Research 10237-246 Raven Press, Ltd.. New York 0 1992 Orthopaedic Reaearch Society
Journol of Orthopedic
Mechanical Properties of Microcallus in Human Cancellous Bone Joanne Blackburn, Richard Hodgskinson, John D. Currey, and Jennifer E. Mason Department of Biology, University of York, York, England
Summary: Until now, the mechanical properties of the microcalluses that form in human cancellous bone have been unexplained. We measured the microhardnesses of microcalluses in cancellous bone, of the trabeculae within the microcalluses, of the trabeculae adjacent to microcalluses, and of trabeculae lacking microcalluses in a human tibia and femur. We observed no important differences between materials at the four different sites. Because the microhardness of bone is very closely related to its stiffness, this finding indicates that microcalluses are likely to stiffen the trabeculae in which they are formed, e v e n though they may surround unhealed fractures of the cancellous trabeculae. Key Words: Cancellous bone-Hardness-Microcallus-Stiffness.
sult as a response to repeated impulsive loading of cancellous bone, the microcallus being a remodeling response. In nonosteoporotic bone, the stiffening of the cancellous bone structure associated with accumulation of callus material would lead to a loss of protection of the overlying cartilage through a reduction of the shock-absorbing quality of the subchondral bone, the result of this loss of protection being osteoarthrotic degeneration of the cartilage. They further suggested that the same cartilage degeneration would not occur in osteoporotic bone because any increase in bone mass due to formation of microcalluses would not increase the stiffness of the already depleted bone structure sufficiently. Later, Vernon-Roberts and Pirie (16) concluded that microcalluses could either be buttresses or be associated with trabeculae that had already fractured. They described four morphologic types of microcallus: rounded nodular, fusiform, angulated, and arched bridge. They showed that in the lumbar spine microcallus prevalence increased with age and concluded that the number of calluses was indicative of the degree of osteoporosis. Sections through callus material nearly always revealed discontinuities of the underlying trabeculae, tending to confirm that microcalluses formed in response to fracture (16).
Nodular accretions (‘ ‘microcalluses”) in cancellous bone tissue have been described by several groups of investigators in the past 20 years. Darracott and Vernon-Roberts (5) described them as “birds’ nests” and speculated on their role as buttresses. They showed the material of the birds’ nests to be woven bone. Todd et al. (15) observed that microcalluses sometimes surrounded a fracture of the trabecula and postulated that microcallus is analogous to the callus formed during long bone fracture healing. They described microcalluses as occurring in bone showing a range of pathologic conditions (osteoarthrosis, rheumatoid arthritis, subcapital fracture, and degeneration of the articular cartilage); they suggested that the initial lesion was fatiguc fracture related to these conditions. They also suggested that when the microcallus did not appear to be associated with trabecular fracture consolidation had already occurred or the thickening was only a response to abnormally high stress in the trabecular struts. Radin et al. (12) suggested that fracture may reReceived January 23, 1991; accepted September 4, 1991. Address correspondence and reprint requests to Professor John Currey, Department of Biology, University of York. York YO1 SDD, England.
