A study of bone remodeling using metal-polymer laminates

A study of bone remodeling using metal-polymer laminates zyxwvutsrq zyxw zyxwvutsrq zyxwv J. A. Szivek", G . C. Weatherly", R. M. Pilliart, and H. U. Camerod Department of Metallurgy and Materials Science, University of Toronto, Toronto, Ontario, Canada M5S l A 4 Bone remodeling due to stress-shielding has been studied using a model system consisting of metal-polymer laminated fixation plates securely fixed to canine femurs. The plate stiffness was controlled by varying the ratio of metal facing to polymer core thickness in the laminate design while secure fixation to bone was achieved by providing a porous bone interfacing surface for the ingrowth of bone from the periosteal sur- face. Observations of laterally and medially placed plates indicated resorption in the area of the periosteal and endosteal bone surfaces respectively, for the higher stiffness composite plates used. The results indicate that plate stiffness greater than approximately 70 GPa (axial) and 6 N m2 (flexural) will result in extensive bone remodeling in the canine femur after a six month implantation period. INTRODUCTION Resorption of cortical bone beneath metal fixation plates has been reported by a number of investigator^.'-^ This phenomenon has been attributed to "stress shielding," a reduction in stresses below physiological levels as a result of stress transfer to a higher stiffness metal plate fixed to the surface of the bone. In order to evaluate the effects of implant stiffness on bone resorption because of stress shielding, Woo et al.4 bilaterally plated the femurs of dogs using conventional stainless steel and carbon fiber-reinforced poly(methy1 methacrylate) plates for periods of nine and twelve months. Post-sacrifice histology indicated extensive resorption of the femoral cortex under the stiff metal plate while far less cortical thinning was noted under the more flexible composite plate. Bone remodelings was also studied by Moyen et al.,5 using intact dog femurs plated with either standard cast cobalt-chromium alloy plates zyxwvuts * Department of Metallurgy and Materials Science Faculty of Engineering, University of Toron to, Toronto, Ontario, Canada M5S 1A4. t Faculty of Dentistry, Univcrsity of Toronto, Toronto, Ontario, Canada M5G 1'26. t Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. 5 The term "remodeling" as used throughout this text refers to structural changes caused by altered stressing of the bone as opposed to its normal connotation to describe dynamic bone turnover under normal physiological stress conditions. zyx Journal of Biomedical Materials Research, Vol. 15,853-865 (1981) 0 1981 John Wiley & Sons, Inc. CCC 0021-9304/81/060853-13$01.30 zyx SZIVEK ET AL. 854 or thinner, more flexible Ti alloy plates. They concluded that only the stiffer cobalt chromium alloy plates caused cortical bone thinning, predominantly by endosteal resorption. A loss of 20 to 30% of the skeletal mass was recorded below the stiffer fixation plates after the six month implantation period. In these studies fixation was achieved by the use of standard bone screws. In order to assure more effective and controlled stress transfer between bone and plate, Pilliar et a1.6 used standard stainless steel plates with porous structured bone interfacing surfaces. Fixation of these porous surfaced plates to the femurs of dogs was achieved by bone ingrowth. This resulted in firm attachment of the plate to the bone, leading to far more extensive bone resorption beneath the plate than other studies had indicated. This exaggerated resorption was attributed to a greater transfer of stress to the metal plate as a result of the strong interface bond between the plate and the underlying bone. The purpose of the present investigation was to study bone remodeling under laminated plates attached by bone ingrowth as a function of plate stiffness and position of placement of the plate (lateral or medial bone surface). It was felt that the firm fixation of the plates to bone by bone ingrowth would result in a reproducible model for studying bone remodeling as a function of plate stiffness and position. EXPERIMENTAL PROCEDURE Materials and methods The facings of the laminates were fabricated from either 316L stainless steel or Ti-6A1-4V sheet. To form the bone interfacing porous surface region, the metal sheets were coated with cobalt-chromium alloy powder particles (for the 316L substrate) or with Ti-6A1-4V powder particles. The powder particles were bonded to the metal sheets using a high temperature reducing atmosphere heat treatment.7 For the stainless steel-cobalt alloy combination, earlier studied indicated that for the six-month implantation period used and the test procedure employed, no detectable corrosion occurred as a result of the dissimilar metal contact. Surgical grade polycarbonate and polypropylene were used as the polymeric core