minute. The extremely fine powder thus produced was then transferred to an electron microscope SPGCinen grid by directly dusting on to the surfaca of the Formvar film.1 All grids were lightly shadowed with gold. 1 Made from drying a 1% solution of polyvinyl formal in ethylene di- Chloride. 20 2. Instrumentation The grids were studied and photographed in an RCA Electron Micro- scope ELU-ZC at magnifications between 5.000 and 10.000. Additional photographic enlargenent resulted in final magnification indicated on the figures. Diffraction patterns were obtained with a Selected Area Aperture which reduces the area contributing to the diffraction pattern to as little as one/uz. Under favorable conditions, it was possible to observe and photograph patterns from single crystals. 3. Mathematical Considerations Determination of the interplanar spacings. which produce the diffraction pattern. may be done most accurately by comparison with an internal standard such as gold. Since the effective wave length of a 50 KV electron beam is very short. the diffraction angle. 0. is also Very small. Consequently. cos 6 is never less than .998 and may be taken as 1.000. This makes it possible to calculate an unknown interplanar Spacing. dx. from the simple relationship diameter X ring without introducing appreciable errors other than those of measurement. The value d Au(100) = 2.344 K. Since considerable ellipticity exists in the patterns. the diameter of the gold ring was measured at the same angle of rotation as a line connecting the spots being measured. No other investigators in this field (or indeed in most electron diffraction work) have routinely used internal standards. nor are error estimates to be found in the literature. Since d values vary rather widely in the literature. it is evident that technical factors have not been always adequately controlled. As an example of methods used in this study. a complete set of individual measurements and calculations from a single plate are found in Table XII in the appendix. Considerable difficulty was experienced in locating the exact center of bright Spots or of a relatively broad ring. An estimate of errors in interplanar spacings resulting from an error in linear measurement of 0.2 mm. and 0.5 nm. on a 2.7 X projection print is given in Table III. Since a measurement error of 0.5 mm. was easily possible. the difference between d values calculated from a single pattern may be explained solely on the basis of errors in linear measurements. Bow 1. Preparation of Materials As in the previous study, the biologic materials were chick embryos incubated at 38° C. from six days to 21 days; and also two week old chicks. The embryonic femurs were removed and then freed of adhering soft tissue by gentle movement on hard surface filter paper. This pro- cess also removed excess tissue fluid. In studies where the material was run "wet". the femur or a small longitudinal section was placed in a glass capillary tube and sealed with a micro burner. Material run “dry“ was first placed in a 100° C. oven for 24 hours. Material designated as ED ashed was obtained by the extraction method described previously. The wet and dry embryonic femurs from six to eleven days were in- troduced directly into thin walled glass capillary tubes suitable for 0? 22 TABLE III CALCULATION 01" moss INVOLVED IN ESTIMATION OF SPOT POSITION ON AN EIECI‘RON DIFI'BACTI ON PATTIRN‘ Region Linear Error Error in d R O 0.5 A 0.2 mm. 0.50 f .00 0.5 mm. 1 .09 2.6 I 0.2 mm. 2.60 1 .02 0.5 mm. 1 .04 1.5 X 0.2 m. 1.500 3,001; . 0.5 m. 1.010 ‘Celculatione based on measurements of e 2.7 x projection.print of the original pattern. TABLI IV CALCULATION 01‘ 133035 INVOLVED IN ESTIMATION 0! LINE POSITION ON AN X—RAY DIIFRAC‘I'ION PATTERN Region Linear Error Error in d 3 8.0 3 0.1 mm. 8.00 g .05 6.0 X 0.1 m. 6.00 g .03 0.0 2 0.1 mm. n.00 _t_ .02 3.0 I 0.1 an. 3.00 i .01 2.5 I 0.1 mm. 2.500 t .005 2.0 3 0.1 m. 2.000 g .003 1.53 0.1 mm. 1.500 1 .002 .“ 23 x-ray diffraction.1 All other bones were ground and introduced as a fine powder. 2. Technical Aspects Capillary tubes containing the various bone preparations were placed in the cradle of the powder camera and oscillated through 20° at the rate of one complete Oscillation per minute. the tube always remaining at the exact center of the camera. Exposures were made on Kodak No—Screen x-ray film usually for four hours. Nickel filtered copper K-alpha radiation was used with the tube being Operated at 35 KV and 20 MA. The camera has an effective film diameter of 14.32 cm.. so that with normal film shrinkage. the angle of diffraction. 9. may be ob- tained either by multiplying the distance (in cm.) between two corres- ponding lines by a factor of 2 or by multiplying the distance from the center of the undeviated beam to a line by a factor of h. A graphic solution of Bragg's Equation. nit: 2 d sin 8. for each value of 9 gave the desired interplanar spacings in Xngstroms.(Parrish and Irwin. 1953). Test runs on standard materials2 showed this calibration to be in ex- cellent agreement with published values.3 Idne positions were measured to the nearest millimeter and estimated 1 Glaskapillaren fur rdntgenographische Aufnahmen nach Ibbye—Scherrer were obtained from Caine Sales Co.. Chicago. Illinois. having a diameter of 0.5 mm and a wall thickness of 0.01 mm. 2 Checks on calibration of powder camera made with silica. sodium chloride. and potassium chloride. Values for sodium chloride and potassium chlor- ide are compared with ASTM (1950) values in Table XIV. 3 A11 Figures reproduced in this thesis are slightly larger than the Original film. by a factor of 1.08. w- .W“ ‘Jw 24 to the nearest 0.1 millimeter. with repeated measurements not varying more than i 0.1 mm. for relatively sharp lines. Some difficulty was experienced in accurately determining the exact center of broad lines as they appear in the dry and ED ashed bone. A calculation of the error produced in the interplanar value by an error of 0.1 mm. in the linear measurement is given in Table IV. Relative intensities were estimated by use of a convenient film comparator constructed by a method similar to that of Klug and Alex- ander (1954). A strip of film was wrapped in aluminum foil and exposed to a weak x-ray beam collimated with several narrow slits. The exposure time was varied so that a suitable range of fihn densities resulted. Visual matching of line densities with the standard allowed relative densities to be established. Since it was noted that preosseous embryonic bone when dried con- tained a mixture of irystalline MCI and KCl. a rough estimate of the relative amounts of each was thought to be of value. Therefore. an equal parts (by weight) mixture of NaCl and KCl was prepared and its diffraction pattern studied. It was observed that the 3.13 K KCl line and the 2.81 3 NaCl line had about equal relative intensities. Since the intensity of the diffracted beam is directly proportional to the number of diffracting crystals. the relative amounts of NaCl and KCl can be estimated from the ratio of the relative intensities of their strongest lines. However. it must be recognized that this method pro- vides only a very rough estimate of relative concentration because an error (which may occasionally be as high as 20%) may be involved in the estimation of the relative intensities. In addition. only a very l 25 small part of the femur is in the path of the beam. There may be real local variations in the Na/K ratio. Therefore, quantitative chemical analyses of sodium and potassium was carried out by flame photometric methods. C. General Conventions Imolving mgjfzaction Data All measurements are reported after conversion to interplanar spacings in Xngstroms. Unit cell dimensions are easily calculated using the standard formula for hexagonal crystals. This reduces to a = 2d for the 110 plane or a = “d For the 220 plane and c = d for the 001 plane or c = 2 d for the 002 plane. These