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Benaissa et al. (2) produced some evidence that the nodular type occurs predominantly in areas of compressive loading and suggested that the particular form of the microcallus is determined by a reaction to the rubbing together of the ends of the fatigue-fractured trabecula. Freeman et al. (8) and Watson (17) reported similar relationships between the level of osteoporosis and microcallus incidence in the head of the femur. Watson (17) and Freeman et al. (8) agreed that the presence of sufficient numbers of microfractures could eventually lead to subcapital fracture. Ohtani and Azuma (11) studied the acetabulum, in which microcalluses (assumed by them to be indicative of sites of fracture) were more common in the subchondral bone of the more highly stressed regions than in non-weight-bearing sites. More recently, Fazzalari et al. (7) reassessed the role of microfractures in osteoarthritis of the hip. They disagreed with the conclusion of Radin et al. (12) that microcalluses stiffen cancellous bone and so initiate joint degeneration, because they noted fewer microfractures in osteoarthrotic bone. Instead, Fazzalari et al. (7) proposed that microfractures, by inducing remodeling changes, have a beneficial role in the maintenance of joint structure, the reduction in number of microfractures, and hence of microcalluses in osteoarthrosis, being an indication of a breakdown in the remodeling process of the bone material. Therefore the role, if any, of microcalluses in diseased or aged cancellous bone or in cancellous bone associated with diseased joints is still debatable. The extreme views would be that microcalluses are either agents responsible for onset of degenerative joint disease or are the manifestation of the bone repair process responsible for maintenance of cancellous bone and therefore of the associated joint structure. The literature on this subject barely addresses the mechanical properties of the callus material itself. Clearly, we must know these properties if we are to understand fully the effect of microcalluses on cancellous bone. In this study, we used the results of microhardness testing to infer some of the mechanical properties of the callus material. MATERIALS AND METHODS Specimen Origins Microcalluses were taken from two areas: the proximal tibia of a 45-year-old woman weighing 50
J Orthop Res, Vol. 10, No. 2 , 1992
kg and the distal femur of a 62-year-old man weighing 90 kg. To minimize the risk of pathogens from the material, each specimen was first fixed for a minimum of 20 h in a solution of 4% formaldehyde at room temperature. Before the fixation process and subsequently, between fixation and study, all material was stored frozen at - 20°C. Material Removal and Cleaning Specimens were defatted by a combination of tumbling in excess 2: 1 chloroform/methanol solution and exposure to high-pressure water jet (9). With more fragile specimens, the water pressure was lowered to prevent trabecular damage and loss. This cleaning process was repeated until all intertrabecular material had been removed. Cleaned specimens were refrozen at - 20°C. Callus Embedding We were able to identify callus material in the sectioned material with a binocular microscope. Each recognized callus was assigned to one of the morphologic groups described by Vernon-Roberts and Pirie (16). The temporal relationships between the different types of callus are not clear, although Fazzalari et al. (7) suggested that the fusiform type represents a later stage of the nodular type. There is no hard evidence that this is the correct temporal sequence, however. We found that the callus material is difficult to distinguish from material of normal trabeculae (if indeed it is different) when the structures are of the fusiform, angulated, or arched bridge type. For this reason, we studied only the rounded nodular callus type (which was by far the most frequently recognized type). This is the classic birds’ nest and its ease of identification removed the possibility of incorrectly selecting non-callus material for testing. We removed callus trabeculae from the sections, with associated trabecula attached, using a fine scalpel blade, and embedded it in Metset mounting resin (FT type), which is transparent on curing. This transparency allowed the tiny calluses to be viewed in the embedding resin. Specimens were ground with a series of carborundum papers of increasingly fine grit until the surface of the microcallus just coincided with the plane of the resin surface. We then polished the specimens further with alumina paste while following the erosion of the cal-
MICROHARDNESS OF MICROCALLUS