materials. Metal facing-polymer core bonding was achieved by forming a porous region on the polymer interfacing metal sheet surface and subsequently pressure bonding this to the polymer core using the pressure molding conditions indicated in Table I. Intrusion of the polymer into the porous metal surface layer during pressure molding resulted in a micromechanical bond at the metalpolymer interface (Fig. 1). By this method a laminated composite plate was formed without the use of chemically active adhesives. Plates of several different stiffnesseswere produced using this procedure and were modeled on standard six-hole fixation plates. Table I1 indicates the relative stiffnesses of the plates fabricated, the thickness of each component and the facing and core materials. BONE REMODELING 855 TABLE I Pressure Molding Conditions for Bonding Metal Facings to Polymer Core zy Material Characteristics Polycarbonate Polypropylene Molding Parameters Platen preheat temperature Pressure during molding Time to melting of polymer surface Mold cooling time Mold cooling technique 240-250 "C 0.7 MPa 7-8 min 4-5 min water quench 18O-19O0C 0.7 MPa 7-8 min 3-4 min water quench Laminated plates were produced for both mechanical testing (plate characterization) and for animal implantation. Both outer surfaces of the plates used for mechanical testing were smooth. A set of smooth surfaced plates for animal implantation was also produced. These plates were attached to bone by screws only, and therefore, served as a control set in our experiments to compare the effect of fixation by bone ingrowth to normal screw fixation on bone remodeling. All plates were gas sterilized prior to implantation. zyxwvuts zyxwvutsr Figure 1. Scanning electron micrograph of the metal-polymer interface showing the interdigitation of the polymer i n the porous metal surface structure (250X). TABLE I1 Description of Laminated Plates Fabricated Facing Description Approx. Thickness Material (mm) 316L Ti-6A1-4V 316L Ti-6A1-4V 1.3 1.5 1.o 1.o Core Description Approx. Thickness Material (mm) Polypropylene Polypropylene Polycarbonate Polvcarbonate 1.5 1.0 3.0 2.0 Plate Type high stiffness medium stiffness low stiffness low stiffness zyx SZIVEK ET AL. 856 zyxw Characterization of plates In order to evaluate the stiffnesses of plates used in the animal studies, smooth surfaced plates of the same configuration were tested axially in compression and by three point bending using an Instron Universal Test Machine. Tests were carried out at two strain rates, 9 X 1W6and 75 X s-l. It was felt that tests over this range of strain rates would correspond to the strain rates encountered during gait. The strains were measured using strain gauges attached to the outer surface of the plates. Animal testing Eight adult mongrel dogs were used in this study. Plates were attached using standard aseptic surgical procedures to the intact femurs of the dogs and for all plates initial fixation was achieved using self-tapping bone screws. For all the animals a porous surfaced plate was attached to one femur and a smooth surfaced plate of similar stiffness was placed on the contralateral limb. Plates were attached as nearly as possible to either the lateral or medial aspect of bone. The animal test protocol used is outlined in Table 111. All the animals were observed to be active shortly after surgery. A normal gait appearance and the retention of muscle mass suggested that the dogs were actively loading the operated limbs. Following surgery the animals were radiographed at regular intervals. After sacrifice at 6 months, the femurs were excised and placed in formaldehyde prior to dehydration and embedding in poly(methy1 methacrylate) for histological sample preparation. A transverse section was cut from the center of each bone-fixation plate sample and the remainder of the sample was sectioned longitudinally. The transverse sections were thinned for examination by transmitted light microscopy, while the longitudinal sections were polished and viewed using reflected light microscopy. A computer aided analysis of the percentage porosity observed in the cortical bone below the higH stiffness plates was carried out. For this purpose, part of the program reported by Nagurka and Hayes* for determination of bone sectional properties was used. The calculations were performed on a Tektronix 4051 Minicomputer with a Graphics Tablet and Digitizer. The four segments zyxwv TABLE 111 Implantation Protocol Laterally plated Medially plated Number of Stiffness Test Animals Facing and Core Material Plate Type Facing Thickness 316-L-Polypropylene Ti-6Al-4V-Polypropylene 316L-Polycarbonate Ti-6Al-4V-Polycarbonate thick thick thin thin high medium low low 2 316L-Polypropylene 316L-Polycarbonate thick thin high low 1 1 1 2 1 BONE REMODELING 857 of each longitudinal section were enlarged to facilitate accurate definition of the regions of resorption. The cortical