Rapid determination of cancellous bone mineral loss in ovariectomized rats by a subtraction technique.код для вставкиСкачать
THE ANATOMICAL RECORD 230:169-174 (1991) Rapid Determination of Cancellous Bone Mineral Loss in Ovariectomized Rats by a Subtraction Technique ENASS ESKANDAR, DONALD B. KIMMEL, AND THOMAS J. WRONSKI Center for Hard Tissue Research, Department of Medicine, Creighton University, Omaha, Nebraska (E.E., D.B.K.); Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32091 (T.J.W.) ABSTRACT We present a rapid technique for determining cancellous bone mineral changes in small experimental animals. We used the distal centimeter of the right femur from ovariectomized (OX) (N = 30) and shamovariectomized (ShOX) (N = 28) rats, aged 90 days a t surgery and killed a t times from 125-540 days postsurgery. We used dual photon absorptiometry to scan the segment three times: intact, after parasagittal splitting, and after removing all cancellous bone. We equated the difference between the second and third scans to cancellous bone mineral content (Cn.BMC). To validate this, we compared i t with histomorphometrically determined bone volume (BVITV) of the proximal tibia1 metaphysis of the same rat. Parasagittally splitting the segment removed no detectable mineral. OX rats had 40% less Cn.BMC than ShOX rats. However, OX rats had 80% lower BVITV than ShOX rats. The subtraction technique not only makes a rapid, reasonable assessment of cancellous bone loss in OX rats but permits a smaller sample size than histomorphometry. The histomorphometric technique finds a greater difference between OX and ShOX rats because it examines a region where cancellous bone loss is more marked than does the scanning technique. The current technique measures bone of not only the central secondary spongiosa but also the juxtacortical region and the primary spongiosa, where OX-related differences are less prominent. The principles of this subtraction technique proved workable. However, for the future, we recommend a two-scan technique using a dual energy X-ray scanner. It is likely to take only 20-30 min per specimen to assess cancellous bone mineral. A frequent clinical symptom of postmenopausal osteoporosis is vertebral crush fractures, often associated with low cancellous bone strength (Cummings et al., 1985). Part (Weaver and Chalmers, 1966; Mosekilde and Mosekilde, 1988) but not all (Kleerekoper et al., 1985) of cancellous bone’s strength is due to its mineral. Studying cortical and cancellous bone mass separately in vivo in humans is possible by several quantitative computed tomography (CT) techniques (Jones et al., 1987; Cann, 1988; Liliequist et al., 1979; Ruegsegger et al., 1981). Cody and Flynn (1989) even subdivide spinal cancellous bone measured in vivo into regions containing 1.6 cm’. Separating cancellous and cortical bone is necessary because their rates of loss may be different (Meier et al., 1984; Riggs et al., 1986). Investigators using experimental animals to test regimens to increase cancellous bone mass could benefit from a quick, accurate technique. The above-described CT techniques are often unavailable or lacking sufficient resolution for measuring small animal bones. However, investigators using small animals have the advantage of harvesting and dissecting apart bones for analysis. They measure bone quantity by weighing and ashing (Yamazaki and Yamaguchi, 1989; Nottestad et al., 1987; Gong et al., 19641, neutron activation (Parks 0 1991 WILEY-LISS, INC. e t al., 1986), photon absorptiometry (Kiebzak et al., 1988; Kimmel and Wronski, 1990), or quantitative histology (Kimmel and Jee, 1983). Physically separating and quantitating cancellous and cortical bone remains troublesome, but necessary (Nottestad et al., 1987; Gunness-Hey and Hock, 19841, if the analysis is to approach the level of in vivo human applications (Jones et al., 1987; Cann, 1988; Liliequist e t al., 1979; Ruegsegger et al., 1981). The purpose of this work is to report and validate a quick, reliable technique for measuring cancellous bone mass in small experimental animals. MATERIALS AND METHODS Experimental Design We obtained 58 female Sprague-Dawley rats 85 days of age weighing roughly 210 g (Charles River Co., Cambridge, MA). They were caged individually and fed ad libitum in a room with a 12 h r on112 h r off light cycle. We randomized them by weight into experimental and control groups. At age 100 days, the rats were Received September 14, 1990; accepted November 5, 1990. 