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Inflammation-mediated osteopenia in the rat: The effects of artificial granuloma and sham operation on cortical and trabecular bone
Bone, 6, 461-465 (1!385) Printed in the USA. All rights reserved.
Copyright
8756-3282185 $3.00 + .OO @ 1985 Pergamon Press Ltd.
Inflammation-Mediated Osteopenia in the Rat: The Effects of Artificial Granuloma and Sham Operation on Cortical and Trabecular Bone J. PFEILSCHIFTER,’
H.W. MINNE,’
E. ENZMANN,’
B. KREMPIEN,’
and R. ZIEGLER’
Qepartment of internal Medicine VI, Endocrinology, University of Heidelberg, D-6900 Heidelberg, 2/nstitufe of Pathology, University of Heidelberg, O-6900 Heidelberg, West Germany. Address for corresoondence and reprints: Dr. H.W. Minne, Department Luisenstr., 5, D-6990 Heidelberg, West Germany.
Recent studies have established that generalized loss of trabecular bone occurs in the growing rat following day-today inflammatory irritation for a period of 3 wk. We can now demonstrate that there are similar effects on bone in milder but more prolonged chronic inflammation (14 wk). Thus, there were significant decreases in trabecular bone mass as well as in cortical bone after weekly subcutaneous injections or implantations of nonspecific irritants. Osteopenia, induced by a single but extensive inflammatory lesion, remained apparent even 14 wk after induction. This indicates that inflammation-mediated osteopenia is at least incompletely reversible. A less pronounced but similar reduction of cortical and trabecular bone was observed in rats following sham operation. This might be of importance in all animal studies on bone metabolism that include surgical procedures. Cortical Bone-Serum
of Internal Medicine VI, Endocrinology,
University of Heidelberg,
investigated the effects on cortical bone in both intact and parathyroidectomized rats. IMO may be due to a generalized host reaction irrespective of the nature of inflammatory substances. As a consequence, even sham-operated animals might be affected. We therefore compared the bone mass of animals after implantation of foreign body granulomas with the bone mass of sham-operated and untreated rats.
Abstract
Key Words: Inflammation-Osteopenia-Trabecular
West Germany.
Material and Methods Female rats (strain Chbb Thorn, Thomae Co., Biberach; FRG) weighing 180 to 200 g at the beginning of the study were used. Some of the animals had been parathyroidectomized 3 to 4 wk prior to the experiments. Parathyroidectomy (PTX) was performed under light ether anesthesia by electrocoagulation. A decrease in total serum calcium below 6.5 mg/dl proved successful PTX. Rats were fed a standard rat chow (Altromin Co., Lage; FRG) and water ad libitum.
Bone-
Calcium Experimental
design
Fifteen PTX and fifteen intact rats were subcutaneously injected with a 1 ml saline suspension of 400 mg magnesium silicate weekly for a period of 14 wk. Thirty PTX and fifteen intact control rats received weekly injections of the same volume of saline. Pellets of sterile compressed cotton (diameter 0.8 cm) were subcutaneously implanted in PTX rats. In one group of 10 animals, 8 pellets were implanted simultaneously, and the rats were then kept for 14 wk without further treatment. In another group of 14 animals, implantation of one pellet was performed weekly for a period of 8 wk. The skin of the back was shaved and washed, and the pellets were inserted into the subcutaneous fascial space via a 1 cm incision after creation of a subcutaneous pocket by blunt dissection. The incision was closed with two metal clips. In a third group of PTX rats the same surgical procedure was performed but no pellets were implanted. Nine PTX rats without further treatment were used as controls. Blood samples were obtained from the orbital plexus under light ether anesthesia at different intervals. Serum calcium was determined by atomic absorption spectrophotometry. The rats were fasted overnight prior to the collection of the blood samples. After sacrifice the right femur, the left tibia, and fourth lumbar
Introduction Previous studies have demonstrated the occurrence of bone loss in rats following Inonspecific inflammation (Minne et al., 1984; Pfeilschifter et al., 1984). This inflammation-mediated osteopenia (IMO) developed independent of PTH and vitamin D metabolites. Elone rarefaction was generalized and appeared as a decrease of trabecular bone volume and bone ash weight. In parathyroidectomized rats bone loss was associated with significant increase in serum calcium. The effect of inflammation on bone metabolism by daily injections of magnesium silicate had been examined for a period of 2 to 3 wk. The present study was performed to determine whether more prolonged inflammatory irritation would lead to similar changes in bone mass and if there is any restitution of bol-te mass after inflammation-mediated bone loss in the rat. Previously, only the effects of inflammation on trabecular bone had been studied. We have now 461
J. Pfeilschifter et al.: Inflammation-mediated
462