bone below the plate was divided into two halves, namely, the periosteal envelope extending from the plate surface to the midline of the cortex and the endosteal envelope extending from the midline to the endosteal surface of the cortex. Percentage porosity calculations were carried out for each region. In preparing the longitudinal samples for these measurements, care was taken to section the bone-plate complex as nearly as possible through the longitudinal axis of the plate. This insured that the region of most extensive resorption invariably found along that axis was used in the analysis. zy zyxw zyxwvu zyxw zyxwvuts zyxw zyxw RESULTS Elastic property measurements The results of the axial modulus and bending stiffness measurements for the different laminate geometries used in the animal tests are shown in Figure 2. The theoretical axial and bending stiffnesses included in this figure were determined from the following relationships: Axial modulus: E, Bending stiffness: EI = 2E,(zu,ti/ + VrEr 12 + wmt,d2) + E,(w,t;/ 12) = V,,E, (1) (2) where Ec,m,p = elastic modulus of composite, metal, polymer; Vm,,= volume fraction of metal, polymer; I = area moment of inertia; t,,, = thickness of metal, polymer; w,,~ = width of metal, polymer; and d = distance between neutral axis and centroidal axis of metal facings. As can be seen in Figure 2, the experimentally determined stiffnesses were in good agreement with the theoretical predictions. It is evident from Figure 2 that an increase in the polymer core to metal facing ratio resulted in a greater reduction in the axial modulus ( E ) , than in the bending stiffness (EI). Although not critical for our model studies this would be a desirable design for a clinical fracture fixation plate since it would allow the maintenance of a relatively high bending stiffness (useful for fracture stabilization) while providing a lower axial plate stiffness. Animal studies Radiography proved to be an insensitive technique for the study of changes in bone structure below the plates. Only post-sacrifice histology could, therefore, be used to assess bone remodeling. Transverse histological sections from the midlength region of the plated bones were examined for both bone ingrowth (porous surfaced plates) and for evidence of bone remodeling (resorption and formation). The interface region of a high stiffness porous surfaced plate and the underlying bone is shown in Figure 3. Osteocytes are visible up to and within 858 zyx zyxwvutsr SZIVEK ET AL. z zyx zyxwv zyxw - -f v) v) W z LL 3 1 6 L S S Polymer Laminate zyxwv zyxwvutsrqpo 9- N . . 1 0 Strain r a t e A Strain rate 7 5 p s t r a i n Spstrain, T I - B A I - ~ V Polymer Laminait 8- 8 7- Straln r a t e \Strain gpstrain, rate 75 psirain, 6-- LL 5I- -I 4- a 5x 3- 2 2- w 1- (b) __ - 3 1 6 L S S ____ T i - 6 A I - 4 V 0 1 I 0 1 P o l y m e r L a m i n a t e s (theoretical) Polymer Laminates (theoretical) I 2 1 3 4 P O L Y M E R CORE T H I C K N E S S ( m m ) Figure 2(a). Axial modulus of laminates as a function of polymer core thickness. (b) Bending stiffness of laminates as a function of polymer core thickness. the porous surface region of this plate. Similarly, ingrowth of bone was observed into at least some regions of the porous surface layers for all the porous surfaced plates. For some of the specimens fibrous tissue was observed toward the edges of the implants (specifically for lower stiffness plates). However, there was sufficient area of bone ingrowth for all the plates to ensure that the BONE REMODELING zy zyxwvuts 859 zyxw Figure 3. Interfacial region of high stiffness laminate and bone showing bone ingrowth into the porous surface layer of the laminate (250X). zyxwvuts plates were rigidly fixed. Any attempt to physically remove the plates resulted in either their delamination or failure through the bone. The interface itself remained intact. Effective stress transfer, thus, was assured between bone and all the different stiffness composite plates. For the laterally plated femurs the most severe resorption occurred below the thick stainless steel faced plates, i.e., the high stiffness composite plates. This is demonstrated in Figure 4(a). In the transverse section of the bone plated with a high stiffness laminate [Fig. 4(a)] it is seen that resorption has occurred directly below the plate at the periosteal surface. The area of bone resorption extends only part way through the cortex with dense cortical bone being retained at the adjacent zone toward the endosteal surface. The longitudinal section indicates that the bone resorption occurred below the plate from the most proximal to the most distal screw. Regions of bone formation are also evident below this high stiffness plate. Extensive new bone was formed below the edges of the plate and at the periosteal surface of the diametrically opposed cortex [regions A and B, respectively, in Figure 4(a)]. Only minor amounts of intracortical