J. Anat. (1977), 124, 1, pp. 193-203 193With 4 figuresPrinted in Great Britain

Microradiographic and histological examination of thesplit-line formation in bone

J. C. BUCKLAND-WRIGHT

Anatomy Department, Guy's Hospital Medical School,London Bridge, London, SE1 9RT

(Accepted 16 July 1976)

INTRODUCTION

The split-line technique was devised by Benninghoff (1925) as a method of deter-mining the alignment of osteones and the lamellar organization in bones. Thepattern of lines thus produced was interpreted as representing the direction of thestress trajectories (Benninghoff, 1925; Bruhnke, 1929; Henkel, 1931; Dowgjallo,1932; Siepel, 1948; Tappen, 1953, 1954, 1957, 1964, 1969b, 1970, 1971). This inter-pretation of the split-lines has not been accepted by other investigators (Ahrens, 1936;Pauwels, 1950; Evans & Goff, 1957; Isotupa, 1972) who considered that the linesrepresented the direction of growth in bone. The problem of interpreting the natureof the split-lines is further compounded by the fact that their formation has beenattributed to different features of the structural organization of bone, and in particu-lar the alignment of either the osteones and lamellae (Benninghoff, 1925; Siepel,1948; Tappen, 1953, 1954), or the lamellae and cement-lines (Tappen, 1964), orthe vascular canals and immature osteones (Moss, 1954; Evans & Goff, 1957;Isotupa, 1972).These differences in opinion as regarding the nature of split-lines seem to be

based on an incomplete understanding of the formation of the split in bone. Thispaper presents the results of tests carried out on skull bones to determine therelationship between the formation of the split and the structural organization ofthe bone.

MATERIALS AND METHODS

Five adult female cat skulls were prepared for the split-line studies. Each skullwas sectioned in the mid-sagittal plane and the flesh was stripped from the left side.Stereo-projection microradiographs were taken of the skulls on Cronex NDT 55fine grain X-ray film using a modified Intercol (XX 50) X-ray machine (Ely, 1972), ata magnification of x 2. The skulls were decalcified in 5 % nitric acid, rinsed andstored in 70% alcohol. The splits were produced by puncturing the bone with amounted needle. Where the direction of the split was obvious the needle was left inposition and used to lengthen the split by pulling in the direction of least resistancein the manner described by Siepel (1948) and Tappen,(1953). To facilitate examina-tion and photography India ink was inserted into the splits. The radiographic appear-ance of the split-lines in the decalcified skulls and the relation of the splits to thearrangement of the trabeculae and large vascular canals in the bone were assessed bytaking projection microradiographs of the skulls on Cronex NDT 55 X-ray fflmusing a micro-focal X-ray unit (XM 30 M) (Ely, 1972) at a magnification of x 4. Asmall protractor, prepared by photographic reduction, was used to measure the

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J. C. BUCKLAND-WRIGHT

Fig. 1. Split-line preparation of five adult female cat skulls.

difference in alignment between the trabeculae and/or vascular canals, and the split-lines.The effect of puncturing the decalcified bone with a mounted needle was studied

on blocks cut from the five skulls. The blocks were approximately 1 cm2 in areaand were selected from two regions of the skull, one composed largely of cancellousbone, the other of compact bone. They were mounted under a dissecting micro-scope, and the responses of the bone to being punctured by two different needles,one with a shaft diameter of 05 mm and the other of 1 0 mm, were monitored. Theneedles were inserted into the main body of the block as well as at its margin. Theslabs of bone were paraffin-embedded, serially sectioned and stained for routinehistological examination. Blocks containing split-line patterns from the differentparts of the skulls were prepared in the same way. The sections from both sets ofblocks were examined under a microscope using normal and polarized lighting.