lus closely with a binocular microscope until we judged that a significant area of the callus had been exposed. We washed and dried the specimens for a short time at room temperature. The embedding resin provided general support for the tissue but did not infiltrate it to any extent. The blood channels were not infiltrated, nor were the osteocyte lacunae. As a result, we consider it most improbable that the resin would have had any effect on the measured hardness. Microhardness Testing Microhardness of microcalluses was determined with a Leitz Wetzlar “Miniload” hardness tester (060-366.002; Ernst Leitz GMBH, Wetzlar, Germany). An indenting load of 50 pond was used with descent time for the diamond of 15 s (1 pond = 1 g weight). Once it reached the lowest position, the diamond was allowed to impress the material for 10 s more before being raised. The average of the two diagonal lengths for each indent was calculated and converted to the Vickers hardness number using standard tables. Typically, after each set of indentations was made, the callus was again polished, exposing a fresh surface, and more microhardness values were determined. The indented surface always had a sufficient exposure of callus material to allow us to make the indents well away from the callusiresin interface. Initially, material was tested wet (allowed to rehydrate fully and then blotted surface-dry before testing). However, the outlines of the indents produced in wet specimens, especially of callus material, were often difficult to make out with reflected light microscopy because water wept back into the impression; therefore, we deemed it preferable to test all material dry. This is not ideal, because previous investigators have demonstrated the greater hardness of dry as opposed to wet bone (1,181. We made measurements to discover the magnitude of this effect (described below); dry bone yielded measurements -20% greater than wet bone. Nevertheless, because we are interested essentially in the relative hardnesses of “normal” trabecular bone and callus material, we consider use of dry bone satisfactory. We must assume that drying affects the hardness of cortical bone and callus in the same way. Five types of site were tested: microcallus material, trabeculae surrounded by callus, trabeculae outside the callus, trabeculae not associated with
239
miqocallus, and nearby cortical bone. Usually, distinguishing callus material from trabecular material lying within the callus mass was easy, because there was a clear interface between the two (as shown in Fig. 1C). Where there was not a clear interface, the two types could usually be distinguished because trabeculae showed a clear lamellar structure, with the lamellae all lying in the same direction. In some areas, we could not distinguish trabecula from callus with certainty. We made no measurements in such areas. Finally, for comparison, we tested a 12-week-old fracture callus from the tibia of a sheep, with cortical bone attached. The material had been dried but not fixed. Effect of Specimen Preparation on Measured Values of Microhardness Preparation of the material used in this study involved five steps: formalin fixation, degreasing in chloroform/methanol, drying, embedding and, finally, grinding and polishing. Any or all of these procedures may have an effect on the measured value of hardness. Although we are primarily concerned with dijj5erences between microcallus and ordinary trabecular bone, it is important to have some idea of the magnitude of the changes that these procedures may have caused. Formalin Fixation
The effect of formalin fixation on the hardness of bone is unclear. Although Weaver (18) suggested that prolonged storage in formalin causes a 20% increase in bone hardness, Amprino (l), quoting Rossle and Lexer, stated that brief fixation in formalin has no effect on hardness. We fixed our bones only briefly. Degreasing with Solvent
Indents produced in degreased bone have sharper outlines and are therefore easier to measure accurately and consistently. The sharpness of the indents presumably results from the absence of fat at the measuring site and from absence of grease on the indenting diamond during serial tests. To our knowledge, no information is available on the effect of this defatting process on the hardness of bone.
J Orthop Res, Vol. 10, No. 2 , 1992
240 A
FIG. 1. Scanning electron micrographs of two polished, etched calluses, showing the effect of serial polishing and etching (A and B), and the interface between callus and trabecular material (C). A: Callus polished and etched in 1N HCI for 5 rnin. Arrow shows the fracture. Bar = 100 prn. B: The same callus as in A repolished and etched for 10 rnin more in 10% hypochlorite solution. The section is now deeper in the callus, and the fracture (arrows) is more apparent. Bar = 100 pm. C: Another callus polished and etched in 1N HCI for 25 min. A fracture is evident crossing the trabecula and ending at the obvious interface with the callus material (arrows). Bar = 100 km.
C
J Orthop Rrs, Vol. 10, N o . 2 , 1992
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MICROHARDNESS OF MICROCALLUS Drying
We observed during previous work on bone that indents produced in dry bone have much clearer outlines and are therefore easier to measure accurately. Bone used in this study had been dried at 40°C after being degreased in chloroform/methanol. Previous investigators assessed the effect of drying on the hardness of bone. Amprino (1) reported that bone dried at 38°C showed an increase in Vickers hardness from 50 to 63 (-25%). The increase in hardness became greater with an increase in drying temperature. Weaver (18) reported that the combined effect of drying bone and embedding in methylmethacrylate was to increase hardness by 3040%; the indents produced were “much clearer and permanent.” To assess the effect of testing dry bone in this study, specimens of cancellous and cortical bone were first tested in their dry state and then after a period of rehydration. Currey (3), although not investigating hardness, showed that several mechanical properties of bone (Young’s modulus, bending strength, energy absorption) were barely affected by drying and subsequent rehydration. Embedding