simplifications naturally do not apply to NaCl or KCl. III. PRESENTATION OF DATA A. glggtgon Qiffnagtiog Studies Two distinct types of fragments were consistently seen on the grids when viewed in the electron microscope. One type consisted of rather dense fragments. which varied in size down to less than 0.1;”. They were apparently too thick to allow passage of the electron beam or were insufficiently crystalline to give a diffraction pattern. Many of these particles may be seen in Figure I. In addition to these "dense" parti- cles. thin well-formed crystals may also be seen in a variety of sizes and shapes up to several micra. The source of these thin crystals may not be bone. necessarily. since they have been seen on some grids which had not been I'dusted" with the bone preparation. Good diffraction patterns have consistently been obtained from these crystals with the use of the Selected Area Aperture (See Figure 2). The average values for the six largest spacings are given in Table V. A complete summary of all measurements made is given in Table XIII in the appendix. TABLE V AVERAGE VALUES FOR SIX IARGEST SPACINGS OT CRYSTAL OBSERVED WITH CAI» CULAIED VALUES FOR TEE "a" AXIS 102 no 200 120 + 210 300 220 d d K d K d K d R d K Spacing n.57 2.62 2.30 1.739 1.525 1.329 Calm "a" 5.28 5.2» 5.31 5.31 5.28 5.32 In all the patterns that were produced by the thin crystals. no Fig. / E/eclron Microgmp/rs of Powdered Bone Fly. 2 Electron attraction Patterns from Min Crystals in HgJ 29 spots were ever seen other than those whose calculated spacings appear in Table V for the range covered by the Table. Additional spacings from greater diffraction angles all fit into the hexagonal pattern established by the larger spacings, and can be assigned diffracting planes consistent with those assumed for the given spacings. In some cases. several crystals were obviously contributing to the diffraction pattern. each giving its own characteristic hexagonal single crystal pattern (Figure 2). Since considerable individual variation existed. no good estimate of relative intensity of diffraction Spots was made. In general. how- ever. as may be seen from Figure 2. the spots assigned to the (300) plane are the most intense with the 110 and 220 next in order. This is of little value for comparisons with x-ray diffraction data. since the x-ray values are based on powder patterns with random orientation of the crystals. which obviously is not the case here. Evan though the crystals here described must be regarded as a con- taminant. identification would be of considerable interest. If we assume that the innermost spots visible in the diffraction pattern are due to the 100 plane of a hexagonal crystal. an 'a" axis of 5.29 K may be calculated.1 Since the patterns observed were always hexagonal, it nust be assumed that the "c" axis of the crystal was always parallel to 1 Formula used to calculate spacings from hexagonal crystals. d(hkl)= /% (h2.ah:.k2>+(_§_) 30 the electron beam. This makes it impossible to obtain a value for the "c" axis with present equipment. Search of the available literature (Frevel and Rinn. 1953; American Society for Testing Materials. 1950; Donnay and Nowacki. 1954) reveals only very few possibilities. Among these. CaNaPOu. a = 5.2“, c = 7.14 fits the value calculated from the observed crystal pattern rather well and would seem likely to be present. B. Ra ato St 1. Data from Dry [Embryonic Femurs of Six to Ten Days Incubation Diffraction patterns were obtained from dry early embryonic femurs beginning with six days and continuing through ten days of incubation. A total of twenty-three patterns were measured and all lines present identified. Bones were obtained from two different settings of eggs (designated as series "a" and "b‘). However. since no differences were observed between the two series. both series were averaged to- gether. The complete tabulation of individual lines is found in Tables XV to XX in the appendix. The diffraction patterns suitable for reproduction are found in Figures 3 and h. and Figures 9 and 13 in the appendix. The interplanar spacings for the lines appearing on the diffrac- tion patterns for six. seven. and eight day femurs were calculated and compared with values in the ASTM File (1950). It was found that they result from a mixture of NaCI and KCl. No additional crystalline com— POnents could be identified. Therefore. a diffraction pattern was made from an equal parts mixture of NaCl and KCl. The results were in ex- sag §em 3.3 363:8 335‘ massaged. EEK Sufism 5&3th balk M 6Q ‘ bu t 5me 3.3.» \ob % mvé on E Sea ES en a _ . is Gk fieewtmas Z l M . . .3ng natesbnsm Eek 5&er toaoEkG \okuk 9 9% at Q SEQ $8 at \ Rd be C $me .36.» \o x . Rim p~~~--*"-*"‘-"~ me \ CRYSTALLINE COMPONENTS OF DRY EARLY EMBREENIC CHICK FEMURS 33 TABLE VI m -a- ~'- A- o - “v. ...-o .<..-. Age Pattern (in days) Series Number ._, "Apetite" NaCI K01 _ 6 a 1342‘ - 309% 703% 13-58 80% 20 7 a B—ua 30% 70% 3-50 30% 70% 3-56 20% 80% 'b 13-60 5% 95% 3—62 - 0% 100% 3-67 0% 100% 8 a 3-44 - 30% 70% B-u9 - 50% 50% 3-51; - of of 3-57 - 5% 95% 1) 3—61 - 30$ 70% 3.6u - 10% 90% 9 a B-hj 4 3.1.8 + - 3-55 + - + b 13-63 — _ 3.66 + - 10 a B-hé + - - 3-147 4 - - b 3-65 + - 3-68 + - ””980 25% 75{_-__ ‘ neSative 4 present ‘_‘ ’All "B” numbers in this and subsequent tables refer to the x-rey diffraction pattern from a certain bone preparation. .059 vacuums one op penguin mowfimfiope—H a n moH.H n moH.H m ees.a m one.a m aes.H m aHm.H m mam.a Hes 0H oHa.H oH moa.a oH oHa.H 0H oaa.H «we 0H omw.a oH mmm.H oH smm.a ca nmm.H mmm 0H amm.a oH omm.H oH ode.fl oH wmm.H 9H m Hmo.~ m owed m 034 can we nom.m om omN.~ 0H mom.m oH on~.m fly HNH OOH ow.~ 00H ow.m ooa om.m 00H om.m cam on 0H.m om oH.m ma QH.m om mo.m moo on me.n o: Ne.m om ms.m om ms.n HHH 0H mm.m m Hw.m oH mm.m om ow.m as; HH\H a e HH\H a e HH\H « e HH\H M e xmecw toflaaa .awmtopa awe am a He has ca has m mmot azmmga ed. mng Hun EOE EZH MU§< HH> MAE; 35 cellent agreement with the ASTM values (Table XIV). Since the 3.13 R line of mi and the 2.81 1! line of NcCl had equal relative intensities. the relative amounts 0" crystalline NaCl and KCl present in each dry bone were estimated. The results appear in Table VI. As can be seen from the Table. all but one of the embryonic femurs from age six to eight days contained crystalline KCl and only three lacked detectable amounts of crystalline NaCl. as seen from the diffraction patterns. Apatite could not be detected in any femur of this age group. After nine days of incubation. an osseous cylinder is usually seen when the femur is dried. The presence of "bone" is confirmed by x-ray diffraction as in all cases except 3-63. the typical apatite pattern was observed. However. a remarkable event occurred concomitantly. There is a marked reduction in crystallizable sodium and potassium as evidenced by the complete absence of NaCl lines from all spectrograms and the presence of rather faint K31 lines in only one of the 23 plates studied. This indicates either an actual loss of theSe ions or their presence in a non-crystalline complex. No crystalline calcium compounds. other than the typical "apatite". have ever been detected in bone. Further. there is no detectable change in lattice parameters at any time after the crystals are first laid down (see Table VII). Chemical analyses for sodium and potassium found in embryonic femurs after seven to ten days of incubation are summarized in Table VIII. 36 TABLE VIII CHElflCAL ANALYSES ON EMBRYONIC FEMURS Sodium Pota ssium Total Bone Day No. Used wgi.* x/ mg. mEq/lOO gm. 37 mg. mEq/loo gm - 7 6 o.u mg 12 so 10 3o - 8 6 0.8 31 130 15 no + 9 u 0.9 20 9o 11 30 + 10 3 1.1 26 110 11 3o It Weighed dry on a balance with sensitivity : 0.1 mg. + Bone present - preosseous Since the weight of the samples was so small. considerable error could have been introduced from weighing error alone. Only enough sam— ple was available for one analysis per group. The difference observed cannot be regarded as significant. but the order of magnitude of the results is in agreement with the literature (Eichelberger. et a1.. 