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Split-line formation in bone

_s 002 cm (b)

Fig. 2(a) A print from the projection microradiograph of the anterior facial region of thedecalcified skull D, showing the differences in orientation between the split-lines (s) and thetrabeculae (t) and vascular canals (c).Fig. 2(b) Diagram of the same region in which the dense areas of the decalcified bone arestippled and the undecalcified apex of the canine tooth is inked in. The black lines in the frontalprocess of the maxilla represent the split-lines seen in (a). The short arrows indicate the align-ment of the trabeculae and vascular canals in the bone.

RESULTS

Split-line patterns in the five cat skullsThe configuration of the split-line patterns in the cat skulls is illustrated in Figure

1. Although the general arrangement of the split-lines appeared similar, examinationof these patterns showed that their alignment was not always the same in the differentskulls. Those in the frontal process of the maxilla either passed directlyfrom above thealveolar process to the maxillo-frontal suture (skulls A, B, C, Fig. 1), or coursedobliquely across the frontal process of the maxilla (skulls D, E, Fig. 1). Other areaswhere there were differences in the split-line pattern were in the orbital plate of thefrontal, the frontal and parietal bones and to some extent in the zygomatic bone.Comparison of the orientation of the split-line patterns with the trabeculae and

vascular canals recorded in the projection microradiographs of the skulls showedthat the variation in the alignment of the split-lines was greater than that in thestructural organization of the bone. The orientation ofmany of the split-lines did not

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correspond to that of the trabeculae and vascular canals, and in fact the lines oftenappeared to transect these structures.

Projection microradiographic examination of the decalcified skullsProjection microradiography of the decalcified skulls recorded the same structural

features of bone as were visible in the projection microradiographs of the undecalci-fied material. The particular advantage of this technique was that the split-lines wererecorded as linear interruptions in the skull's surface, and appeared as irregularlight lines in the prints of the radiographic plates (Fig. 2a).As both the split-lines and the osseous structures were visible on the same plate,

a direct comparison of their respective alignments was possible. Of 200 measure-ments made on the skulls, 47 % showed no correspondence between the split-linesand vascular canals and/or trabeculae. The mean and standard deviation of theangles measured was 20° ± 290. The large standard deviation indicated that therewas a wide range of angles at which the split-lines transected the underlying structures(Fig. 2). Where the orientation of the split-lines did agree with that of the boneorganization, this was in areas of the skull in which the vascular canals and/or tra-beculae were aligned parallel to one another. These points are illustrated in Figure2; (a) is a print from a projection microradiograph of the anterior facial region ofskull D. At the bottom of the print is the undecalcified apex of the canine toothand associated trabecular bone. In this region spherules of calcium salts can be seen.The nasal bone is situated to the left and the anterior orbital margin to the right.The trabeculae in the frontal process of the maxilla, above the apex of the canine,appear as narrow dark grey bands, and the vascular canals as narrow light greybands. Both are aligned in the direction indicated by the arrows in Figure 2b, inwhich the dense regions of the anterior facial bones have been shaded'and-the split-lines drawn in to facilitate their recognition in the adjacent X-ray plate. This plate(Fig. 2a) shows that the split-lines correspond to the direction of the underlyingstructures in bone only in the anterior region of the frontal process of the maxilla.This figure is representative of the differences generally found in the orientation ofthe split-lines and of the osseous structures observed in the skulls.

The decalcified bone's response to a pointed needleAlthough the decalcified bone reacted differently to being punctured by the two

different sized needles, the pattern of split-line formation was similar in the two cases.With the needle of smaller diameter (0-5 mm) there was a ready penetration of theneedle into the bone through the parting and/or cutting of the collagen fibres, untilapproximately half the diameter of the needle had been introduced. No furtherpenetration was achieved by pressure on the needle. The compressible nature of thedecalcified bone was indicated by the closure of the large vascular and Haversiancanals. The penetration of the needle in the compressed bone did not appear to berelated to the nature of the underlying structures. As the needle penetrated the bonethe circumferential lamellae close to the surface were raised into a pressure ridgearound the needle. The radial pressure exerted by the needle resulted in the suddenrupturing of the lamellar bone in the pressure ridge. This rupture formed the split,which extended along the bone surface as well as deep to the tip of the needle.Further introduction of the needle resulted in a slight increase in the length of thesplit, the depth of which corresponded to that of the needle.