All our cancellous bone specimens were embedded in methacrylate resin. Evans et al. (6) measured the change in hardness of cortical bone after embedding and found a statistically significant but small increase (4%). Weaver estimated that the combined effect of drying and embedding produced an increase of 3040%, although data from other investigators show that the effect of drying probably accounts for a large proportion of this. Measuring the hardness of cancellous bone without first embedding it is effectively impossible for two reasons. First, one cannot produce a sufficiently flat polished surface of “free-standing” cancellous trabeculae. Second, cancellous struts deform under the weight of the indenting needle and produce extremely irregular and unreliable impressions. Grinding and Polishing
We ground and polished the embedded bone surfaces to produce surfaces of sufficient optical smoothness to allow measurement of the pyramidal indent diagonals. Ramrakhiani et al. (13) used silver
sputtering of polished bone sudace to increase contrast after polishing. They measured a slight increase in hardness due to polishing. (We calculate from their data a mean difference of -6% for indenting loads >50 8.) How these investigators measured the hardness of unpolished surfaces, which would be very rough, is not clear. The effect of polishing was not tested in our study. Scanning Electron Microscopy
We performed scanning electron microscopy on some microcalluses whose microhardness had not been tested. Specimens were polished and subjected either to mineral etching (in 1N hydrochloric acid) or organic etching (in 10% sodium hypochlorite). Etching times varied between specimens but typically lasted 10 min. We used these procedures in an effort to show more clearly the lamellar structure of the bone. After etching, we dried the specimens at 70”C, sputter-coated them with gold, and viewed them in an ISI-100A scanning electron microscope (International Scientific Instruments).
RESULTS Our results were derived from 475 hardness determinations from 23 different sites including seven different microcalluses. The main results are shokn in Tables 1 and 2. We first assessed the size of various potential artefacts (Table l), and then compared microcalluses between each other and with trabeculae (Table 2). Effect of Formalin Fixation We did not test any unfixed human bone. We did, however, examine the effect of formalin fixation on bovine cortical and cancellous bone. We tested 10 polished specimens of cortical and trabecular bone, both wet and dry. We fixed the bone for 24 h in 4% formaldehyde at room temperature, and made 10 measurements, again both wet and dry, in the same area of the specimens. None of the four comparisons (trabecular bone and cortical bone, wet or dry) was significant at the 0.05 level. Two tests showed an insignificant decrease in hardness, and two tests showed an insignificant increase in hardness (Table 1). Effect of Degreasing with Solvent
To assess the effect of chloroform/methanol on bone hardness, we tested samples of bovine cortical
J Orthop Res, Vol. 10, No. 2 , 1992
242
J . BLACKBURN ET AL. TABLE 1. Effect of different treatments on measured hardness Specimen treatment
Trabecula outside callus
Effect of fixation Bovine A W, unfixed 34.5(10)8.3[0.97, NS] A W, fixed 38.2(!0)9.3[+ 111 A D, unfixed 42.9(10)9.8[1.48, NS] A D, fixed 37.9(10)9.8[- 121 B W,unfixed B W, fixed B D, unfixed B D, fixed Effect of solvent C W, unfixed c w , solv C D,unfixed C D, s o h Effect of drying (on unfixed) Bovine A W, unfixed 34.5(10)8.3[2.07, 0.0541 A D, unfixed 42.9(10)9.8[ + 241 B W, unfixed B D, unfixed C W, unfixed C D,unfixed Effect of drying (on fixed or degreased) A W, fixed 38.4(10)9.3[0.15, NS] A D, fixed 37.9(10)4.7[ - 11 B W, fixed B D, fixed c w, solv C D, solv
Cortical
40.9(10)3.9[0.54, NS] 39.6(10)6.2[-31 43.3(10)5.2[1.45, NS] 47.5(10)7.5[+ 101 40.9(10)10.8[1.53, NS] 47.2(10)7.1[+ 151 52.5(10)5.6[0.14, NS] 52.7(10)3.5[0.0]
40.9(10)3.9[1.16, NS] 43.3( 10)5.2[+ 61 40.9(10)10.8[3.08, 0.011 52.5(10)4.6[+28]
39.6(10)6.2[2.55, 0.0211 47.5(10)7.5[+20] 47.2( 10)7.1[2.17, 0.0491 52.7(10)3.5[+ 121 Callus
46-year-old woman D W, fixed D D,fixed E W, fixed E D, fixed
36.4(10)8.0[3.69,
Journol of Orthopedic
Mechanical Properties of Microcallus in Human Cancellous Bone Joanne Blackburn, Richard Hodgskinson, John D. Currey, and Jennifer E. Mason Department of Biology, University of York, York, England
Summary: Until now, the mechanical properties of the microcalluses that form in human cancellous bone have been unexplained. We measured the microhardnesses of microcalluses in cancellous bone, of the trabeculae within the microcalluses, of the trabeculae adjacent to microcalluses, and of trabeculae lacking microcalluses in a human tibia and femur. We observed no important differences between materials at the four different sites. Because the microhardness of bone is very closely related to its stiffness, this finding indicates that microcalluses are likely to stiffen the trabeculae in which they are formed, e v e n though they may surround unhealed fractures of the cancellous trabeculae. Key Words: Cancellous bone-Hardness-Microcallus-Stiffness.