1952. and Everett. l9h8). 2. X—ray Diffraction by "Wet" Femurs The scattering of x-rays by the water present in femurs from six to eleven days was so great that it was impossible to detect any pattern at all, even though apatite was clearly present in dry bone after the eighth day. However. a longitudinal section of a wet 21-day femur showed a typical apatite pattern which was indistinguishable from dry hone (See Fig. 1b). This observation has been reported by Reed and Reed (1912). ..\ e-...———~._../“"_ I V 37 3. Comparison of Values from Dry and Ethylenediamine ashed Twenty-One Day Vemurs Ignited at High Temperatures As has been previously reported (Klement and Trémmel. 1932) heat— ing dry bone causes a marked sharpening of the apatite lines and also an increase in the number which appear on the diffraction pattern. This has generally been attributed to the removal of organic material and a growth of the bone crystals. This phenomenon was also observed (See Fig. 5). There does not seem to be an diange in lattice parameters when the specimens were heated up to four hours at 500° C. or for two hours at 600° C. However. when heated for six hours at 600° 0.. there was a complete conversion t0/90a3(PQn)2 (See Fig. 7. Table X). Heating at 700° C. for 6 hours produced the same pattern seen at 600° C. (Fig. 8). Twentyaone day femurs which were ashed with ethylenediamine (ED) showed a pattern which was practically indistinguishable from dry 21 day bones. There was no increase in the number of lines, no shift in the position of those present. nor was a sharpening of the lines ob- served (See Fig. 5 and Table XXII). This indicates that there is no apparent alteration of the lattice parameters by the ED ashing procedure. nor has there been the crystal growth seen in heat ashing. The only observable difference was the loss of two broad bands seen at about 10 R and u.5 K in all dry bones which are probably due to the organic components of the bone (Clark. 1931). These broad bands are also seen in diffraction patterns from dry preosseous femurs. from dry Periosteum and from the epiphyseal cartilage of a 21 day femur. (Fig. 1a). deem been? QM poo AG .8 tomteQEou m. 6?. . \Q . MLJ_.' ,- “-.‘ doom .8ch 9w pee \uQ Baum}. as Baum b fit .3 v Noose Na 938 QM xi sEQ Es.» so & ..QQ N . 6 oQQh RC Page 9% \ $me .33.» Rob \N ., MN..Q estates a 8.8 RE. em as be t tattoo a at .3 e 96% S sets on 3 see 3% stem and .3 m ...o. see «we at 3 see he... as aw 1; QNIQ it .. . «an o .68. awe be 3 tea «at s a A ‘ ... .owd .l ‘ |||I|aIS\ .ehaookéaa ox beauties.» atom been? QM .26 \\Q \o «Eaton. 3.59th Ask - k m fit as o o o . 8k ‘ e. l 1 1. .3 o 96% we note em 3 sea «at seam and . at o ..o. 8o Q at S tee E8 so aw : . mud 14.)..‘2.¢222222.l.¢27~2222\11111Ml‘vin OBSERVED VALLES FOR BONE TABLE IX 42 APATITE AITER HEATING COMPARED TO VALUES CALe CULATED FOR BONE APATITE BASED ON LATTICE PARAMETERS “fin-...- -..-...-- " w 135. ED P a r: s e 112'" Iigbinaon d 3 d 3 1/11 hkl d i d i 9.18‘ .---'..”‘ _—' I -..-.__._1_. 8.09 100 8.0u5 8.230 001 6.959 6.85o 5.2u' 5 101 5.263 5.269 n.73 2 110 4.645 b.737 n.27' 2 n.0u ' 5 n.08 70 200 n.023 n.102 3.8h 5 3.90 20 111 3.862 3.902 201 3.u83 3.522 3.113 3.1414, 80 002 3.181 3.1139 3.15' 3.18' 10 3.07 3.08 20 210 3.0a1 3.099 2.797 2.82 100 121 2.786 2.826 112 2.785 2.782 2.708 2. 71 90 300 2.681 2.73:; 2.618‘ 2.62‘ 50 2.516 2.52 10 301 2.503 2.5u1 2 2 220 2.322 2.368 2 003 2.320 2.303 2 2.28u 122 2.290 2.293 30 2.253 2.25 60 310 2.232 2.27u 2 221 2.203 2.239 5 10 2.1u1 2.1» 10 131 - 2.12u 2.159 2 10 2.057 2.06 10 113 2.076 2.06n 5 2.00 10 400 2.011 2.050 5 1.989“ 1.01 1.933 1.965 no 1.936 1.92 70 222 1.932 1.950 5 10 1.885 1.89 30 132 1.878 1.898 5 1.860‘ no 1.835 1.83 70 123 1.84» 1.8u3 1.800 1.80 no 231 1.78u 1.815 1.776 1.77 no u10 1.756 1.789 1.750 1.75 no 00!» 1.7110 . 1.720 1.71u 1.75 no 1u1 1.702 1.733 1.6u0 1.64 20 223 1.6u1 1.6u7 V‘— lattice parameters. aprien and Frondel (19147). I“revel and Rina (1953). aluee’ihiéE—do not fall in the range of calculated values based on These values appear in Table )(. Observed Calculated from a 3 9.29. c 3 6.96, c/e = 0.7h9 Ehobinson (1951). Calculated from a = 9.u7, c = 6.88. c/a = 0.727 [#3 After heating the dry and ED ashed bone for a period of one hour at 500° C.. a large additional number of lines may be seen in both preparations. However, more lines are observed on the spectrogram of heated dry bone and they appear to be much sharper than those of the heated ED ashed bone. (See Fig. 5. and Tables XXI and XXII). Heating for periods of two and four hours at 500° C. further sharpens the lines in both cases but the difference between the dry and Efi)ashed bone is still apparent (Fig. 6). Heating for two hours at 600° C. produced material which gave essentially the same patterns as at 500° 6.. however. two minor differ- ences were noted in the ED preparation. The lines were somewhat sharper than at 500° C. for four hours. and there was a slight. but definite. shift of the strongest line at 2.79 R (see Fig. 7). This shift is from a value of 2.82“ K (Table III, B—26) for heated dry bone to 2.790 X (Table XXII, 3-27) for the heated ED ashed bone. The difference of .o3n R is greater than the estimated error of measurement in this region but is less than the range of values in the literature given for bone ”apatite" (See Table IX). A calculation of the 'c” axis based on the 002 plane indicates little or no change in the "c" axis (6.84 K'- 6.83 K) Using these values of “c3 “a" calculated from 2.824 K for the 121 plane is 9.“? for "dry". heated bone, and from 2.790 K. "a" = 9.3h K for ED ashed. heated bone. Additional heating of both dry and ED ashed bone gave a material which is identified as fiCa3(P0u)2 or whitlocldte. The diffraction Patterns appear in Figure 8. Calculated Spacings from these four Exitterns are compared with observed data in the literature (See Table X). n n 25' Z.‘ - TABLE X OBSERVED VALUES FOR WHITLOCKITE COMPARED TO VALUES IN LITERATURE Literature1 Ashed Bone2 Heated Bonej d 2 1/11 d 2 1/11 d 3 1/11 9.28 - 8.17 10 8.06 20 6.50 23 6.46 20 6.44 10 5.65 20 5.23 47 5.18 40 5.19 15 “.33 7 “-33 5 4.06 28 4.03 15 3.92 5 3.79 3 3.50 20 3.44 40 3.42 48 3.34 10 3.34 5 3.32 5 3.19 72 3.18 70 3.17 20 3.10 2 3.00 10 2.99 5 2.99 2 2.87 100 2.857 100 2.76 25 2.737 10 2.769 30 2.69 18 2.667 2 2.60 77 2.596 70 2.622 15 2.53 20 2.538 5 2.576 15 2.41 19 2.387 10 2.490 2 2.26 20 2.248 10 2.19 18 2.181 10 2.15 17 2.14? 10 2.169 5 2.07 13 2.063 5 2.094 2 2.05 20 2.01 15 2.019 10 1.98 20 1.987 10 1.988 5 1.93 40 1.922 20 1.911 5 1.88 30 1.872 15 1.86 30 1.866 5 1.82 18 1.813 2 1.809 20 1.77 30 1.763 2 1.71 60 1.717 25 1 Average value from ASTM File #2436 d 3-0701, 2437 d 3-0702. 2435 (1 1-096“. 2468 d 2-0783. 2409 d 3-0692. and Prien and Frondel (194?) 2Awerage values of B-28. 3.29. 3-71. and 3.72. (See Table xx) spacings from heated dry bones which cannot be assigned to apatite. (See Table 1X). . gear... «4.. we: ...-v ,- JIu-‘Ww ' _.a_——_- I15 The primary pattern seen in bones heated for several hours at 500° C. and at 600° C. is that of an apatite. This fact is readily observed when Table IX is consulted. In addition. the dry bone heated for only one hour at 500° C. showed an additional 18 lines which did not fall in the range of values for apatite. With the exception of only four. these lines may be assigned to ,6Ca3(P0u)2 (See Table x). on the other hand. ED ashed bone when heated for one hour at 500° C. showed only seven lines that did not fall in the range of apatite; and of these. only three very weak lines could be assigned to flCa3(P0u)2. The same observations were made also for heating periods of two, and four hours at 500° C.. and for two hours at 600° C. It is clearly evident that FCa3(P04)2 is much more readily produced by heating dry bone in a furnace than when ED ashed bone is heated. There are several lines present on both the heated dry bone and heated ED ashed bone which cannot be assinged to either apatite or whitlockite. These lines represent interplanar spacings of 9.23 3. 