Bone Vol. 17, No. 4 October 1995:431433 ELSEVIER
Alterations to the En Bloc Basic Fuchsin Staining Protocol for the Demonstration of Microdamage Produced In Vivo D. B. B U R R and M. H O O S E R Departments of Anatomy and Orthopedic Surgery, Biomechanics and Biomaterials Research Center, Indiana University School of Medicine, Indianapolis, IN, USA
Materials and Methods
En bloc staining with basic fuchsin has become the method of choice to demonstrate bone microdamage produced in vivo. Several alterations have recently been made to the protocol. This technical note presents the new protocols, which include staining through a graded series of alcohols under vacuum, and eliminating the original evaporation stage in the staining protocol. Reasons for variations in staining quality can be related to inadequate dehydration, failure to stain under vacuum, the source of the basic fuchsin, or the solubility of basic fuchsin in water. The most common reasons for over- or understaining are the time left in stain, and the density of the bone. Limitations to the technique include the fact that probably not all cracks are stained by the technique, and the technique is not useful for studies that involve bone with highly variable densities in a single section. (Bone 17:431433; 1995)
Specimens to be bulk stained should be preserved for 48 h or longer in 70% ethanol (ETOH) or 10% cold neutral-buffered formalin. For cortical bone, it may be necessary to cut the ends of the bone off, leaving a length of 1-5 cm, to allow stain to penetrate. Specimens of either trabecular or cortical bone should be rinsed under running water to remove fatty marrow. The major change to the protocol is that specimens are stained in 1% basic fuchsin in a graded series of alcohols under vacuum, but thereafter they do not need to be left in solution until the alcohol has evaporated. The use of graded alcohols provides for adequate dehydration of the specimens and provides a more even stain. Staining under vacuum allows better penetration of the stain, and eliminates the need for the evaporation procedure. Basic fuchsin from J.T. Baker (Phillipsburg, NJ, Cat. No. B660-03) is made up in a series of 1% stock solutions in 70%, 80%, 90%, or 100% ETOH. Basic fuchsin does not easily go into solution and should be stirred for several hours or overnight. Following preservation in 70% ETOH, specimens should be placed in the following solutions under a 20-psi vacuum:
Key Words: Microdamage; Bone; Histomorphometry.
1. 2. 3. 4. 5. 6.
It has been proposed that the initiation and accumulation of bone microdamage contributes to bone fragility in osteoporotic women, independent of the loss of the bone mass, increasing the risk of fracture. 4"8'H 13,18 En bloc staining with basic fuchsin as a method to separate in vivo from artifactual bone microdamage was first proposed by Frost 7 and later validated by Burr and Stafford. 2 This procedure has become the method of choice to demonstrate the accumulation of bone microdamage. Several different groups have used the technique successfully to demonstrate damage accumulation either in vivo as the result of mechanical loading, ~'~4 as the result of drugs that suppress bone turnover, 6'15 or from ex vivo mechanical tests. 5'9'10'16'19 Current concern over the effects of osteoporotic therapies which use drugs that prevent bone loss by suppressing bone remodeling, and thereby allow the accumulation of microdamage that may increase bone fragility, has made use of the technique more widespread. Because of the number of inquiries regarding the specific protocol we use, and recent changes that we have made to improve the technique, we feel it necessary and important to describe our current methodology.
Times for each solution depend on the size and density of the bone. A good rule of thumb is that the same times are used for bulk staining as those used to dehydrate a specimen in preparation for embedding. We generally use the following times for different kinds of bone: (a) rat cortical bone or human rib---2 h/step; (b) human femoral h e a d - - 2 - 3 days/step; (c) dog femoral head/neck---4 h/step; and (d) dog radius (cortical)--3 days/step. Times can be adjusted using these as guidelines. However, it is important to note that bone that is rapidly turning over or poorly mineralized will require substantially reduced stain times. If the vacuum is not used, longer staining times can be substituted with some sacrifice to the penetration of the stain. If specimens are not received in 70% ETOH, they can be placed in 1% basic fuchsin in 70% alcohol, with the solution changed once, before continuing the protocol. If there must be breaks in the staining protocol for any reason, specimens can be held indefinitely in the current alcohol concentration without stain or vacuum. Cortical bone specimens can be sectioned immediately after
Address for correspondence and reprints: David B. Burr, Ph.D., Department of Anatomy, MS 259, 635 Barnhill Drive, Indiana University School of Medicine, Indianapolis, IN 46202. E-mail: [email protected]
iupui.edu. © 1995 by ElsevierScience Inc.