sult as a response to repeated impulsive loading of cancellous bone, the microcallus being a remodeling response. In nonosteoporotic bone, the stiffening of the cancellous bone structure associated with accumulation of callus material would lead to a loss of protection of the overlying cartilage through a reduction of the shock-absorbing quality of the subchondral bone, the result of this loss of protection being osteoarthrotic degeneration of the cartilage. They further suggested that the same cartilage degeneration would not occur in osteoporotic bone because any increase in bone mass due to formation of microcalluses would not increase the stiffness of the already depleted bone structure sufficiently. Later, Vernon-Roberts and Pirie (16) concluded that microcalluses could either be buttresses or be associated with trabeculae that had already fractured. They described four morphologic types of microcallus: rounded nodular, fusiform, angulated, and arched bridge. They showed that in the lumbar spine microcallus prevalence increased with age and concluded that the number of calluses was indicative of the degree of osteoporosis. Sections through callus material nearly always revealed discontinuities of the underlying trabeculae, tending to confirm that microcalluses formed in response to fracture (16).
Nodular accretions (‘ ‘microcalluses”) in cancellous bone tissue have been described by several groups of investigators in the past 20 years. Darracott and Vernon-Roberts (5) described them as “birds’ nests” and speculated on their role as buttresses. They showed the material of the birds’ nests to be woven bone. Todd et al. (15) observed that microcalluses sometimes surrounded a fracture of the trabecula and postulated that microcallus is analogous to the callus formed during long bone fracture healing. They described microcalluses as occurring in bone showing a range of pathologic conditions (osteoarthrosis, rheumatoid arthritis, subcapital fracture, and degeneration of the articular cartilage); they suggested that the initial lesion was fatiguc fracture related to these conditions. They also suggested that when the microcallus did not appear to be associated with trabecular fracture consolidation had already occurred or the thickening was only a response to abnormally high stress in the trabecular struts. Radin et al. (12) suggested that fracture may reReceived January 23, 1991; accepted September 4, 1991. Address correspondence and reprint requests to Professor John Currey, Department of Biology, University of York. York YO1 SDD, England.