4.33 3. 2.769 K. and either 2.662 X or 2.576 3. They may be due to a contaminant.to some intermediate in the conversion of apatite to whit- lockite. or to minerals containing Na, K. Mg or Cl. Quite evidently. the technique is sufficiently refin ed to detect. although not to iden- tify. constituents other than apatite or whitlockite. ‘5. Values Obtained from Ashing Young Embryonic Femurs Young femurs of eight to ten days were ashed at 600° C. f‘or six hOurs in either porcelain or platinum crucibles. Extremely small qtlantities of ashed material were recovered after ashing. however. enough was obtained for suitable x-ray diffraction patterns to be made. Spacings from these patterns appear in Table XXIV and the patterns in Figure 15. A mixture of NaC1 and KCl when heated for six hours gave essentially the same x-ray diffraction as was seen in ashed eight to ten day femurs (See Fig. 15). IV. DISCUSSION OF RESULTS A. Electron_2if§gac§lpn Sigdies The techniques of electron diffraction impose rather severe limi- tations in choice of methods employed for preparation of biologic mterial. All specimens must be thoroughly dry and thin enough to per- mit penetration by the electron beam. Considerable heat is also gener- ated by bombardment of the sample with electrons. Early attempts to visualize bone mineral and tooth crystals usually employed fragmentation or thin shavings. By these methods. dry or ashed calcified material may be used without additional treatment. By grinding young teeth between watch glasses and dispersing in collodion. Boyle. Hillier. and Davidson (1946) were able to obtain electron diffrac- tion patterns from enamel fragments which showed crystal orientation. A few years later. Robinson and Bishop (1950) used high pressure auto— claving and ultrasonic dispersion to prepare bone for electron micro- scopy. By this method they were able to make a reasonably accurate estimate of crystal size. Then Robinson and Watson (1952) showed bone CI‘YStals 1;} m on thin sections of n-butyl—methacrylate embedded human rib cortex. They also showed the typical apatite diffraction pattern from the thin section. I In each of these cases. the bone specimen was exposed to a large electron beam. (No Selected Area Aperture was used). The exact portion of the grid which gave rise to the diffraction pattern could not be .- ‘ t N. . ‘ ‘ visualized and was certainly very large. since single crystal patterns did not appear. Instead. the image formed was the typical circular powder pattern. often showing orientation of the crystal population. Using these methods. bone "apatite". which is stable under rather extreme conditions and present in high concentration. was the only cry- The data are not sufficiently pre- stalline material ever identified. cise to allow conclusions as to the exact type of apatite. If some other crystalline component is present inbone to a much lesser extent. its contribution to the diffraction pattern must be so slight that it has remained undetected by the simpler techniques employed to date. 01‘. it is possible that the conditions used may have destroyed or altered other crystalline components which might conceivably be present. In order to reduce the chance of these two possibilities in the present work. dry bone was ground to a fine powder and transferred direct- 1y to the film of the specimen grid. In addition. the size of the area contributing to the diffraction pattern was reduced by means of a Selec- ted Area Aperture. so that the pattern observed was produced primarily by a single crystal or small group of crystals. Under these conditions. apatite was never seen but only the unknown "contaminant". When the grid was placed in the lower stage of the electron microscope (which would then expose a larger area of the grid to the electron beam) no It is apparent that apatite. even when ground The pattern was ever seen. extremely fine. has a remarkable stOpping power for electrons. dense particles in Figure 1 must he apatite with its associated collagen. Even the finest of these. however. does not permit enough of the inci— dent electrons to pass through to give diffraction patterns of sufficient L “9 contrast to the surrounding beam to be photographed. Very short film exposures are used in electron diffraction (seconds to a minute or two) in contrast to the long exposures in x-ray work ( 4 hours to days). Increasing the exposure time merely weakens the contrast of spot or ring to background. since the background then becomes extremely black. If. in addition. the beam is reduced in size by the Selected Area Aper- ture. the number of electron diffracted (and thus available to initiate an image in the time of exposure) is very low. One crystal. the "contaminant'I frequently almost invisible in the conventional electron microscope field. gave very excellent single cry- stal diffraction patterns. It was never present in amounts exceeding about 5% of the total material on the grid (estimated from plates such as Figure l) and was not visualized by lower stage diffraction proce- dures. It was remarkably transparent to electrons (as evidenced both by its lightness in the conventional field and its strong diffraction Pattern a property which in itself is sufficient to differentiate it from the apatites. Identification of this material. which gave the diffraction Patterns seen in Figure 2. would be of considerable interest. Appar- ently only the hkO spacings are seen which makes positive identification in1D<:~ss:'1.ble. Further difficulties arise from the fact that relative int ensities such as reported in ASTM (1950) are not of much value. Sines they are dependent on random orientation of the crystals. There- f °1‘e. the only reasonable approach to the problem is to calculate a Value for the "a" axis. having assumed that the crystal is hexagonal 1 . “G that the innermost spots observed are due to the 100 plane. This 50 "a" value then may legitimately be compared with "a" values for hexagonal crystals. There are surprisingly few crystalline materials listed in the literature whose "a" values correspond to the value a = 5.29 calculated for the crystals seen in Figure 1. Donnay and Nowacki (1951;) and Frevel and Rinn (1953) have listed the "a"and "c" values for many hexa— gonal crystals. Some of those whose I'a" values are closest to the cal— culated value are given in Table XXV. If an error has been made in the assumption that the innermost spacings observed are due to the 100 plane. and are due rather to the 200 plane. an additional number of compounds with an "a" axis of about 10.58 might be implicated. These also are given in Table 10W. However. this is unlikely because it would then be necessary to explain the systematic absence of all odd numbered spac- ings. From Table XXV. it is obvious that the crystalline ”contaminant“ could be unequivocally identified if its "c' axis could be determined. In theory. this could be accomplished by tilting the ”3" axis of the crystal away from the normal to the electron beam until hkl. hkz or hk3 planes gave refractions which could be picked up and identified f rom the photographic plate. The simplest way to do this would involve the use of an internal standard and a good goniometer. operating. of course. inside the vacuum system and sufficiently well-constructed so that the crystal under examination could be visualized constantly d“ ring rotation and focusing. To construct a universal goniometer which would permit any minute crystal. no matter where located on the grid, to be rotated around one of its axes without leaving the focal .- “.7.