vertebra were removed and cleaned from adherent soft tissue. The femur was rehydrated in distilled water in vacua for 24 h, weighed without removal from water (WUW) and then again in air (WIA) after blotting. The difference between WUW and WIA measured the bone volume according to Archimedes’ principle. The bone was then ashed at 600°C for 24 h and the ash weighed. In prior experiments concerning IMO, correlations between bone density and bone calcium content had been made. Both parameters correlated positively with a correlation coefficient of r = 0.92. Bone density was calculated as ash weight/bone volume. The proximalmost 1 cm of the tibia was cut off the shaft with a razor blade and embedded in methylmethacrylate without prior decalcification (Schenk, 1965). A longitudinal section was cut to the midsagittal plane with a precision bone saw, and from there sections of 3 pm thickness were prepared on a Jung Model K microtome (Jung Co., Heidelberg; FRG). The body of the embedded fourth lumbar vertebra was cut in half by a transverse section, and 3 pm sections were prepared. Sections were stained according to a modified Goldner method (Goldner, 1938; Schenk et al., 1969) and then subjected to quantitative histologic analysis. A method reliant on a Merz grid was employed for quantitative estimation of the trabecular bone (Merz, 1967; Merz and Schenk. 1970; Kimmel and Jee, 1980). A 1 mm band 1.75 mm distal to the epiphyseal cartilage was analyzed from cortical to cortical bone by point counting. Using a x 400 magnification, this area comprised about 90 to 100 fields. The percentage of trabecular bone was calculated as: Number of points over trabecular bone x 100 Total number of points The area of trabecular bone in the fourth lumbar vertebra was defined by two parallels tangential to the vertebral canal and perpendicular to the ventral bordering of the vertebral canal. Using a x 400 magnification, this area comprised about 60 to 80 fields. The cortical area of the fourth lumbar vertebra was determined either by point counting or by planimetry using a half-automated image analysis system (Videoplan; Kontron Elektronik, Eching; FRG). Cortical bone was expressed as percent of the total vetebral body. The area of the cross section of each vertebral body was measured and expressed as percent area of total vertebral area of control animals. Statistical analysis was performed using the Wilcoxon rank sum test, Q was determined to be 0.05 before the study. All data were collected using the single blind technique to reduce bias of interpretation. The data are expressed as the mean + the standard deviation (f SD).
osteopenia.
Results All groups
of animals weight steadily.
appeared
healthy
and
active
and
At the end of the experiment there was no significant difference in body weight between treated and untreated rats. Body weight of the magnesium silicateinjected rats was corrected by subtracting the weight of the injected material. Parathyroidectomy caused a slight retardation in growth of the rats. Liver and kidney appeared normal in both histology and fresh weight per body weight, but spleen was enlarged and showed activation of the lymphatic nodules in all animals treated with irritants once a week. The bone marrow of these animals was markedly hypercellular with an increase in mature neutrophils. gained
Trabecular bone and ash weight A significant decrease in mean femur ash weight per volume of bone occurred in PTX (7%) as well as in intact animals (9%) treated with magnesium silicate. Trabecular bone volume of the tibia and of the fourth lumbar vertebra was decreased in the same animals (Table I). Similar changes were found in PTX rats that underwent implantation of one pressed cotton pellet once a week. The amount of ash per volume of femur was reduced by 6%, the trabecular bone volume of the tibia by 29%, and trabecular bone volume of the fourth lumbar vertebra by 17%, as compared with these same parameters in the PTX controls. Similar changes were also found in rats that underwent the same surgical procedure but actually did not receive cotton pellets, i.e., in sham-operated animals. Again the trabecular bone volumes of the tibia and of the fourth lumbar vertebra were reduced, though to a lesser extent, whereas the amount of ash per volume of femur was not significantly changed (Table II). Bone rarefaction produced by single implantation of eight cotton pellets was seen even 14 wk after implantation. We found the trabecular bone volume in the tibia metaphysis decreased by 40% and a 10% less ash weight of the right femur. However, ash weight per volume of femur, as well as vertebral trabecular bone, failed to show any significant differences.
Table I. Effect of weeklv iniections of maonesium silicate or of single implantation of cotton pellets on bone parameters after 14 wk
Groups PTX (n=30) PTX-magnesium
Tibia Trabecular Bone (%)
Vertebra Trabecular Bone (%)
Vertebra Cortical Bone 1%)
639 (i-30)
18.8 (k5.8)
15.1 (f3.2)
25.9 (1t3.6)
102.3 (+9.9)
99.9 (+9.5)
Femur Ash Weight/ Volume (pg/mm?