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Benaissa et al. (2) produced some evidence that the nodular type occurs predominantly in areas of compressive loading and suggested that the particular form of the microcallus is determined by a reaction to the rubbing together of the ends of the fatigue-fractured trabecula. Freeman et al. (8) and Watson (17) reported similar relationships between the level of osteoporosis and microcallus incidence in the head of the femur. Watson (17) and Freeman et al. (8) agreed that the presence of sufficient numbers of microfractures could eventually lead to subcapital fracture. Ohtani and Azuma (11) studied the acetabulum, in which microcalluses (assumed by them to be indicative of sites of fracture) were more common in the subchondral bone of the more highly stressed regions than in non-weight-bearing sites. More recently, Fazzalari et al. (7) reassessed the role of microfractures in osteoarthritis of the hip. They disagreed with the conclusion of Radin et al. (12) that microcalluses stiffen cancellous bone and so initiate joint degeneration, because they noted fewer microfractures in osteoarthrotic bone. Instead, Fazzalari et al. (7) proposed that microfractures, by inducing remodeling changes, have a beneficial role in the maintenance of joint structure, the reduction in number of microfractures, and hence of microcalluses in osteoarthrosis, being an indication of a breakdown in the remodeling process of the bone material. Therefore the role, if any, of microcalluses in diseased or aged cancellous bone or in cancellous bone associated with diseased joints is still debatable. The extreme views would be that microcalluses are either agents responsible for onset of degenerative joint disease or are the manifestation of the bone repair process responsible for maintenance of cancellous bone and therefore of the associated joint structure. The literature on this subject barely addresses the mechanical properties of the callus material itself. Clearly, we must know these properties if we are to understand fully the effect of microcalluses on cancellous bone. In this study, we used the results of microhardness testing to infer some of the mechanical properties of the callus material. MATERIALS AND METHODS Specimen Origins Microcalluses were taken from two areas: the proximal tibia of a 45-year-old woman weighing 50
J Orthop Res, Vol. 10, No. 2 , 1992
kg and the distal femur of a 62-year-old man weighing 90 kg. To minimize the risk of pathogens from the material, each specimen was first fixed for a minimum of 20 h in a solution of 4% formaldehyde at room temperature. Before the fixation process and subsequently, between fixation and study, all material was stored frozen at - 20°C. Material Removal and Cleaning Specimens were defatted by a combination of tumbling in excess 2: 1 chloroform/methanol solution and exposure to high-pressure water jet (9). With more fragile specimens, the water pressure was lowered to prevent trabecular damage and loss. This cleaning process was repeated until all intertrabecular material had been removed. Cleaned specimens were refrozen at - 20°C. Callus Embedding We were able to identify callus material in the sectioned material with a binocular microscope. Each recognized callus was assigned to one of the morphologic groups described by Vernon-Roberts and Pirie (16). The temporal relationships between the different types of callus are not clear, although Fazzalari et al. (7) suggested that the fusiform type represents a later stage of the nodular type. There is no hard evidence that this is the correct temporal sequence, however. We found that the callus material is difficult to distinguish from material of normal trabeculae (if indeed it is different) when the structures are of the fusiform, angulated, or arched bridge type. For this reason, we studied only the rounded nodular callus type (which was by far the most frequently recognized type). This is the classic birds’ nest and its ease of identification removed the possibility of incorrectly selecting non-callus material for testing. We removed callus trabeculae from the sections, with associated trabecula attached, using a fine scalpel blade, and embedded it in Metset mounting resin (FT type), which is transparent on curing. This transparency allowed the tiny calluses to be viewed in the embedding resin. Specimens were ground with a series of carborundum papers of increasingly fine grit until the surface of the microcallus just coincided with the plane of the resin surface. We then polished the specimens further with alumina paste while following the erosion of the cal-
MICROHARDNESS OF MICROCALLUS