- we" 51 gflane. would be difficult to say the least. An attempt was made by Dr. H. h. Bendlerto do this by insertion of wedges after an initial pattern was photographed. The same crystal could not be relocated. however. and the attempt was unsuccessful. In other words. this promising tech- nique had to be abandoned for reason of inadequate instrumentation. In viewing Figure 1. a striking similarity is seen between the micrographs shown there and micrograph of precipitated synthetic hydroxyl- apatite with recognizable crystals of secondary calcium phosphate also present. (Figure lZ-B. Page 80. Hodge. 1950) Since bone cannot necessarily be regarded as the source of the thin crystals seen in Figure l. the data presented cannot be directly applied to the bone problem. However. the method is useful and renewed efforts should be made to eliminate the contaminant and further studies should be con— ducted using preosseous embryonic femurs as a source of biologic materi- 81. Bo X- fato 1- Discussion of Data Obtained from Ery Embryonic Femurs After Six to Ten Days Incubation. Two general approaches have been made in the study of calcification. 535: fine. One utilizes primarily the study of solubility products of mterials present and of hypothetical intermediates in an effort to decixie (usually by a process of subtraction) what actually "precipitates". shBar-and Kramer (1928a). Logan and Taylor (1937). (1938). Huggins (15137). Logan (1940). Hodge (1950). Another group studies local fac— t <3rs such as enzyme systems or organic complexes ("metachromasia"). which might be involved in calcification. Robinson. (1923). Sobel (1950). Gutman and Yu (1950). Marks and Shorr (1950). Although significant advances have been made from both approaches. elucidation of the actual sequence and spacial arrangement of the ions going down as apatite re- mains a problem. particularly since there is no apparent reason for them to stop "precipitating" when the crystal is only a few unit cells in size. The study on six to ten day embryonic femurs was undertaken. there- fore. to see if any information could be gained bearing on the mechanism of calcification. It is at about the ninth day of incubation that an osseous cylinder first begins to form around the shaft of the embryonic femur. By running diffraction patterns of wet embryonic femurs. an attempt was made to study the bone under conditions as nearly normal as possible. However. the large amount of scatter. presumably from the water present in these young femurs. made it impossible to see any diffraction lines. This is a considerable disappointment since it is difficult to believe that the "osseous" cylinder is not crystalline when first laid down. even though it is formed in the highly aqueous and only semi- solid "gel" which is its cartilaginous anlage. Nonetheless. diffrac- tion lines were not observed although the method and instrumentation "as adequate in the case of 21 day wet femurs. The water content of puppy articular cartilage is 761$ grams/kg (Eichelberger. 1952) and in the adult rat skeleton is 561 grams/kg (white and Rolf. 1955). Libby (pers. com.) reports up to 743 grams/kg in 21 day embryos. The 11 day femur could scarcely have contained "Dre than 900 grams/ kg. Consequently. the non-detectability of apatite L in 11 day wet "bones" must be due either to the masking effect of an extra 100-200 mg/kg (15 - 307‘ more H20) or actually to the lack of appreciable crystallinity in the bone first deposited. or, of course to the concurrent effect of both factors. In the absence of a defini- tive study of this point. it is well to point out the definite possi- bility that what first is laid down as bone may not be crystalline until water is removed. either physiologically as the bone "matures' or artificially as the bone. removed from the embryo. dries out. The negative diffraction results should perhaps be expected. since hydrated crystals are usually (not just exceptionally) different in crystal habit. unit cell dimensions and all the other usual crystal characteristics. Further. materials such as the penicillins. are amorphous when hydrated and ibrm regular crystals only when water is lost. The potential significance of this point can scarcely be over- emphasized since all the diffraction work on bones has been done either on mature bone. which has been physiologically dehydrated. or on .Ybunger bone which has been dried before examination, at the least in air. at the worst in a ruffle furnace. and in the case of electron microscOpy and diffraction by preliminary embedding in a non-aqueous Plastic followed by examination in a vacuum system. The distinction (long ago drawn by the histologist and pathologist) between "calcifi— cation" of a tissue and "ossification" may have to be reviewed in modified form for the case of normal bone development. If this possi- bility were ever rigorously proved. the existing literature on the bone a‘Dfitites would relate to secondary phases in bone deve10pment and not t0 the primary (first) processes. __w. “-7, Diffraction methods are available for the study of "non—crystalline" materials (radial distribution analysis). These have been developed largely in connection with research in polymers. most of which have. at best, a very low order of crystallinity at lower temperatures. Studies of collagen by small angle scattering are also pertinent in this connection. The results obtained when the femurs were dried were also not anticipated. NaCl and KCl crystallized out of preosseous cartilage. but as soon as a detectable amount of bone apatite appeared. no NaCl was seen and a marked reduction occurred in crystallizable KCl. Chem- ical analyses showed that there was no significant decrease in the total amount of Na and K present. Consequently. it is assumed that after apatite started to form. both cations were present in a form which pre- vented them from crystallizing as the chlorides. Even though considerable variation exists in the chemical analyses for Na and K of the femurs from 7 to 10 days, as might be expected from the possible error in weighing. the average values fit very well With those reported for preosseous articular cartilage (Eichelberger. 91" a1.. 1952) and cartilage (Everett. 19148). (See Table XI. Total N3 and K) The potassium values are somewhat high as compared with literature values. Some interesting calculationsmay be made regarding the amount of Na and K that might be free to crystallize upon drying. using the data. calculations. and assumptions of Eichelberger. et a1. They had previously established that a constant ratio of Na. K. and Cl is associated with the collagen present in tendon. and assumed lawman-T42 " 55 TABLE XI CAICUI‘TED AMOUNTS OF SODIUM. POTASSIUM, AND CHLORIDE ASSOCIATED WITH SEVERAL POSSIBLE BINDING MECHANISMS IN CARTILAGE1 Eichelberger2 Everett3 Preosseou Osaifying Articular Cartilage Cartilage Cartilage Cartilage Sodium free a 160 151‘ 57‘ 0 CS Na 656 719 7b6 892 fibre Ba 92 106 9? 108 Total 908 976 900 1000 Potassium free 8 28 133‘ . - cellular K 216 217 217" 300 Total 224 2&5 350 300 Chloride free 169 179 190‘ 0 fibre 95 109 100 111 Total 236 288 290 111 'Meximum “M1nimum 1All values in table are mlq./kg solids 2Eichelbergor at al.. (1952) . 3m:m,(1943) ‘ Awerege of seven and eight day femurs sAverege of nine and ten day femurs Sodium associated with chondroitin sulfate 56 the I'a‘tio to remain fixed in cartilage. For K and Cl. this leaves a certain amount "free". There then must have been enough "free" 'sodium t° be associated with the excess free chloride. The remaining sodium "“St be bound and can be accounted for on the basis of chondroitin sulfate content. These calculations were also applied to the cartilage values of Everett. and the preosseous (7-8 day) and ossifying cartilage ( 94.1.0 day) of chick femurs. Since epiphyseal cartilage of 21 day chick femurs (similar to articular cartilage) did not show detectable amounts °f crystalline NaCl and K01 by x—ray diffraction, the free Na and K as calculated from data of Eichelberger. et aLmust not be the same 1‘”action of Na. K and Cl which was found to crystallize when 7 and 8 day embryonic femurs were dried. This then suggests some binding other than by collagen or chondroitin sulfate. However. it cannot be defi— “1‘3er stated that the portion called "fibre" Na, K and C1 could not crystallize on drying and contribute to the diffraction pattern. Several possibilities may be presented to explain this phenomenon °f binding Na and K in bone. Boyd and Neuman (1951) showed equal bind- ing of Na. Ca. and Ba ions by veal costal cartilage. and correlated this with the so“ content. 