170 E. ESKANDAR ET AL Scanning and Analysis Procedure ovariectomized (OX) (N = 30) or sham-ovariectomized (ShOX) (N = 28) under ketaset/xylazine anesthesia by a dorsal approach as described by Waynforth (1980). We sacrificed them a t 125,180,270,360, and 540 days postovariectomy. Bone loss in the proximal tibia1 metaphysis after estrogen depletion in rats reaches a steady state by 100 days (Wronski e t al., 1989). At autopsy, we removed the right tibias and femurs. We placed the femurs immediately into 70% EtOH. We placed the tibias in formalin for 48 h r and then moved them into 70% EtOH. We blind coded all specimens before any manipulation. We used a Norland 2600 Dichromatic Bone Densitometer (Norland Corp., Ft Atkinson, WI; Software Version 2.23W), previously validated a s accurate for small animal bones (Kimmel and Wronski, 1990). We placed the bone specimens in a n 18.5 cm2 weighing dish on a 2.5-cm-thick piece of Plexiglass to provide the appropriate soft tissue equivalent. We used a scan width of 1.9 cm and a line and point “resolution” of 0.4 mm a t 1 m d s e c scan speed. Each scan lasted 10-12 min, including set-up. We analyzed the raw data averaging over 13 “points.” Strategy for Measuring Cancellous Bone Quantity (Cn.BMC) Cancellous Bone Quantity Measured by Histomorphometry (BV/TV) We used a subtraction technique to find cancellous bone quantity. First, we removed the distal 1cm of the femur, a region containing both cancellous and cortical bone and measured its mineral content (BMC). Next, we split it parasagitally and remeasured its BMC. Finally, we removed the cancellous bone tissue from both halves and remeasured BMC of the remaining piece. We determined mineral lost to opening the bone as the difference between BMCs of the first and second scans. We determined cancellous mineral content (Cn.BMC) as the difference between BMCs of the second and third scans. To validate Cn.BMC as a bone mass index, we compared Cn.BMC of the distal femur to BV/TV of the proximal tibia in each animal, as reported by Wronski et al. (1989). We dehydrated the proximal 1 cm of the tibia in graded alcohols and embedded i t in modified methyl methacrylate (Wronski et al., 1989). We cut 4 pm frontal sections with a Reichert-Jung 1150 microtome, from a region midway through the bone in a n anteroposterior direction. We stained them with the Goldner (1938) method. Finally, we measured cancellous bone volume (BV/TV) in a region located 1mm distal to the growth cartilage-metaphyseal junction, which measured 2 mm transversely by 3 mm proximodistally (Wronski et al., 1989). Specific Steps for Mineral Determination 1. First, we cut the femur transversely 1cm from its distal end with a Hyperflex diamond disk (Brassler USA, 940-220, Savannah, GA) in a straight dental handpiece. The diamond disk was 22 mm in diameter and 0.15 mm thick. The time taken to slice each bone was about 1 min. 2. We scanned the piece by dual photon absorptiometry (DPA) (see Scanning and Analysis Procedure) (scan 1). 3. Next, we cut the 1 cm piece parasagitally into two halves with the diamond disk. We cleaned the halves of bone“crumbs” in a n ultrasonicator for 30 sec and reapposed the pieces in their anatomically correct relationship. The time taken to slice and reappose each bone was about 1 min. 4. We scanned the reapposed pieces (scan 2). 5. Next, we removed the cancellous bone from each half with a slowly rotating (100-200RPM) round burr (Brassler USA, No. 4, 014, Savannah, GA) and a discoid-cleoid dental carver (G.C. International, 3-COOO5, Scottsdale, AZ). We worked under a 2 x magnifier, removing all cancellous bone distal to the growth cartilage-metaphysealjunction but leaving the cartilage intact. We left a smooth endocortical surface. We again cleaned both halves in the ultrasonicator and reapposed the pieces as in step 3 (scan 3). The time taken to remove the cancellous bone and clean the pieces was 5-10 min. We reapposed the two halves and scanned them a third time. Much of this step was done during other scans. 