osteoporosis are seen below the medium stiffness Ti-6A1-4V faced laminate [Fig. 4(b)]. Again evidence of additional bone formation is apparent beneath the edges of the plate and peripherally on the opposite cortex. Less new bone formation is observed in this case as compared to that observed below the higher stiffness plates. The transverse and longitudinal sections of a bone plated with a low stiffness stainless steel faced laminate indicate that negligible remodeling (resorption and formation) occurred and both the cortical thickness and density were retained throughout the region below the plate [Fig. 4(c)]. For the rnedially plated femurs the most extensive remodeling is also ob- 860 zyx zy zyxwvutsrq SZIVEK ET AL. (a) (b) zyxw (C) Figure 4(a). A transverse section of a bone plated on the lateral aspect with a high stiffness 316L facing laminate. Oseoporotic bone is clearly evident at the periosteal surface of the plate below the center of the plate. Regions A and B are regions of bone formation (3X). (b)A transverse section of a bone plated on the lateral aspect with a medium stiffness Ti-6A1-4V laminate. Some intracortical osteoporosis is visible below the plate. Regions of bone formation can also be seen below the plate and on the diametrically opposite periosteum (3X). (c) A transverse section of a bone plated on the lateral aspect with a low stiffness 316L laminate. No resorption has occurred below this plate. Cracks in the polycarbonate core are an artifact of the histological preparation process (3X). served below the high stiffness plate [Fig. 5(a)]. Resorption below this plate, however, occurred at the endosteal surface of the bone and throughout most of the cortex with dense cortical bone being retained near the periosteal surface. Bone formation at the plate edges and on the lateral periosteal surface is also evident. No obvious regions of resorption are seen below the low stiffness laminate, as seen in Figure 5(b) and, although a thin bone spur appears to have formed posteriorly, this is not extensive. Observations of the contralateral limbs that were plated with smooth surfaced plates attached by screws only demonstrated negligible remodeling of bone in all instances, regardless of plate stiffness. This result is consistent with our earlier studies on higher stiffness, all-metal plates? in that there was much less resorption under the plates attached using screws only. The quantitative analysis of the porosity below the midline regions of the high stiffness plates is shown in Figure 6. Porosity below the two laterally plated cases is greater in the periosteal envelope than in the endosteal envelope. In the first laterally plated case, it should be noted that although the difference in percent porosity is not very great the average percentage porosity for the whole section is very high. For the medially plated bone the porosity is significantly higher in the endosteal envelope than in the periosteal envelope. BONE REMODELING zyxwvuts zy 861 (a) Figure 5(a). A transverse section of a bone plated on the medial aspect with a high stiffness 316L laminate. Gross endosteal resorption is evident below the center of the plate. Extensive bone formation is also visible below the edges of the plate and on the periosteum of the diametrically opposite cortex (5X). (b) A transverse section of a bone plated on the medial aspect with a low stiffness 316L laminate. N o visible resorption has occurred below this plate. The cracks in the polycarbonate core of the plate are an artifact of the histological preparation process (5X). zy DISCUSSION During gait, the femur is stressed as a result of axial, bending, and torsional components of the applied Ioad and muscle forces. The bending component a ) LATERAL b) C) LATERAL MEDIAL P - P E P E P zyxwvut zyxwvuts Cortical Bone Percent b e l o w Plate Porosity P e r i o s t e a l Envelope E -Endosteal Envelope -- I 65.3 % 57.4 % 7.9 % 55.5 % 30.9 % 24.6 % 22.5 % 40.3 % 70.0 % L-! A% - Cortical Bone - Resorption Cavities Figure 6. A schematic diagram of porosity in the cortex below high stiffness plates for laterally and medially plated cases. Percent porosity is shown for the periosteal envelope and endosteal envelope of each section. The A% indicates the difference in porosity between the two regions. SZIVEK ET AL. 862 will result in a variation of stress across the cortex with a maximum tensile stress occurring toward the lateral periosteal surface and a lower level of stress at the endosteal surface. On the medial surface a maximum compressive stress acts at the periosteum with a lower amplitude stress on the endosteal surface. The axial stress component imposes a compressive stress on the femur. Superposition of the bending and axial stress components results in an anterior-lateral displacement of the neutral surface, i.e., the plane on which bending stresses are zero. In addition, a torsional stress component, which