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(b) (c)

(d) - (e) v,Fig. 3(a) Diagram of a block of bone cut from the decalcified skull showing in section (1) largevascular canals, (2) Haversian canals and (3) circumferential lamellae. (b) Pressure applied bythe needle of larger diameter to the block compressed the bone (indicated by arrows) and closedall the spaces. (c) The penetration of the needle did not appear to be related to the nature ofthe underlying structures. As the needle passed deeper the circumferential lamellae were drawninto the hole. (d) The pull applied to the needle (represented by the arrow) caused the bone'ssurface to be pushed up into a number of ridges. (e) With the increase in pull on the needle acurvilinear split formed deep in the bone. It extended horizontally in front of the needle and verti-cally to the bone's surface as well as to the tip of the needle.

The response of the bone to a needle of larger diameter (1 0 mm) was different inthat the needle did not penetrate the bone immediately. This was because of therelatively larger tip of the needle, which caused the decalcified bone to be compressed,thus closing the spaces in the bone (Fig. 3a, b). As with the smaller needle, penetra-tion was independent of the osseous organization. As it passed deeper the layers of

J. C. BUCKLAND-WRIGHT

the circumferential lamellae were drawn by the needle into the hole being formed(Fig. 3 c). It was only when the needle was pulled along the line of least resistancethat the split formed.The formation of the split-line was similar when produced by the two different

needles. When a pull was applied to the needle the pressure produced a ridge in thesuperficial layers of the bone immediately in front of the needle (Fig. 3d). As thepull was increased the split-line was formed in one of two ways. Either the splitformed at the surface of the bone by breaking across the fibres of the circumferentiallamellae, as well as passing deep into the bone, or the crack front ran ahead of thelamellae, deep to the circumferential lamellae. The widening split caused the crackto extend vertically beyond the tip of the needle, as well as to the bone's surface, bybreaking across the lamellar fibres. The shape of the split tended to be curvilinearand it frequently took an oblique course through the bone (Fig. 3 e), resulting, in themajority of instances, in part of the split passing between the circumferential lamellaeand underlying bone causing the two to separate. In general, the facility with whichthe split-line was produced depended on the complexity of the bone's structuralarrangement. The needles were pulled readily through regions where the trabeculaeand vascular canals lay parallel to one another, and not so readily where the organ-

ization was more intricate.

Histology of the split-line formationExamination of the serial sections of the splits and split-lines confirmed that the

crack front extended both horizontally and vertically. In the former plane, the crackran preferentially along cement lines, between lamellae, and occasionally alongHaversian or large vascular canals. The removal by decalcification of the groundsubstance in the cement-lines and between the lamellae rendered these interfacessusceptible to the propagation of cracks. The latter did not pass preferentially alongthe Haversian and large vascular canals as their concentric lamellae offered a resis-tance to the split.

In the vertical plane, the cracks extended in the bone by breaking across thefibres of the intervening lamellae, thus uniting the several spaces formed in thehorizontal plane; they also traversed the lacunae, Haversian and large vascularcanals. The rupturing of the collagen fibres frequently gave the broken surface a

step-like, or an irregular oblique, appearance. The split-line was frequently seen toterminate in either Haversian or large vascular canals, which probably acted as

'energy sinks' (Gordon, 1968) for the cracks.In regions of compact bone where there were few or no Haversian and large

vascular canals, the split in the bone caused nearly all the lamellar interfaces toseparate, producing a ladder-like appearance. With increased pressure from theneedle the intervening lamellae were broken. In those regions of compact bone in

Fig. 4 Histological sections (10,tm thick) stained with van Gieson, showing four stages in theformation of the split in the bone.(a) The crack front of the split opens up spaces in cement-lines (1) and interlamellar inter-faces (2), and enlarges existing vascular spaces (3).(b) These spaces are joined by the crack extending along the lines of discontinuity in the bone(4) and by breaking across intervening lamellae (5).(c) The crack passes towards and enters vascular spaces by rupturing lamellae (6).(d) And finally reaches the surface by breaking across the collagen fibres of the circumferentiallamellae (7) to produce the split-line. The crack is seen to extend deeper in the bone (8).