6. Finally, to find Cn.BMC, we subtracted BMC of scan 3 from BMC of scan 2. We assumed t h a t bone marrow contained negligible mineral. Statistical Methods We applied a paired t test when comparing scans 1 and 2 or scans 2 and 3 of the same femoral specimen. We applied a t test of means when comparing ShOX and OX rats (Sokal and Rohlf, 1969). To find the minimum detectable change in Cn.BMC and BV/TV, we applied this formula from Snedecor and Cochran (1969): where N is the sample size, (Z, - ZP)’ is a multiplier reflecting the probabilities of Type I and Type I1 errors (a = .05 and @ = 0.2), with a value of 7.9, aD2 is the stochastically determined variance of the differences, and s is the absolute minimum detectable difference. All values are expressed as mean SD. * RESULTS We estimate the time for this three-scan analysis at 40-50 min per bone. Mineral Lost to Parasagittal Cut (Difference of Scans 1 and 2) For OX rats, BMC changed from 115.5 -t 9.6 mg to 113.8 t 9.4 mg between the first two scans (Fig. 1).For ShOX rats, BMC changed from 130.7 -t 16.1 mg to 129.5 2 17.7 mg between the first two scans (Fig. 1). These changes within the OX and ShOX groups of rats were not significant. Cn.BMC (BMC of Cancellous Bone) (Difference of scans 2 and 3) In OX rats, BMC decreased from 113.8 -t 9.4 mg in the second scan to 99.7 2 11.2 mg in the third scan (Fig. 1).In ShOX rats, BMC decreased from 129.5 2 17.7 mg in the second scan to 106.2 -t 16.2 mg in the 171 RAPID CANCELLOUS BONE DETERMINATION Distal Femoral Cancellous Bone Endpoints BMC Cn.BMC L BV/TV (fSEM) (Mg) (*SEMI 35 - 30 ‘“1 n T ap 2 5 re1 125 b n 20 -5” 15 0 5 10 5 ShOX m1 1 2 1 1 3 Fig. 1. Distal femoral bone mineral content (BMC) in all scans. There is no difference between the first two scans of the distal 1 cm of the femur in either group. However, BMC was significantly less in scan 3 than in scan 2 in both OX and ShOX rats (bothP < ,001). Since the difference of scans 1 and 2 represents the mineral lost during a parasagittal slice of the bone with a thin diamond disc, we conclude that scan 1 can be omitted. OX, ovariectomized rats; ShOX, shamovariectomized rats; e, P < .001. third scan (Fig. 1). These changes within the OX and ShOX groups of rats were significant (P < .001). Comparison of Cancellous Bone Quantity in ShOX and OX Rats By subtraction technique The difference in BMC between the second and third scans is distal femoral cancellous bone mineral (Cn.BMC). Cn.BMC of OX rats was 14.1 2 6.1 mg, 39% less than the ShOX rats’ 23.2 2 5.8 mg ( P < .001) (Fig. 2). By histomorphometry Cancellous bone volume (BVITV) of the proximal tibia in OX rats was 4.8% 2 4.2%, 80% less ( P < .001) than the ShOX rats’ 24.2% 2 10.3% (Fig. 2). The difference between Cn.BMC (mg) and BV/TV (%) in the ShOX rats was -1.0 2 9.4, whereas the difference between Cn.BMC and BV/TV in the OX rats was greater at 9.5 2 6.2 ( P < .001). Cn.BMC detected a significantly smaller difference in bone mass than did BVITV. By percent of distal femoral mineral in cancellous bone In OX rats, 12.5% 2 5.6% of the mineral in the distal centimeter of the femur was in cancellous bone. In ShOX rats, 18.0% -t 4.1% of that mineral was in cancellous bone. The difference between OX and ShOX rats was 31% (P < .001). Minimum Detectable Difference To find reliably a 20% change in Cn.BMC of intact rats, that is, from 23.2 to 27.8 mg, the minimum group size would be 12. On the other hand, to find a 20% change in BVITV of intact rats, from 24.2% to 29.0%, 0 Cn.BMC BV/TV ShOX OX Fig. 2. Cancellous bone mass endpoints in distal femur of ovariectomized and sham-ovariectomized rats. Cn.BMC is the difference of scans 2 and 3 for the OX and ShOX groups of rats. There is less cancellous bone in OX rats than ShOX rats whether measured by Cn.BMC or BVITV. However, the histomorphometric measurement shows a much greater difference than the subtraction technique. It probably measures a n area of the metaphysis where cancellous bone loss is most marked. Cn.BMC, cancellous bone mineral content of distal femur by subtraction technique; BV/TV, bone volume of proximal tibia1 metaphysis by histomorphometry; ShOX, sham-ovariectomized rats; OX, ovariectomized rats; e, P < .001. the minimum group size would be 36. We have made additional calculations in Table 1. DISCUSSION We present a quick, accurate technique for