0n the basis of this. they felt chondroitin Sulfate was the binding agent. Other workers have presented the idea that the sodium present in bone may well be "occluded, adsorbed or interstitially crystallized". (French. at a].. 1938. Hodge. et al.. 19193). This adsorption may be due to a hydration layer as preposed by Neuman (1952). Neuman, et a1. (1950) studying fluoride uptake by bone salt jg; v'tgo. assumed that the carbonate present in bone shared a monovalent 5? bond withcalcium and that the second carbonate valance was occupied by Sodjdlm. i.e. - Ca - 0 - 002 - Na. Bergstrom (195%) also found that the changes observed following acidosis suggest that all of the Na and K in bone is present as Ca - O — CO2 - Na(K)~. It is also pOSSible that heterionic exchange may be responsible f‘n‘ the binding - especially of sodium. Na and Ca both have approxi- mateh the same ionic radii (about .98 3). therefore. sodium can and d°es replace Ca in the apatite. Potassium. on the other hand, with an iOnic radius of 1.33 X is usually considered too large to fit into the hole left vacant by a calcium ion (Hendricks. 1952. and Neuman and Neunan. 1953). Citric acid may also be involved in the picture of Na and K bind- ing. (Shear and Kramer. 1928b); however. this possibility does not seem t0 be as plausible as some of the others. Actually, very little is known about the position of citrate of bone (Armstrong. 1950). Dickens. (1911.1) determined the citrate content of embryonic chick bones and alsa computed the equivalent Ca to citrate ratio. The citrate content fails steadily from about 31.0 rug/100 gm embryo at 3-1/2 days to 9.h “‘8/100 gm embryo at Zldays. The Ca: citrate ratio goes from 3.3:1 to 130:1 for the same periods. Between 8 and 12 days. there is very little Change in citrate content. the period during which calcification first takes place. Whatever the mechanism of this binding of Na and K, which prevents their crystallization on drying when there is a demonstrable amount of apatite present. it must not be the only method by which Na and K are bound. As is seen from Table XI. dry preosseous cartilage contains about 90 mEq/lOO gm sodium and 22 mEq/loo gm potassium as (Eicllelberger. et a]... 1952.). whereas young bone contains much less 0f bOth. Na and K. (LL mEq/lOO gm Na; 3.1: ItlEq/lOO gm of K. Bergstrom 1952). Therefore. in the nine day embryonic femur. which has a small amount of apatite present. some of the sodium must be associated with the new mineral phase while most of it still remains bound by the car— tilage. It is not clearly understood how the small amount of bone apatite present at nine days could bind sufficient quantities of Na and K to reduce the crystallizable portion below the limits of detection. Since young bone contains only about one-tenth that of cartilage. How- ever. some mechanism associated with the bone apatite must be responsible for the binding of the "crystallizable" portion because the cartilage present is probably binding Na and K as it did in the preosseous femur. This would tend to rule out binding of the crystallizable portion by chondroitin sulfate and favor some process of adsorption in the apatite laattics. This view is not inconsistent with that of Neuman and Berg- 3111mm. since they assume that the carbonate is attached to the calcium (31‘ the apatite . 2. Studies on the Nature of Bone Mineral In general. most investigators agree that the solid phase of bone may be regarded as a slightly impure basic calcium phosphate (Eisenberger. Lehrman and Turner. 19140). Differences in opinion arise regarding the exact position of the "foreign" ions, and whether or not they are important in the mineralization process. Since rather extensive substitution into theapatite lattice. or adsorption of ions by apatite does not alter the lattice parameters appreciably. it is difficult or impossible to show the percentages of substitution by x-ray diffraction. In addition. the diffraction pattern from fresh bone has very broad lines which makes accurate estimation of the line position even more difficult (Taylor and Sheard. 1929). The broad lines indicate that the crystal size in bone is extremely small. a finding which has recently been confirmed by electron T’VICI‘OSCOPY (ROb‘inson. 1951). In order to sharpen the lines present on the diffraction pattern. so that more exact determination of lattice constants can be made. it has been customry to heat ash bone at high temperatures. It has uSnailly been assumed that this procedure does only two things. eliminate Sc"#11:.tering by water and organic matter and induce growth of the cry- Stale. A number of techniques have been more recently devised to rid the bone of its organic material so that the mineral phase could be better Studied (Neuman and Neuman. 1953). In each case it can be shown that the ashed material resulting from the process is altered somewhat from the original mineral phase. The question is. however. which alterations are to be avoided and which are desirable. In other words. which por- tions of the bone composition are basic constituents and which have no bearing on the behavior of the system. Ethylenediamine (ED) was used by Arnold (1952) to remove the or— ganic portion of bone. and thus to obtain information regarding the rela- tive amount of injected Can5 organically combined. By using the method of ED ashing reported by Arnold. the organic material was removed from 21 day chick femurs. As shown earlier. the only difference between the diffraction patterns for dry bone and from ED ashed bone was the absence of certain broad bands. probably due to the loss of collagen after ED extr"=‘<3‘tc:’l.<)n (Clark, 1931, Wyckoff and Corey. 1936). After one hour heating at 500° C.. interesting differences begin to show on the diffrac- tion patterns. Lines from the heated dry material are much sharper than those of the heated ED ashed material. The reason for this prob- ably is due to the presence of organic matter in the dry material. which when heated to 500° C.. burns and causes a local rise in temper- ature (Dallenagne. 1952). Even at this time. some whitlockite lines were identified. Whitlockite usually requires temperatures above 500° C. 1‘ or its formation. although a few instances of low temperature for- mation have been reported (Trautz. 1955). After additional time at 500° C. or when heated for two hours at 500° 0.. there is noticeable shift of the strongest line (2.79 K) on the heated ED ashed bone when compared to the heated dry bone. By study- ing the diffraction patterns as they appear in Figures 5. 6 and 7. it can be seen that with heating dry bone. the b$ad line of dry bone gradually is resolved into three lines: 2.8214- (100)1. 2.770 (30). and 2.718 (60) (Table XXI. 3.26). On heating ED ashed bone. this same broad band will resolve into sharp lines sbmewhat differently. The 8‘lirongest line 2.790 (100) (Table XXII. 8.27) appears at a position Which had previously been the exact center of the broad band. whereas the strongest line of the heated dry bone appears at a spacing larger than the exact center of the broad band of the unheated bone. Recent precise work by Perdak, W. as reported Posner (1955) gives Relative intensities 61 the lat“Lice parameters of hydroxylapatite as a = 9.’420 i 0.001 i3; C =~ 6$385 2*. 