Vertebra Area of Cross Section (Int.= 100%)
Vertebra Marrow Cavity (Int. = iOO%p
593 (f30)”
10.9 (f4.1)’
11.4 (f2.3)”
21.8 (k3.3)”
101.3 (*3.4)
104.5 (k9.5)
silicate (n = 15) PTX-cotton
618 (f27)
10.2 (f4.0)”
12.7 (f2.3)
23.7 (f 1.3)'
100.5 (f9.7)
101.3 (+10.4)
pellets (n = 10) Intact
637 (+40)
20.8 (f4.9)
17.9 (f3.8)
24.3 (k3.6)
100.0 (i9.0)
100.0 (19.7)
582 (f27)b
14.5 (k3.8)
13.4 (+_2.5)b
21.3 (f2.1)b
111.4 (+12.4)b
115.2 (+14.9)b
(n=l5) Intact magnesium silicate (n = 15)
Data expressed as mean + SD. aSignificantly different from PTX control. bSignificantly different from intact control (P Clnt. = Intact (control) animals’ values.
< 0.05).
b
J. Pfeilschifter et al.: Inflammation-mediated
Table II. Effect of weekly implantation
of pressed cotton pellets or of sham operation on bone parameters
Femur Ash weight/ Volume Groups PTX (n=9) PTX sham op. (n= 14) PTX cotton pellets (n = 14)
463
osteopenia.
Tibia Trabecular Bone (%)
(I*g/mm?
Vertebra Trabecular Bone (%)
Vertebra Cortical Bone (%)
after 8 wk.
Vertebra Area of Cross Section (PTX= 100%)
Vertebra Marrow Cavity (PTX= 100%)
634 (+39)
30.3 (k6.2)
18.3 (k2.2)
26.4 (f3.9)
100.0 (k7.5)
100.0 (*9.4)
607 (+30)
24.3 (f4.8)a
16.4 (f2.8)a
23.1 (&4.4)a
100.0 (k7.4)
106.0 (k6.2)
595 (&38)a
21.4 (k6.5)”
15.1 (k2.8)”
21.3 (+3.2)a
105.7 (f7.5)
115.6 (k9.4)”
Data expressed as mean * SD. “Significantly different from PTX control (P ~0.05)
Cortical bone Measurements of the cortical area of cross sections from the fourth lumbar vertebra clearly demonstrate the differ-
ences in cortical bone between untreated and IMO rats at the end of both experiments. The data are presented as percent cortical bone area of the vertebral body in relation to the total area of vertebral body (Tables I and II). In animals that were subcutaneously injected with magnesium silicate, the cortical area decreased by 16% in PTX and 13% in intact animals when compared with PTX or intact controls. PTX rats that received one cotton pellet once a week showed a similar decrease of 19%. Sham-operated animals at least had a 12% less cortical area at the end of the study. The cortical bone of those PTX animals that underwent single implantation of eight pellets was significantly decreased by 9% even after a period of 14 wk. Marrow cavity and total bone area Whereas the marrow cavity was found enlarged in IMO PTX rats and in IMO intact rats, only the latter had an enlarged total area of the vertebral body. In all PTX groups no significant changes in total area of body were found as compared with untreated controls. Serum calcium One month after parathyroidectomy concentration was 5 1:; 6 mg/dl.
athyroidectomized
table
the mean serum calcium
in animals that were parand then subcutaneously injected with
magnesium silicate once a week, serum calcium began to rise after two injections and thereafter was only slightly elevated as compared with PTX controls. However serum calcium of PTX controls did rise from 5 to 8 mg/dl during the experiment. By 14 wk the serum calcium levels of IMO rats could not be distinguished statistically from those found in untreated PTX animals (Table III). In those animals that were parathyroidectomized and then received a single subcutaneous implantation of cotton pellets, an initial rise of serum calcium to 7.6 mg/dl occurred within the first 2 wk. However, by 4 wk the serum calcium values of these rats were low again and remained so for the remainder of the experiment. In intact rats treated with magnesium silicate, serum calcium concentration was slightly decreased throughout the experiment.
Discussion Cortical and trabecular bone of the growing rat undergoes extensive modeling by continued bone formation and bone resorption. Thus, the growing rat bone offers an excellent opportunity for the short-term study of various conditions thought to influence bone formation or bone resorption. For the same reasons it may be difficult to draw conclusions about long-term effects from short-term experiments, especially since bone formation and bone resorption may be
Ill. Effect of weekly injections of magnesium silicate or of single implantation of cotton pellets on serum calcium concentration. Groups
Serum Calcium in mg/dl Day 0
PTX (n=30) PTX magnesium silicate (n=15) PTX cotton pellets (n=lO) Intact (n = 15) Intact magnesium silicate (n = 15)
14
28
55
100
4.8 (kO.9) 4.6 (10.6)
6.2 (kO.7) 6.7 (*I 0)”
6.1 (k1.0) 6.8 (*1.2)
7.1 (fO.9) 8.5 (f0.7)a
7.9 (f0.8) 8.0 (kO.9)
4.5 (L-0.8)
7.6 (f0.9)a
6.4 (kO.7)
6.7 (k1.0)
7.9 (*0.7)
9.6 (f0.4) 9.6 (f0.3)
9.6 (L-0.7) 9.6 (f0.2)
10.0 (+0.3) 9.6 (+0.2)b
10.0 (f0.5) 9.8 (kO.6)
10.1 (kO.4) 9.7 (&0.3)b
Data expressed as mean + SD. aSignificantly different from PTX control. bSignificantly different from intact control (P < 0.05).