1% basic fuchsin in 80% ETOH. Change solution. 1% basic fuchsin in 80% ETOH. Repeat steps 1-3, 1% basic fuchsin in 90% ETOH. Repeat steps 1-3, 1% basic fuchsin in 100% ETOH. Rinse in 100% ETOH for 1 h to remove excess stain; the specimen can be shaken or swirled occasionally during rinse.
8756-3282/95/$9.50 SSDI 8756-3282(95)00241-5
D.B. Burr and M. Hooser En bloc staining with basic fuchsin
Bone Vol. 17, No. 4 October 1995:431-433
staining, or may be embedded in methylmethacrylate (MMA). Trabecular bone specimens are always embedded in MMA. If specimens are to be embedded in plastic, immerse in 100% M M A for 4 h at 20 psi before proceeding with routine embedding.
Results and Discussion Problems and Solutions Investigators using this technique have encountered several difficulties.
Uneven staining. Uneven staining can be caused by several factors. The most c o m m o n cause is inadequate dehydration of specimens during the staining procedure. This is one reason we refined the technique to use basic fuchsin in a graded series of alcohols. A second cause for uneven staining is the failure to stain specimens under vacuum. In this case, the unevenness will characteristically not be random, but regions adjacent to periosteal, endocortical, or haversian surfaces will stain more darkly. A third cause for uneven staining is the source of the basic fuchsin. We have found basic fuchsin from the J.T. Baker Co. to provide the most adequate results. Fourth, it is possible that sectioning in water, even after embedding, can cause loss of stain in some areas. This is because basic fuchsin is water soluble, but is not removed by alcohol. However, we have not found this to be a serious problem. Finally, specimens that have been seriously damaged, even in the absence of microscopically visible microdamage, may present a nonuniform appearance (Figure 1). This has nothing to do with the technique used but may be the result of submicroscopic damage. 3 Overstain or understain. The most common cause of overstaining or understaining is the time in solution. It is wise before beginning a new study to test stain a few specimens and adjust the times to achieve the most adequate result. The best result is when canals, osteocyte lacunae, and canaliculae are darkly stained but the bone matrix is unstained or only lightly stained (Figure 2). This appearance makes microdamage quantitation much easier. Microdamage in specimens that are stained too darkly is difficult to detect. We have found that basic fuchsin purchased from Sigma pro-
Figure 2. Cross-section of dog femur, 80 Ixm thick, stained en bloc with basic fuchsin. Haversian and Volkmann's canals and osteocyte lacunae are stained darkly, but bone matrix remains virtually unstained. Microdamage (arrows) is clearly visible throughout this section. Original magnification x49. duces a more darkly stained specimen. This is why we recommend stain from the J.T. Baker Co. Understaining can also be caused by improper dehydration or by the failure to stain under vacuum. The density of the bone is a major determinant of the stain intensity. Therefore, staining times for poorly mineralized bone should be reduced, whereas staining times for highly mineralized plexiform bone (horses, cows) may need to be increased.
Limitations of the Technique The technique presents several limitations in quantitating bone microdamage. First, basic fuchsin penetrates by diffusion, so that while all of the microcracks that are stained are known to have been produced in vivo, not all cracks may be stained. It is not certain that the stain diffuses to all corners of a given specimen. Therefore, quantification of microdamage is probably underestimated. En bloc staining with basic fuchsin is not adequate to allow the visualization or quantification of submicroscopic damage, but is only useful with light microscopic techniques. Other techniques utilizing lead acetate staining have been developed for use with electron microscopy.17 Finally, and as mentioned previously, bone that presents a series of different densities is difficult to stain using this technique because those areas of low density can be overstained whereas those of high density may be understained. For example, detecting microdamage around a prosthetic implant can be problematic because it is difficult not to overstain this poorly mineralized bone.
Figure 1. Cross-section of dog femur, 80 ~xmthick, stained en bloc with basic fuchsin. Note the apparent unevenness of the stain in the center of the micrograph compared to surrounding regions. The arrow points to a small microcrack which may not be visible in adjacent sections. Original magnification x49.
The use of en bloc staining with basic fuchsin has proven to be a useful technique for the demonstration of microdamage produced in vivo. However, it can be a difficult technique to use because it is so sensitive to the density of bone being stained. As density of bone is not always known, can be variable within a specimen, and varies according to the source of bone and turnover rates, the exact staining times using this technique can vary somewhat. We have presented guidelines that are as specific to the kind of bone as possible. However, it may be necessary for
B o n e Vol. 17, No. 4 October 1995:431-433
investigators using this technique to stain some test samples prior to applying the technique to experimental material. We have also found it important to adhere closely to the protocol presented, otherwise significant difficulties can occur.