measuring cancellous bone mineral in bones of small experimental animals. We used i t to find differences in cancellous bone mass between groups of sham-operated and ovariectomized rats and validated it by comparing its results to histomorphometric studies of the proximal tibia of the same animal. There was no significant difference between scans 1 and 2 for either group. The only difference between the specimen in scans 1and 2 was that a parasagittal slice was made through its central part. We conclude that making this slice with a thin diamond disc removes a n insignificant amount of mineral. It is suitable to begin this subtraction technique without a scan of the intact piece. Advantages This technique is faster than histomorphometry. Under the best of circumstances, quantitative histology, with its embedding and sectioning, would take 4-6 weeks for a 40 r a t experiment. With this technique, the time to find Cn.BMC is less than 1 h r per specimen. Even allowing time for errors, a 40 rat study can be completed within 1week. One might envision quickly screening for cancellous BMC changes with this technique, saving the more comprehensive histomorphometric studies of cell endpoints for experiments in which a certain difference exists. 172 E. ESKANDAR ET AL. TABLE 1. Sample size to find changes in cancellous bone quantity by subtraction and histomorphometric techniques' Variable Cn.BMC(ShOX) Cn.BMC(OX) BV/TV(ShOX) BVITV(0X) Baseline Mean SD 23.2 5.8 14.1 6.1 24.2 10.3 4.8 4.2 210% 49 148 143 605 Sample size to find change of t20% *30% 250% 12 6 2 37 16 6 36 16 6 151 67 24 'Abbreviations: Cn.BMC, cancellous bone mineral content of distal femur; BV/TV, bone volume of proximal tibial metaphysis; ShOX, sham-ovariectomized rats; OX, ovariectomized rats. It seems straightforward. All techniques for determining cancellous bone mineral in small animals must rely on anatomic dissection and separation of cancellous and cortical bone (Nottestad et al., 1987; Gong e t al., 1964; Parks et al., 1986; Gunness-Hey and Hock, 1984). However, most require saving the marrow and small pieces of removed cancellous bone so that they can be evaluated. Saving those small pieces can be difficult, particularly when few exist. The nondestructive nature of absorptiometry makes possible this subtraction technique, which does not require saving the small pieces. The mineral assessment technology seems widely available. Past studies of mineral content in cortical and cancellous bone have relied on ashing or neutron activation for mineral analysis. Ashing would not work for the subtraction technique, and neutron activation is available only at several locations worldwide. Photon absorptiometry is now widely available and well validated for small animal bones (Kiebzak et al., 1988; Kimmel and Wronski, 1990). Dual energy X-ray absorptiometry, with its fourfold reduction in scan time and improved precision over DPA, seems likely to be even better (Stein et al., 1988). Shortcomings Less difference between cancellous bone of OX and ShOX rats We detected a 39% difference in Cn.BMC of OX and ShOX rats but a n 80% difference in BVITV of OX and ShOX rats. The change found with Cn.BMC was significantly less than that found with BVITV (P< .001). The histomorphometric change in cancellous bone mass of the proximal tibia for OX rats was greater than that seen with our technique. Four possible reasons for these discrepancies exist. 1. The technique of removing cancellous bone probably removed some cortical bone at the endocortical surface. We used 2 x magnification, which we believe is too low to ensure that the burr does not occasionally gouge the endocortical surface. In the future, we recommend doing all work with the round burr and discoid-cleoid, under a dissecting microscope at 5-10 x . 2. The technique of removing cancellous bone removed cancellous bone from the primary spongiosa, but the histomorphometric method did not study the primary spongiosa. If the difference of primary spongiosa mineral content between OX and ShOX rats is less than in the secondary spongiosa, the subtraction technique will reveal a smaller difference. 