0.001 A’. whereas Altschuler. Cisney and Barlow (1953) give values of a = 9.1413 i .002. C r: 6.875 5; .002 for hydroxylapatite. These Precise values were both obtained from supposedly pure hydroxylapatite and yet the lattice parameters differ. Thus. precise values obtained in two different laboratories differ significantly. It is difficult to C’OmIDv'clre values from this thesis with those in the literature for this reason. When the diffraction lines are broad and diffuse as they are in untreated bone. considerably greater range is encountered in reported vaers (See Table Di). Obviously. further descriptive work is less im- portant than theoretical analysis of the changes in the broad bands When treated in various ways. In general. all the shifts in lines observed on ED extracted bone when heated indicate a decrease in the "a" axis. with little or no Ch ange 1n the "c' axis. By calculating the "3" axis from the (300). (200). and (310) planes for both dry and ED extracted bone when heated (See Table IX). a shift from a = 9.142 R for the dry to a = 9.36 K for the ED extracted bone was seen. Based on the (002) plane, only a slight change is noted in the ”c" axis (6.81» K - 6.83 3). By using these c values to solve the hexagonal formula for "a". based on the (121) plane. ”a“ shifts from 9.45 K to 9.33 l. The whole question of line sharpening when bone preparations are heated must be studied from a crystallographic viewpoint to establish whether measurements of the center of broad lines give a true value for lattice parameters. V. SUl'l-Lifii AND CONCLUSIONS Eiy means of electron microscopy and electron diffraction, young bone"prepared by drying. heat ashing. and ethylenediamine ashing was Studied. Two distinct types of fragments were observed. The dense fra'EIITlentswere assumed to be bone particles. although no diffraction patterns were ever obtained. Thin crystals of indeterminate source §h£nwed good electron diffraction patterns. always in a hexagonal form.‘ ASsuming the crystals to be hexagonal and assigning the innermost spots the hkl value of 100. an "a" axis of 5.29 X may be calculated. Without being able to tilt the crystals so as to obtain additional spacings. the "0" axis cannot be determined. With better instrumentation. the crystal SDecies could urxioubtedly be established. There is no evidence of any precursor crystalline material present ill bone prepared under the described condition. However. the diffraction Ifirtterns from single crystals are evidence of the efficacy of the method. X-ray diffraction patterns from dry preosseous embryonic femurs Showed the presence of crystalline sodium and potassium chloride. When apatite was detected in the femurs. no crystalline NaCl was detected and KCl‘was found in only one sample. Since there was no significant change in total sodium.and potassium.over the seven tO'tenlday period during which ossification occurs in the chick femur. some "binding" mechanism associated with the presence of apatite is indicated. Several methods of binding of ions are discussed. with their possible signifi— cance in binding "free" sodium and potassium in preosseous and ossifying 63 bone. No diffraction pattern was seen when young wet femurs were ex- posed to x-rays. This was probably due to scatter by the high water content. Since 21 day "wet" femurs produced good diffraction patterns. the possibility is suggested that bone as it is first laid down is non- crystalline or very poorly crystallized. The bone mineral might then crystallize upon "dryirg". either non-physiologically upon removal. or physiologically by maturation. This point bears further investigation. Upon ashing at 600° C.. embryonic femurs of eight to ten days showed a diffraction pattern similar to that of a mixture of NaCl and x01 heated at 600° c. Nofl-Ca3(P0u)2 was seen in the diffraction pattern from the ashed femurs even though apatite was present in the dry samples of nine and ten day tenure. Since only a minute amount was recovered from each femur. this may be simply a lack of detection. Twenty-one day embryonic chick femurs prepared by etmrlenediarmine ashing were shown to give a diffraction pattern indistinguishable from dry bone of the same age. It was concluded that the process of ED extraction did not cause a significant change in lattice parameters and did not induce lattice growth. ED ashed bone would serve as an excellent preparation for further study on crystal size by electron microscOpy. Upon heating. dry bone produced a much sharper diffraction pattern and was more readily converted to #:1390192 than ED extracted bone. This. can be explained on the basis of incineration of the organic material in dry bone which would cause a local rise in temperature abOVe that of the furnace. The flCa3(POu)2 produced upon further heat- ing was the same from both preparations. L Before conversion to whitlockite. x-ray diffraction patterns from ED extracted bone showed a shift in the "a" axis toward a smaller Spacing as compared with the dry bone. The calculated value of a = 9.42 X, c = 6.84 K for heated dry bone corresponds closely to the values of hydroxylapatite.whereas the value of a = 9.36 X for heated ED extracted bone corresponds more closely to carbonate apatite. Manna Dr. H. M. Bendler. Department of Physics and Astronomy. suggests that the contaminant seen by electron microscOpy and diffraction may be 3- species of clay mineral of the mtmrillonite family. such as nontronite. “Orrin-chin gives observed and computed spacings identical with those seen for the contaminant. «...—f f“ 3mm: A TAN-35 .. ‘u- ‘ . . magi-433...! 4' L ' TABLE XII EXAMPLE OF INDIVI DIAL MEASUREMENTS IRON AN EIECTRON DIFFRACTI ON PAT’I‘ERNI =-~; WWW ---w v-".‘;m—-Wmfi—v-"—cmw--. ‘-~ ~--- - -..-.--J“- -a"- Aungie of2 Gold ~-_._”_ Crystal Diffraction spots _-____~-_ “”83““ Bflfiiijzniil‘il 21a- d 1 918-- 51.1....“ 0° 16.1 8.3 n.60 16.5 2.30 24.5 1.5u8 60° 15.9 8.1 n.62 16.2 2.31 2n.2 1.5u8 120° 15.2 7.9 11.53 15.7 2.28 23.1; 1.530 30° 16.1 1a.3 2.65 28.5 1.330 90° 15.5 13.6 2.68 27.3 1.336 150° 15.5 13.8 2.6u 27.5 1.330 20° 16.1 21.8 1.740 1u0° 16.1 21.8 1.7u0 80° 15.7 21.0 1.760 100° 15.3 20.7 1.740 luo° 15.6 21.0 1.750 150° 15.9 21.u 1.750 1 Pattern used for the example in #503-D appearing in Fig. 2 Measurement! were made on a.proJection print. x 2.7. 2Angle of rotation is determined oinply from a reference lineo drawn through a pair of’pointe which then is designated as 0° 3Linear measurement: are given to the nearest 1/50th inch. 1.; Calculated diameter Au ring - diameter x ring d8u(100) 2 2.3% 3 from the relationship: “it . m..." weeps cote-no hobo one: 558%. page on .uuneaoudeeea wen we awoke: 23 a 93m...“ some dance :5 em .03 one emu an? new cord: no doggone on» near ounce—c.3303 0923 we emanate on» one use» :5 a.“ wnaudomne condo» :4. men; mmmé one; end med Rs .9234 HR .H new A and 8.ch .. ._ 3d ems "Team .. ace 2 mmng numam .. .. own A com A on. .H mm .~ cm .a cued». .. .. saw.“ an... «inn .. e2. 9. can .H “on; Se.” and nod R5 «Jon .. .. «me; New; 3...; and cod a: ennom _. ace d can A new A 3a; and 8d 8.: enema .. .. Nam; mam.” mi; and $.m 3.: one? .. .. «mm; was; med «in? a8 .3. 3 7 awn; Sn; S... 4.8.. e32 an a3: a 6 and «mi 0.8: .. .. .S.N amp: :wa Baa 73 m oea.H N o¢:.H m Nam.a o“ OHN.N oH oNN.N ca OHN.H oH ONN.H 0H nnm.N o” mam.” ca mam.” n wn0.H o“ cam.“ oH oam.a ON mam.a OH oNN.N o” ooN.N oH NNN.N ooH om.N ooH om.N ooH om.N ooH an.N ma oa.n ma oH.n om n:.n o: N:.n on n:.n ow N:.n on mm.m “\H < . HH\H 4 e HH\H « e HH\H « c 0 o Noun mw-m Nana wanm 95:": unhowmg Ha M3 2H9 soak nflzu KHN Ham; ’21-‘43" ...