lus closely with a binocular microscope until we judged that a significant area of the callus had been exposed. We washed and dried the specimens for a short time at room temperature. The embedding resin provided general support for the tissue but did not infiltrate it to any extent. The blood channels were not infiltrated, nor were the osteocyte lacunae. As a result, we consider it most improbable that the resin would have had any effect on the measured hardness. Microhardness Testing Microhardness of microcalluses was determined with a Leitz Wetzlar “Miniload” hardness tester (060-366.002; Ernst Leitz GMBH, Wetzlar, Germany). An indenting load of 50 pond was used with descent time for the diamond of 15 s (1 pond = 1 g weight). Once it reached the lowest position, the diamond was allowed to impress the material for 10 s more before being raised. The average of the two diagonal lengths for each indent was calculated and converted to the Vickers hardness number using standard tables. Typically, after each set of indentations was made, the callus was again polished, exposing a fresh surface, and more microhardness values were determined. The indented surface always had a sufficient exposure of callus material to allow us to make the indents well away from the callusiresin interface. Initially, material was tested wet (allowed to rehydrate fully and then blotted surface-dry before testing). However, the outlines of the indents produced in wet specimens, especially of callus material, were often difficult to make out with reflected light microscopy because water wept back into the impression; therefore, we deemed it preferable to test all material dry. This is not ideal, because previous investigators have demonstrated the greater hardness of dry as opposed to wet bone (1,181. We made measurements to discover the magnitude of this effect (described below); dry bone yielded measurements -20% greater than wet bone. Nevertheless, because we are interested essentially in the relative hardnesses of “normal” trabecular bone and callus material, we consider use of dry bone satisfactory. We must assume that drying affects the hardness of cortical bone and callus in the same way. Five types of site were tested: microcallus material, trabeculae surrounded by callus, trabeculae outside the callus, trabeculae not associated with
239
miqocallus, and nearby cortical bone. Usually, distinguishing callus material from trabecular material lying within the callus mass was easy, because there was a clear interface between the two (as shown in Fig. 1C). Where there was not a clear interface, the two types could usually be distinguished because trabeculae showed a clear lamellar structure, with the lamellae all lying in the same direction. In some areas, we could not distinguish trabecula from callus with certainty. We made no measurements in such areas. Finally, for comparison, we tested a 12-week-old fracture callus from the tibia of a sheep, with cortical bone attached. The material had been dried but not fixed. Effect of Specimen Preparation on Measured Values of Microhardness Preparation of the material used in this study involved five steps: formalin fixation, degreasing in chloroform/methanol, drying, embedding and, finally, grinding and polishing. Any or all of these procedures may have an effect on the measured value of hardness. Although we are primarily concerned with dijj5erences between microcallus and ordinary trabecular bone, it is important to have some idea of the magnitude of the changes that these procedures may have caused. Formalin Fixation
The effect of formalin fixation on the hardness of bone is unclear. Although Weaver (18) suggested that prolonged storage in formalin causes a 20% increase in bone hardness, Amprino (l), quoting Rossle and Lexer, stated that brief fixation in formalin has no effect on hardness. We fixed our bones only briefly. Degreasing with Solvent
Indents produced in degreased bone have sharper outlines and are therefore easier to measure accurately and consistently. The sharpness of the indents presumably results from the absence of fat at the measuring site and from absence of grease on the indenting diamond during serial tests. To our knowledge, no information is available on the effect of this defatting process on the hardness of bone.
J Orthop Res, Vol. 10, No. 2 , 1992
240 A
FIG. 1. Scanning electron micrographs of two polished, etched calluses, showing the effect of serial polishing and etching (A and B), and the interface between callus and trabecular material (C). A: Callus polished and etched in 1N HCI for 5 rnin. Arrow shows the fracture. Bar = 100 prn. B: The same callus as in A repolished and etched for 10 rnin more in 10% hypochlorite solution. The section is now deeper in the callus, and the fracture (arrows) is more apparent. Bar = 100 pm. C: Another callus polished and etched in 1N HCI for 25 min. A fracture is evident crossing the trabecula and ending at the obvious interface with the callus material (arrows). Bar = 100 km.