imposes a circumferential shear stress, and transverse shear stress components will act on the bone. Superposition of all of these components would define a region of low stress within the femur at every position during the gait cycle. An analysis to determine the exact position of this low stress region in a bone, (with or without plate), would require strain determinations during gait using in vivo strain gauging techniques which were beyond the scope of this study. A qualitative explanation of the observed remodeling is, however, possible and will be presented here. Since the main objective of this study was an investigation of the effect of stress shielding on bone resorption a discussion of the observed resorption below the plates will be dealt with first. The new bone formation noted will be discussed thereafter. zyxwvu zyxwvutsrqpo zyxwvut Bone resorption When a plate is fixed to the surface of the femur, the bone-plate complex can be considered an eccentrically loaded composite column. The redistribution of stress between the plate and the bone will depend on the plate stiffness, its position on the surface of the bone and the nature of the bond between the implant and bone. Secure attachment of a high stiffness plate to the lateral aspect of the femur will result in a reduction in the normal tensile stress component due to bending acting in the cortex just under the plate, as well as a reduction in the axial compressive stress component, torsional stress component and transverse shear component under the plate. The result of these altered stress components is a shift of the low stress region in the lateral direction. As a result, the low stress region occurs near the lateral periosteal surface for the femurs with high stiffness plates fixed to the lateral aspect. The effect of the low stress state in this region is the observed bone resorption under the plate at the lateral periosteal surface and part way through the cortex [Fig. 4 ~ 1 . For the medially attached plate, similar reasoning will indicate a shift in the low stress region. For this case, the neutral axis shift is in the medial direction. Because the neutral axis for the unplated bone is lateral to the centroidal axis, the position of the neutral axis for the medially plated bone is medial to, but closer to the femoral axis. From the observed remodeling for the medially placed, high stiffness composite plate, the resulting low stress region occurs near the medial endosteal cortical bone surface. Again, after six months the resorption was only partly through the cortex [Fig. 5(a)]. BONE REMODELING 863 For lower stiffness plates or poor plate-bone bonding, the stress shielding effect on the bone is lessened. In our study, this resulted in less bone loss. In an earlier study6 using porous surfaced metal plates attached by bone ingrowth to the lateral surfaces of dog femurs extensive loss of cortical bone was also observed. In that experiment, the six-month implantation of the higher stiffness stainless steel plates resulted in resorption virtually completely across the cortex immediately under the plates. The high stiffness composite laminates used in the present study were about 0.5 times as stiff axially and 0.85 times as stiff in flexure as the all-metal plate used in that earlier study. This difference in stiffness was apparently sufficient to result in the partial intra-cortex resorption that was observed. This also allowed us to observe a subtle difference in remodeling for the laterally and medially placed plates. The computer aided porosity analysis of the longitudinal sections verified that this difference occurred along the length of the bone section and was not a sectioning effect noted at the midlength only. The difference in osteoporosis as a function of position in the cortex (i.e., endosteal vs. periosteal envelope) is significant for femurs (b) and (c) in Figure 6. The changes in femur (a) although indicating the same trend were less dramatic. This sample, however, showed the greatest overall porosity suggesting that the load history for this animal was such that the particular plate stiffness used in the experiment masked any differences in remodeling at six months, i.e., the plate stiffness was too high to clearly indicate the differences shown by femurs (b) and (c). This gross overall loss of bone was also noted in an earlier study using higher stiffness (all metal) plates.6 Our observations support the hypothesis that a minimum stress is required to prevent gross bone resorption. Other studies of resorption after prolonged recumbency, immobilization or weightlessness have also indicated that a minimum stress is required to prevent resorption and other physiological change~.