D . B . B u r r a n d M. H o o s e r En bloc staining with basic fuchsin
10. Acknowledgments: S u p p o r t e d b y a W h i t a k e r F o u n d a t i o n g r a n t a n d b y N I H G r a n t R01 A R 3 9 7 0 8 . The authors express their appreciation to Satoshi M o r i a n d M a r k F o r w o o d for their contributions and suggestions a b o u t alterations to the staining protocol.
References 1. Burr, D. B., Martin, R. B., Schaffler, M. B., and Radin, E. L. Bone remodeling in response to in vivo fatigue microdamage. J Biomech 18:189-200; 1985. 2. Burr, D. B. and Stafford, T. Validity of the bulk-staining technique to separate artifactual from in vivo bone microdamage. Clin Orthop Rel Res 260:305-308; 1990. 3. Burr, D. B., Turner, C. H., Naick, P., Forwood, M. R., and Pidaparti, R. M. Does microdamage accumulation affect the mechanical properties of bone? Trans Orthop Res Soc 20:127; 1995. 4. Cooper, C. The epidemiology of fragility fractures: Is there a role for bone quality? Calcif Tissue Int 53(suppl 1):$23-$26; 1993. 5. Forwood, M. R. and Parker, A. W. Microdamage in response to repetitive torsional loading in the rat tibia. Calcif Tissue Int 45:47-53; 1989. 6. Forwood, M. R., Burr, D. B., Takano, Y., Eastman, D. F., Smith, P. N., and Schwardt, J. D. Risedronate treatment does not increase microdamage in the canine femoral neck. Bone 16:643-650; 1995. 7. Frost, H. M. Presence of microscopic cracks in vivo in bone. Henry Ford Hosp Med Bull 8:25-35; 1960. 8. Frost, H. M. Mechanical microdamage, bone remodeling, and osteoporosis: A
13. 14. 15.
review. In: DeLuca, H. F., Frost, H. M., Jee, W. S. S., Johnston, C. C., and Parfitt, A. M., eds. Osteoporosis: Recent advances in pathogenesis and treatment. Baltimore, MD: University Park Press; 1981; 185-190. Fyhrie, D. P., Lang, S. M., Lundin-Cannon, D., and Schaffler, M. B. Quantitative analysis of trabecular damage in crushed human cancellous bone. Trans Orthop Res Soc 18:199; 1993. Fyhrie, D. P. and Schaffler, M. B. Failure mechanisms in human vertebral cancellous bone. Bone 15:105-109; 1994. Heaney, R. P. Qualitative factors in osteoporotic fracture: The state of the question. In: Christianson, C., Johansen, J. S., and Riis, B. J., eds. Osteoporosis, 1987. Viborg, Denmark: Nohaven; 1987; 40-44. Heaney, R. P. Osteoporotic fracture space: an hypothesis. Bone Miner 6:1-13; 1989. Heaney, R. P. Is there a role for bone quality in fragility fractures? Calcif Tissue Int 53(suppl 1):$3-$6; 1993. Mori, S. and Burr, D. B. Increased intracortical remodeling following fatigue damage. Bone 14:103-109; 1993. Norrdin, R. W., Robinson, H. T., Powers, B. E., and Histand, M. B. Evaluation of microdamage in canine trabecular bone. Trans Orthop Res Soc 18: 198; 1993. Schaffler, M. B., Choi, K., and Milgrom, C. Microcracks and aging in human femoral compact bone. Trans Orthop Res Soc 19:190; 1994. Schaffler, M. B., Pitchford, W. C., Choi, K., and Riddle, J. M. Examination of compact bone microdamage using backscattered electron microscopy. Bone 15:483--488; 1994. Sherman, S., Heaney, R. P., Parfitt, A. M., Hadley, E. C., and Dhutta, C. NIH workshop on aging and bone quality. Calcif Tissue Int 53(suppl 1): 1993. Wenzel, T. E., Schaffler, M. B., and Fyhrie, D. P. In vivo trabecular microcracks in human vertebral bone. Trans Orthop Res Soc 19:57; 1994.
Date Received April 25, 1995 Date Revised: June 8, 1995 Date Accepted: June 12, 1995