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Split-line formation in bone

Fig. 4. For legend see opposite.

199

which large vascular canals lay deep to the split-lines, no correspondence wasobserved between their respective alignments. In the cancellous region of bone, thesplit-lines were formed by the joining of adjacent large vascular spaces through theneedle rupturing the smaller intervening trabeculae. In general, split-lines were notreadily formed in areas of dense cancellous bone as the trabeculae tended to containthe needle at its point of insertion.

DISCUSSION

Siepel (1948) remarked that different investigators had obtained quite divergentresults with the split-line method on the human facial skeleton. This variability wasobserved between the five cat skulls employed in the present study, as well as betweenthe baboon skulls figured in Tappen's paper (1970). Tappen (1954, 1970) creditedthis variation to the combined influence of the intrinsic and extrinsic factors actingon the organization of the bone, but he did not provide any evidence to support thishypothesis. In addition to this, the micro-focal X-ray unit has provided, for the firsttime, a method of examining the detailed structural organization of skull bones inboth undecalcified and decalcified material. And it has made it possible to makedirect comparisons between the split-lines and the osseous structures. This has en-abled the differences in the findings of earlier workers (Benninghoff, 1925; Siepel,1948; Tappen, 1953, and others) to be studied in more detail.The split-line patterns on the skulls exhibited gross linear variations in their orien-

tation relative to that of the associated trabeculae and vascular canals. This waslargely due to the structures not being aligned in a single direction, but in a complexthree dimensional configuration. The split-lines are unidirectional, and in generalare continuous over large areas of the skull, and this resulted in their frequent tran-section of the trabeculae and vascular canals. These observations were confirmed bythe dissimilarity of alignment between the split-lines and the osseous structures in47 % of all measurements taken on the microradiographs of the decalcified skulls.Therefore the split-line method cannot be upheld as a reliable means of assessing thestructural organization in cat skull bones.This conclusion is supported by the findingsof Moss (1954) who considered that the split-line method should be rejected on thebasis that it did not take into account the complexities of the organization of bones.The inadequacies of the split-line technique, when compared with recent experi-

mental work (Buckland-Wright, 1975, 1976) demonstrating the direct correspon-dence between the structural organization of bone and the transmission of bitingforces, suggests that the interpretation of the split-lines in terms of the 'force hypothe-sis' (Benninghoff, 1925; Siepel, 1948; Tappen, 1953, 1954, 1964, and others) canno longer be substantiated. These authors were unable to relate the bone histology tothe apparent mechanical interpretation provided by the split-lines. However, thesimilarity in alignment observed between the split-lines and the osseous structureswhen they were oriented in one principal direction would tend to support thehypothesis that the split-lines are indicators of growth trajectories (Ahrens, 1936;Pauwels, 1950; Isotupa, 1972). In developing bones, the split-lines would occupy thevascular spaces between radially growing trabeculae.The experiments carried out with the needles of different diameters showed that

the initial pressure exerted by the needles compressed the decalcified bone. Thesplit was not formed as the needle was passed into the bone, but subsequent to itspenetration, and was due to the forces exerted by the needle in the surroundingtissue. The initial splitting or cutting of the collagen fibres, which occurred when the