~-l~ A determination of the minimum stress necessary to maintain a healthy bone structure would be highly desirable, not only for the design of load-bearing implants, but also for an appreciation of patient treatment during prolonged bed rest. It would also be useful for the definition of gravity simulation conditions required in space to avoid physiological and anatomical changes in man. Such an analysis could not be made in this study because of limited information regarding the exact load-strain history acting on the femur during normal animal activity. Such an analysis should be possible with refined experimentation. Bone formation It was noted that toward the edges of the higher stiffness plate extensive new bone had formed. Additionally, cortical thickening was observed at the ends of these plates. These regions correspond to zones in which stresses are transferred between bone and plate and as such are regions of higher stress SZIVEK ET AL. 864 concentration. The buildup of bone in these regions is thus a response to the increased stresses in the area. The formation of bone on the diametrically opposed periosteum (with respect to the plate position) can also be explained by enhanced stress levels. The shift in the neutral surface caused by the plate, in addition to reducing stresses on the plated surface by reducing the distance from the plate to the neutral surface, would also increase the stresses on the diametrically opposed cortex. The greatest amount of new bone formed (as expected) was on the bones plated with high stiffness plates. The limited number of samples and the large variation from one test animal to the next, however, warrants a note of caution i n any quantitative judgment of this sort. CONCLUSIONS zyxw zyxw zyxwvu zyxwvu (1) A comparison of high stiffness composite implants attached by bone ingrowth to others attached conventionally using screws demonstrated more extensive bone remodeling for the biologically attached implants. (2) This remodeling was strongly dependent on implant stiffness with negligible effects being observed for well-bonded plates with an axial stiffness of about 0.3 times (70 GPa) and flexural stiffness of about 0.6 times (6N m2)that of a stainless steel plate. ( 3 )For implants of a critical stiffness, a difference in remodeling, (principally resorption), was demonstrated for plates attached to the lateral and medial aspects of the femur. The authors are grateful to the Atkinson Charitable Foundation for financial support for the program. The assistance of the Ontario Ministry of Health through a Senior Scientist Award is gratefully acknowledged by one of the authors (RMP). Additionally, we would like to thank Deloro Orthopaedic, a division of Canadian Oxygen Ltd., and the Ontario Research Foundation for assistance with materials supply and fabrication of parts. References 1. T. Slatis, E. Karaharju, T. Holmstrom, J. Ahonen, and P. Paavolainen, "Structural Changes in Intact Trabecular Bone after Application of Rigid Plates with and without Compression," 1. Bone It. Surg., 60-A, 516-522 (1978). 2. A. J. Tonino, C. L. Davidson, P. J. Klopper, and L. A. Linclau, "Protection from Stress in Bone and its Effects. Experiments with Stainless Steel and Plastic Plates in Dogs," 1. Bone It. Surg., 58-B,107-113 (1976). 3. H. K. Uhtoff and F. L. Dubuc, "Bone Structure Changes in the Dog under Rigid Internal Fixation," Clin. Orfkop. Relat. Res., 81, 165-170 (1971). 4. S. L-Y. Woo, Mi. H. Akeson, R. D. Coutts, L. Rutherford, D. Doty, G. F. Jemmott, and D. Amiel, "A Comparison of Cortical Bone Atrophy Secondary to Fixation with Plates with Large Differences in Bending Stiffness,'' 1. BoneJt. Surg., %-A, 190-195 (1976). 5. B. J-L. Moyen, P. J. Lahey, E. H. Weinberg, and W. H. Harris, "Effects on Intact Femora of Dogs of the Application and Removal of Metal Plates," I . Bone It. Surg., 60-A, 940-947 (1978). 6. R. M. Pilliar, H. U.Cameron, A. G. Binnington, and J. Szivek, "Bone Ingrowth and Stress Shielding with a Porous Surface Coated Fracture Fixation Plate," 1. Biomed. Mafer. Res., 13,799-810 (1979). zyxw BONE REMODELING zyxwvut zyxwvut zyxwvu zyxwvu zyx 865 7. R. M. Pilliar, and H. U. Cameron, and I. Macnab, ”Porous Surface Layered Prosthetic Devices,” Biomed. Eng., 10 (4), 126-131 (1975). 8. M. L. Nagurka and W. C. Hayes, “An Interactive Graphics Package for Calculating Cross-sectional Properties of Complex Shapes,” J . Biomech., 13,59-64 (1980). 9. D. P. Cuthbertson, “Influence of Prolonged Muscular Rest on Metabolism,” Biochem. J., 23, 1328 (1929). 10. J. A. Gillespie, ”The Nature of Bone Changes Associated with Nerve Injuries and Disuse,” J . Bone Jt. Surg., 36-8,464-473 (1954). 11. E. R. Morey and D. J. Baylink, “Inhibition of Bone Formation during Space Flight,” Science, 201,1138-1141 (1978). 12. J. E. Deitrick, G. D. Whedon, and E. Shorr, “Effects of Immobilization upon Various Metabolic and Physiologic Functions of Normal Men,” Am. J. Med., 4,3 (1948). 13. B. Issekutz, ”Effect of Prolonged Bed Rest on Urinary Calcium Output,” J . AppZ. Physid., 21, 1013-1020 (1966). Received September 26,1980 Accepted April 3,1981