C
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MICROHARDNESS OF MICROCALLUS Drying
We observed during previous work on bone that indents produced in dry bone have much clearer outlines and are therefore easier to measure accurately. Bone used in this study had been dried at 40°C after being degreased in chloroform/methanol. Previous investigators assessed the effect of drying on the hardness of bone. Amprino (1) reported that bone dried at 38°C showed an increase in Vickers hardness from 50 to 63 (-25%). The increase in hardness became greater with an increase in drying temperature. Weaver (18) reported that the combined effect of drying bone and embedding in methylmethacrylate was to increase hardness by 3040%; the indents produced were “much clearer and permanent.” To assess the effect of testing dry bone in this study, specimens of cancellous and cortical bone were first tested in their dry state and then after a period of rehydration. Currey (3), although not investigating hardness, showed that several mechanical properties of bone (Young’s modulus, bending strength, energy absorption) were barely affected by drying and subsequent rehydration. Embedding
All our cancellous bone specimens were embedded in methacrylate resin. Evans et al. (6) measured the change in hardness of cortical bone after embedding and found a statistically significant but small increase (4%). Weaver estimated that the combined effect of drying and embedding produced an increase of 3040%, although data from other investigators show that the effect of drying probably accounts for a large proportion of this. Measuring the hardness of cancellous bone without first embedding it is effectively impossible for two reasons. First, one cannot produce a sufficiently flat polished surface of “free-standing” cancellous trabeculae. Second, cancellous struts deform under the weight of the indenting needle and produce extremely irregular and unreliable impressions. Grinding and Polishing
We ground and polished the embedded bone surfaces to produce surfaces of sufficient optical smoothness to allow measurement of the pyramidal indent diagonals. Ramrakhiani et al. (13) used silver
sputtering of polished bone sudace to increase contrast after polishing. They measured a slight increase in hardness due to polishing. (We calculate from their data a mean difference of -6% for indenting loads >50 8.) How these investigators measured the hardness of unpolished surfaces, which would be very rough, is not clear. The effect of polishing was not tested in our study. Scanning Electron Microscopy
We performed scanning electron microscopy on some microcalluses whose microhardness had not been tested. Specimens were polished and subjected either to mineral etching (in 1N hydrochloric acid) or organic etching (in 10% sodium hypochlorite). Etching times varied between specimens but typically lasted 10 min. We used these procedures in an effort to show more clearly the lamellar structure of the bone. After etching, we dried the specimens at 70”C, sputter-coated them with gold, and viewed them in an ISI-100A scanning electron microscope (International Scientific Instruments).
RESULTS Our results were derived from 475 hardness determinations from 23 different sites including seven different microcalluses. The main results are shokn in Tables 1 and 2. We first assessed the size of various potential artefacts (Table l), and then compared microcalluses between each other and with trabeculae (Table 2). Effect of Formalin Fixation We did not test any unfixed human bone. We did, however, examine the effect of formalin fixation on bovine cortical and cancellous bone. We tested 10 polished specimens of cortical and trabecular bone, both wet and dry. We fixed the bone for 24 h in 4% formaldehyde at room temperature, and made 10 measurements, again both wet and dry, in the same area of the specimens. None of the four comparisons (trabecular bone and cortical bone, wet or dry) was significant at the 0.05 level. Two tests showed an insignificant decrease in hardness, and two tests showed an insignificant increase in hardness (Table 1). Effect of Degreasing with Solvent
To assess the effect of chloroform/methanol on bone hardness, we tested samples of bovine cortical
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J . BLACKBURN ET AL. TABLE 1. Effect of different treatments on measured hardness Specimen treatment
Trabecula outside callus
Effect of fixation Bovine A W, unfixed 34.5(10)8.3[0.97, NS] A W, fixed 38.2(!0)9.3[+ 111 A D, unfixed 42.9(10)9.8[1.48, NS] A D, fixed 37.9(10)9.8[- 121 B W,unfixed B W, fixed B D, unfixed B D, fixed Effect of solvent C W, unfixed c w , solv C D,unfixed C D, s o h Effect of drying (on unfixed) Bovine A W, unfixed 34.5(10)8.3[2.07, 0.0541 A D, unfixed 42.9(10)9.8[ + 241 B W, unfixed B D, unfixed C W, unfixed C D,unfixed Effect of drying (on fixed or degreased) A W, fixed 38.4(10)9.3[0.15, NS] A D, fixed 37.9(10)4.7[ - 11 B W, fixed B D, fixed c w, solv C D, solv
Cortical
40.9(10)3.9[0.54, NS] 39.6(10)6.2[-31 43.3(10)5.2[1.45, NS] 47.5(10)7.5[+ 101 40.9(10)10.8[1.53, NS] 47.2(10)7.1[+ 151 52.5(10)5.6[0.14, NS] 52.7(10)3.5[0.0]
40.9(10)3.9[1.16, NS] 43.3( 10)5.2[+ 61 40.9(10)10.8[3.08, 0.011 52.5(10)4.6[+28]
39.6(10)6.2[2.55, 0.0211 47.5(10)7.5[+20] 47.2( 10)7.1[2.17, 0.0491 52.7(10)3.5[+ 121 Callus
46-year-old woman D W, fixed D D,fixed E W, fixed E D, fixed
36.4(10)8.0[3.69,