200 J. C. BUCKLAND-WRIGHT

Split-line formation in bone 201needle of smaller diameter was passed into the bone, appeared to haveno direct relationto the formation of the split-line. With the needle of larger diameter greater forcewas required to form the split and to overcome the resistance of the circumferentiallamellae which had been drawn into the hole during the penetration of the needle.The forces exerted by the pull on the needle placed the bone under tensile stress

of sufficient magnitude to overcome the toughness of the decalcified bone. The forceswere dissipated through the rupture of the bone along its lines of weakness. Themechanism of split-line formation did not alter with increasing depth of penetrationof the needle, provided the bone's resistance was insufficient to withstand the pullexerted on the needle.The course of the split-line was determined by the type of osseous structure

lying in front of the needle. In general, the crack extended along the course ofleast resistance in the bone, the crack front tending to pass (a) towards large vascularcanals, which it frequently penetrated and which acted as an energy absorbing regionfor the split (Gordon, 1968), and (b) away from areas of dense compact bone. Histo-logical examination showed that when the pull was applied to the needle the splitinitially caused the points of weakness, such as cement-lines, interlamellar inter-faces, lacunae, Haversian and large vascular canals, to break open. With increasedpressure from the needle, the spaces enlarged and were finally united by the dis-ruption of the intervening lamellae. This mechanism appears to be the same forbones of different structural organization. As the direction of the split-lines dependson the numbers and types of discontinuities present in the bone, it follows that split-line patterns vary not only between different regions of the same bone, but also be-tween the same regions in different specimens, as well as between'different species.The absence of a definite correspondence between any one, or a group, of structuresin the adult skull bones and the formation of a split or split-line, points to the earlierinvestigators being incorrect in adopting specific features of the bone as beingresponsible for the direction of the split. These conclusions place in serious doubtTappen's (1969 a, 1976) interpretation of the weathering cracks in dry bones asbeing indicative of the split-line orientation. There is, in addition, no experimentalevidence of a relationship of a split-line to the tensile and compressive strength ofany particular bone (Evans, 1957).The process of split-line formation described in the present study is very similar

to that observed during the fracturing of undecalcified bone. The fracture frontmoves along cement-lines (Evans, 1973), interlamellar interfaces (Margel, 1971;Pope & Outwater, 1972) and occasionally traverses lacunae and canaliculi (Pope &Outwater, 1972). In transverse fractures, the crack front breaks across the collagenfibres to form a series of steps across the bone (Pope & Outwater, 1972). Thus, thecrack front in both the split-lines and fractures tends to move around discontinuitiesby preferentially following an interlamellar path and then jumping across lamellaeby breaking the intervening collagen fibres.

SUMMARY

Split-line patterns were produced in five adult female cat skulls. These patternswere seen to be different in a number of the skulls. The detailed structural organiza-tion of the bone and the arrangement of the split-lines were studied by projectionmicroradiography. The radiographs showed that the split-line patterns on the skullsexhibited gross linear variations in their orientation relative to that of the associated

J. C. BUCKLAND-WRIGHT

trabeculae and/or vascular canals. In 47 0 of 200 measurements there was no simi-larity in the orientation between the split-lines and the trabeculae and/or vascularcanals.When a pull was exerted on needles of different diameter that had been inserted

into blocks of bone cut from the skulls, splits were produced which passed towardsenergy absorbing regions such as vascular canals, and away from dense compactbone. The histology of a split-line showed that it initially formed at points of weak-ness, such as cement-lines, interlamellar interfaces, lacunae and vascular canals,and caused them to open up and enlarge. These spaces were subsequently united bythe rupture of intervening lamellae.The results indicate that the split-line technique is an unreliable method for analys-

ing the structural organization of bone and that it is unsuitable for determining thetransmission of force in bone. The mechanism of split-line formation appears infact to be similar to that which occurs during the genesis of a fracture.

I thank Dr M. H. Hobdell for bringing to my attention the problems involved inthe split-line technique, and the late Professor Sir Francis Knowles of King'sCollege, London, in whose department this work was largely carried out with thefinancial support of a King's College Award of a Science Research Council Student-ship. The X-ray work was made possible through the kindness and personal gener-osity of R. V. Ely. I am grateful to Dr A. D. Hoves for reading the MS and toMr K. Fitzpatrick for photographic assistance.

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