3. Other work suggests that cancellous bone loss in estrogen deplete rats is most marked in the area of the secondary spongiosa most distant from the endocortical surface (Okumura et al., 1987). BVITV samples only the central section; Cn.BMC measures the whole metaphysis (and probably part of the inner one-third of the cortex). Since the difference of juxtacortical cancellous mineral content between OX and ShOX rats is less than in the central secondary spongiosa, the subtraction technique would be expected to reveal a smaller difference. 4. We measured Cn.BMC of the distal femur, but BVITV of the proximal tibia. Cancellous bone behavior in the distal femur and proximal tibia may differ. If proximal tibial cancellous bone is more sensitive to estrogen loss than that of the distal femur, then our results are as expected. The subtraction technique seems more sensitive than histomorphometry. It can detect a 30% change in Cn.BMC of OX rats with a group size of 16. It can even detect a 20% change in Cn.BMC of ShOX rats with a group size of 12. Using a histomorphometric technique in the same animals would require group sizes of 67 and 36, respectively, to find the same relative changes (Table 1).The reason for the lower sample size is the decrease in variance with Cn.BMC compared with BVI TV. No assessment of cell function This quick technique, which does not permit studies of cell function, is no replacement for histomorphometry and biochemistry. However, since for now bone mass improvement is the critical endpoint in animal studies evaluating osteoporosis interventions, i t may enable a rapid decision about the need for studies of bone cell activity. Poor site specificity The subtraction technique is not adequate to separate adjacent macroanatomic sites within bone. The histomorphometric examination was easily limited to the central secondary spongiosa, a n area interesting for its bone behavior similar to the adult human skeleton (Wronski et al., 1989). In contrast, the subtraction technique measured mineral in both the primary and secondary spongiosa. We believe it also lacked the ability to delineate the endocortical surface properly. Furthermore, the subtraction technique would probably be imprecise for assaying a bone formation response at the periosteum. RAPID CANCELLOUS BONE DETERMINATION Ability to detect poorly mineralized bone With the subtraction technique, detecting woven bone tissue is likely to be problematic because its mineral content is low. This could be critical because several interesting agents, including prostaglandin E, (Jee et al., 1985), fluoride (Eriksen et al., 1985), aluminum (Quarles et al., 19891, and newer bisphosphonates (Fujimoto et al., 19901, and interventions (Rubin and Lanyon, 1985; Burr et al., 1989) have their earliest effects by causing accumulation of poorly mineralized lamellar or frankly woven bone. Physiologic alterations of endocortical surface possibly affecting identification of cancellous bone Duncan et al. (1973) showed that corticosteroids cause endosteal tunneling into the inner one-third of the cortex. This could create a weak bone layer at the endocortical surface that might be penetrated easily with the burr. This would lead to spurious identification of cancellous bone. Some studies suggest that the remodeling activity and loss of bone a t the endocortical surface is the most critical site affected by estrogen depletion (Parfitt, 1989). SUMMARY We present a rapid technique that detects differences in cancellous bone mineral in a relevant small animal model of egtrogen depletion bone loss. Because negligible mineral is lost during opening the bone, we believe that one scan can be eliminated. We now recommend using dual energy X-ray scanning (Stein e t al., 1988) instead of DPA. The following is a n idealized two-scan technique for finding Cn.BMC. Step 1: With a band saw and premeasured 1cm scale, cut off the distal 1 cm of the femur. There is little to choose between the current technique and a similar one t h a t removes the epiphysis a t this point (GunnessHey and Hock, 1984). However, removing the epiphysis could prove more difficult and less reproducible in older rats, which more resemble the aged human skeleton, than in the younger rats usually used with that method (Gunness-Hey and Hock, 1984). With a thin diamond disc, split the specimen parasagitally into medial and lateral halves. 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