—w —_ (7* VALUES OBTAINID 130M ELEVEN AND TVINTY—OND DAY DRY IEMURS 3-30 (11 day) 3-35 (21 day) o d A 1.813 1.708 3.83 3.u2 3.10 2.80 2.266 VALUES OBTAINED FROM DRY 21 DAY FEMURS WITH AND TEMPERATURE 75 TABLE XXI INCREASE! N6 HEATI N0. TI MD 500°C. 600°C. Dry 1 hour 2 hours 0 hours 2 hours B—18 8.20 B-22 3.22 B-26 d 1 1/11 d 3 1/11 a 1 1/11 a 1 1/11 a 3 1/11 9037 ' 9.18 2 8.19 10 8.12 20 8.19 15 8.12 20 6.00 - 6.00 10 6.06 5 6.06 10 5.18 10 5.18 20 5.20 15 5.20 20 0.70 5 0.70 5 0.76 2 0.70 5 0.35 - 0.30 5 0.35 5 0.33 5 0.32 10 0.05 10 0.07 5 0.10 5 0.07 10 3.86 10 3.86 5 3.87 5 3.88 10 3.79 10 3.78 5 3.80 2 3.51 2 3.50 2 3.01 50 3.02 30 3.02 00 3.03 00 3.02 00 3.3“ 5 3-30 5 3.33 2 3.32 5 3.17 15 3.16 15 3.18 20 3.17 20 3.08 15 3.08 10 3.09 15 3.08 10 2.98 2 2.99 2 2.99 2 2.98 5 2.80 100 2.817 100 2.817 100 2.830 100 2.820 100 2.770 30 2.761 00 2.770 00 2.770 20 2.720 60 2.718 60 2.720 70 2.718 50 2.625 10 2.619 15 2.625 20 2.619 15 2.578 5 2.572 10 2.580 15 2.572 20 2.538 2 2.527 5 2.533 10 2.527 20 2.090 2 2.080 2 2.095 2 2.090 5 2.37? 2 2.367 2 2.377 2 2.379 2 2.300 2 2.335 2 2.308 2 2.339 2 2.290 2 2.280 5 2.296 2 2.262 15 2.262 20 2.262 30 2.262 30 2.262 30 2.236 2 2.228 2 2.232 2 2.231 5 2.167 2 2.171 5 2.169 5 2.100 10 2.108 10 2.107 10 2.100 10 2.090 2 2.050 10 2.050 10 2.050 10 2.050 10 2.006 5 2.002 2 2 006 2 2.006 5 1 1.989 5 1.986 2 1.989 2 1.986 5 -926 15 1.900 00 1.901 30 1.938 50 1.901 30 1.913 5 1.910 5 1.911 5 1.910 5 1.892 10 1.890 10 1.890 10 1.875 5 1.866 5 1.872 5 1.852 5 76 TIBLE xxx (Cont'd) VALUES OBTAINED FROM IRY 21 DAY FEMURS WITH INCREASING HEATING, TIME . AND TMEATURE 500°C. 600°C. Dry 1 hour 2 hours Lt hours 2 hours B-18 B-20 B—22 B—ZZ B-26 d 3 1/11 a 3 1/11 d 3 1/11 0 8 1/11 d 1 1/11 1.823 15 1.835 00 1.832 30 1.835 50 1.835 30 1.808 20 1.808 20 1.811 30 1.808 20 1.782 20 1.782 20 1.782 30 1.782 20 1.750 20 1.750 20 1.750 30 1.750 20 1.700 15 1.710 30 1.708 30 1.708 00 1.710 30 1.689 2 1.686 2 1.687 2 1.689 2 1.666 2 1.660 2 1.660 2 1.666 2 1.600 15 1.602 10 1.600 10 1.600 10 1.606 10 1.606 10 1.606 10 1.606 5 1.586 10 1.580 10 7 1.585 5 1.586 5 1.573 2 1.571 2 1.506 15 1.506 10 1.500 5 1.508 10 1.530 15 1.537 10 1.533 10 1.508 5 1.508 5 1.506 5 1.501 1.099 5 1.099 5 1.095 5 1.073 15 1.073 10 1.075 15 1.073 5 1.050 20 1.008 20 1.050 25 1.050 20 1.036 15 1.036 5 1.036 15 1.036 10 ___—.—. - ---- ---- _ ...—0.-..w- ..a..-_- -. -.- TABLE XXI I ING, TIME AND TEIleERATURE VALUES OBTAINED FROM ETHYLENEDIAHINE ASKED FEMURS WITH INCREASING HEAT— 500° T 600° Unheated 1 hr. 2 hrs. 0 hrs. 2 hrs. B—19 B-Zl 8-23 B-25 B-27 d A 1/11 d 1 1/11 0 3 1/11 0 A 1/11 d 1 9.20 5 9.13 5 9.37 2 9.21 8.02 5 8.12 15 8.25 5 7.96 5.18 10 5.35 2 5.18 L . 0.72 5 0.78 2 0.68 0.27 2 0.06 2 0.07 10 0.10 2 0.00 3.83 5 3.82 2 3.83 10 3.87 2 3.83 3.02 00 3.00 00 3.03 50 3.00 20 3.01 3.10 15 3.13 5 3.10 10 3.18 2 3.10 3.07 10 3.06 15 3.08 5 3.05 2.797 100 2.79? 100 2.797 100 2.800 100 2.790 2.718 30 2.692 00 2.718 60 2.705 2.620 15 2.613 15 2.625 10 2.613 2.510 5 2.516 5 2.527 2 2.510 2.293 2 2.275 2.265 20 2.262 25 2 209 25 2.258 15 2.200 2.132 5 2.103 10 2.103 10 2.100 5 2.136 2.058 5 2.058 10 2.050 10 2.061 2 2.050 1.990 5 1.989 5 1.992 2 1.986 1.938 20 1.901 00 1.932 00 1.901 00 1.930 1.890 15 1.881 15 1.890 10 1.881 ' 1.860 1.835 20 1.838 00 1.832 00 1.838 00 1.832 1.803 15 1.795 20 1.805 20 1.797 1.782 15 1.771 20 1.779 20 1.771 1.750 15 1.700 20 1.750 20 1.706 1.712 20 1.717 25 1.712 30 1.715 20 1.712 1.680 2 1.602 10 1.638 10 1.602 10 1.639 1.602 5 1.602 10 1.608 5 1.602 1-579 5 1.579 5 1.5810 2 1.579 1.561 1.531 5 1.530 10 1.529 10 1.530 10 1.530 ; 1°502 5 1.098 15 1.095 10 1.502 10 1.097 3 1 1.071 10 1.066 10 1.071 15 1.068 g -“55 10 1.052 20 1.009 15 1.050 15 1.007 . 1.025 10 1.020 10 1.030 15 1.027 K ”ma _u~,_.-.--“. 5 ~ 4 78 TABLE XXI I I COMPARISON OF VALUES FROM HIGH TMERATURE INCINERATED DRY BONE AND ED AS BED BONE‘ 600°C. 6 hours 700°C. 7 hours B-28 (dry) B—29 (ED ashed) B-71 (ED ashed) B-72 (dry) d 1. 1/11 d 3 1/11 d 3 1/11 d 1 1/11 8.07 15 8.02 20 8.07 20 8.07 20 6.07 15 6.00 20 6.08 20 6.08 20 5.22 30 5.15 00 5.18 50 5.18 00 0.35 2 0.30 5 0. 35 10 0.35 lo 0.00. 5 3.99 10 0.00 20 0.00 20 3.90 5 3.92 5 3.92 5 3.07 50 3.02 00 3.00 00 3.03 00 .3.33 10 3.32 5 3.35 5 3-33 5 3-19 80 3.18 70 3.18 70 3.18 70 3.08 2 3.12 5 3.09 2 3.10 2 ‘3.00 5 2.99 5 2.99 5 ‘2~859 100 2.851 100 2.859 100 2.859 100 2-738 5 2.731 10 2.738 10 2.738 10 2~673 2 2.661 2 2.667 2 2.667 2 2.650 2 2.590 80 2.580 70 2.596 70 2.596 70 2-538 5 2.538 2 2.538 5 2-500 5 2.080 5 2.500 5 2.500 5 2-387 5 2.387 10 2.387 10 2.387 10 2.330 5 2.307 2 2.209 5 2.200 10 2.209 10 2.209 10 2.187 5 2.179 10 2.179 15 2.179 10 2.100 5 2.107 10 2.108 10 2.108 10 2.065 5 2.058 10 2.065 5 2.062 5 2.023 5 2.012 10 2.019 10 2.019 10 1-989 5 1.989 10 1.986 10 1.986 10 1.920 10 1.920 20 1.923 25 1.923 25 1.870 10 1.869 15 1.875 15 1.872 15 1.816 2 1.811 2 1.813 2 1.813 2 1.787 2 1.803 2 1.800 2 1.750 2 1.766 2 1.761 5 1.761 5 1.720 15 1.713 30 1.717 30 1.717 30 1.703 10 1.700 2 1.703 2 1.672 2 1.672 2 1.623 2 1.621 2 1-592 5 1.592 5 1. 502 2 1. 502 2m 1. 502 20 1. 502 20 77g Iues 1n th1“w1"ar th ey'are campgrggbggth I1 3»- averaged an appear in Table x where tereture v ue TABLE XXIV COMPARISON OF VALUES FROM HEAT ASHED EIGHT TO TEN DAY FEMURS WITH HEATED MIXTURE OF SODIUM AND POTASSIUM CHLORIDE ‘—- -..va n. w - ' —— .— w _ —-——~- . u—uomwafi‘ .. HaCl & X61‘ 8 day‘ 8 day“ 9 day“ 10 day" B-73 B-69 B-53 B-52 B-Sl o ,_ 0031 1/11 d 3 1/11 d 3 1/11 a 3 1/11 d A 1/11 0.75 5 0.78 5 0.66 10 0.66 10 0.66 10 0.36 5 0.36 5 0.36 5 0.36 5 0.10 10 0.10 15 3.95 60 3.98 60 3.90 100 3.90 100 3.90 100 3.57 20 3.55 10 3.57 50 3-57 50 3 57 50 3.29 20 3-17 5 3.19 10 3.08 5 3.07 10 2.852 100 2.852 60 2.817 50 2.817 50 2.852 70 2-798 100 2.777 100 2.797 70 2.660 5 2.691 100 2.692 100 2.692 100 2.305 10 2.377 5 2.339 5 2.339 5 2.336 5 2-275 15 2.209 30 2.203 5 2.200 5 2.207 5 2.210 5 2.215 5 2.120 10 2.128 5 2.087 5 2.028 5 1.999 60 1.986 00 1.999 10 1.999 10 2.005 10 1.907 10 1.960 10 1.960 10 1.960 10 1.932 10 1.932 10 1.932 10 1.862 2 1.850 2 1-779 10 1.798 5 1.779 2 1.779 2 1.782 2 1.760 5 1.621 20 1.672 2 1.623 2 1.653 2 1.602 20 1.610 2 1.555 2 1.555 2 1.555 2 1-“97 20 1.092 5 1.090 2 1.502 2 1.395 15 1.390 5 1.393 2 1.395 2 1.000 2 1.303 2 1.303 2 1.300 2 \— ...“ ' Ashed in platinum crucible “ ABhOd 1n porcelain crucible TABEE XXV LIST OF CRYSTALLIRE MATERIALS WITH AN "a” AXIS NEAR 5.29A 0R 10.58A’ ‘— ...- Material a c Reference KAlSiOh 5.17 8.6? Donnay & Nowacki CazSiOn'Ca.3(POu)2 5.21 6.90 " " 1361.101, 5.21 8.76 8 " 20a28104-Ca3(P00)2 5.22 6.91 Frevel a. Rinn 063.20.. 5.20 7.10 8 8 Pb(OB)2 5.26 10.7 Donnay 8 1160.611 mlicaa. 3 layer structure “3 (on. ”2 (11. 810.010 5.3 30.0 8 81300.92 5.38 19.8 8 2ca2810h-C03(Pob)2 5.38 7.05 " ' Nazsol, 5.38 7.26 . 8 fcafil’owzflhitlockite 10.31 37.00 Prevel a. Rinn 9N0280u823a20038101 10.06 21.2 Donnay & Bowacki Kla22(sou)9(co3)zc1 10.50 21.23 Frevel 8 Rinn Nazce(m.1'..r.)82 KBi,P)Ou]3 10.53 05.5 Donnay & lowacki 11.08801. (high temp.) 10.53 5.76 .. 8 30.1104 10.60 5.80 8 8 (083)201108 10.61 7.02 8 8 UhSrPOQ (low temp.) 10.65 5.81 " " KSrPOh (low temp.) 10.70 5.87 ' " ‘— I‘Crystalline metals not included in this list. APPENDIX B FIGURES 8.32:3 6.020602% \QQ .w B 69.3er =§o§§5 Strk m. 5Q .35 \ $me $8.28 38 .0 Form \byx btbxhuh ..Sdkxfi \..\ . .. QR 1% .EaEmk 63335.0 kg N \o 65$on geofitfi .807». 9 st ..Oh m: 3me 636.6 .30 k 83:280. QQQAEEW .GQ m E 65330. uncouthQ .807». \\ 90. .Ch «1 $me 23.23 .56 % .....Sfimk Qtoxufifim 55 m \o 852qu 25:83th .9054 m< 6.2.1 .Qb \ 02:0 .33.» .30 m. wbum m6: .96 \ 5me .336 .36 m. hm. -m .86 3 $5.... «6...... .86 m mVl .6.on \N 8 Q \o 6386.1 6.365221% Bot 65330. entoouhfi \6 56:32.8 Q 5Q be 2 $me «636.360 .96 KC 5me ES.» .36 \\ Qnum . 4 .Q .325... ...oQ \N xo atom 63:6; \6 «$8.1on 5.38th \6 208.3058 3 6...... 2.... S 888 «8% BEN 28 s bu S 0.88 «6.5 .88 x um. I .08 S 6.068... Es. 8.6 ..w .éh \C 3me .36 \N Eoux $336.th ” . NTQ 632$ tom 38.... 5.... 325k «3368.00. .936? Bot xo «5&qu encouxtfi x6 comtoqsou 3 SC .068 8.88 S .8. 26 8.2 .8638 3 w. 08 - o _ . .- a ~WWW-‘. P. s ‘8 3.215: ' 1'33‘.‘ ‘ NM- .36 .8 .86 S .06 S 3&8 38 .86 m .. mm- .92 m 6.8% N .Qb \ 3me ..Bfiu .9 . , harm LITERATURE CITED Altschuler. Z. S.. E. A. Cisney, and I. H. Barlow. X-Ray Evidence of the Nature of Carbonate-Apatite. Am. Mineral. 38: 328 (1953) Armstrong. W. D. 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