464
mechanistically linked in the growing skeleton (Gasser et al., 1972; Miller and Jee, 1977). Previous studies have demonstrated a dramatic loss of trabecular bone in growing rats following daily injections of nonspecific irritants for a period of 3 wk. One aim of this study was to find out whether the growing rat is able to compensate for bone loss in milder, more prolonged inflammation In addition, we examined whether bone loss after 3 wk of daily inflammatory stimulus is completely reversible. Our data suggest that prolonged inflammation is a powerful stimulus of bone loss and that bone rarefaction induced by a single inflammatory lesion remains apparent even after 14 wk. If there is any reversibility in the volume of cortical and trabecular bone, it is only minor. The bone of the growing rat is quite different from the bone of an adult human (Frost, 1973). Therefore, our results may not be representative of the effects of inflammation on the remodeling of human adult bone. Nevertheless, the experiments described may serve as a new model for long-term or even lasting development of bone loss in a biologic system. Rats that were injected weekly with magnesium silicate for a period of 14 wk showed bone loss similar to the rats that received daily injections for a period of 3 wk. This indicates that each inflammatory stimulus might have a cumulative effect on bone. Up to now, we have little information about the minimal inflammatory intensity of a single stimulus needed to influence bone volume. The effects appeared to be relatively independent of whether the changes were produced by magnesium silicate or subcutaneous implantation of cotton pellets. Animals that underwent the same surgical procedure with no implantation of pellets had milder but similar changes in cortical and trabecular bone. We conclude therefore that the foreign body material is not the main stimulus necessary to induce IMO. There were no visible signs of local wound infection, and all operated animals appeared healthy. However, bone loss was associated with enlargement of the spleen and a marked proliferation of bone marrow. Therefore, surgical trauma as well as bacterial contamination may have contributed to the oberved loss of bone in sham-operated animals. Irrespective of the nature of the inciting stimulus, nearly all infections, injuries, and inflammatory processes stimulate mononuclear cell phagocytes to synthesize and release interleukin 1 (Dinarello, 1984). Interleukin 1, therefore, may play an important role in bone loss associated with inflammation. In animal experiments on bone metabolism, particular attention should be paid to all surgical procedures, since the operation itself may have some effect on calcium and bone parameters. Daily injections of magnesium silicate in PTX rats were followed by a return of serum calcium toward subnormal levels (Minne et al., 1984). This study demonstrates that a powerful single stimulus of inflammatory irritation can raise serum calcium concentration in PTX rats toward a subnormal level 2 wk after the onset of the inflammation. However, 2 wk later there is a return of serum calcium toward preinflammatory levels. Thereafter, serum calcium remained unchanged. Weekly injections of magnesium silicate resulted in slightly but significant changes of serum calcium levels as compared with controls. In PTX animals, serum calcium increased to the upper normal range or slightly above the serum calcium of untreated PTX rats. However, we also
J. Pfeilschifier
et al.: Inflammation-mediated
osteopenia.
observed a continuous increase in serum calcium of untreated PTX rats. Calcium had increased from 4.8 to 7.9 mg/dl 14 wk after parathyroidectomy. It is unlikely that the return of serum calcium toward normal levels was due to compensatory function of parathyroid remnants after incomplete parathyroidectomy. Our results are consistent with those of other authors (Kenny, 1962; Kemm, 1976; Peng and Garner, 1979), who also reported a return of serum calcium toward normal in PTX rats. According to Peng and Garner (1979), this phenomenon is associated with an increase of serum calcitonin. Rats with intact parathyroid glands tended to have slightly but significantly lower serum calcium levels after weekly injections of magnesium silicate. These surprising data indicate that the elevated calcium levels of IMO PTX rats are not necessarily due to increased calcium mobilization from bone. Recent studies of the biphosphonate APD (Pfeilschifter, unpublished data) demonstrated that APD is completely able to inhibit inflammation-mediated bone loss without inhibiting the rise of serum calcium in PTX rats. Inflammation may, at least to some extent, raise serum calcium concentration by decreasing urinary excretion of calcium or by increasing the gastrointestinal absorption of calcium. Further work is needed to define the mode of action of inflammation on calcium homeostasis. The present results provide further confirmation that IMO occurs to the same degree in PTX and intact rats. The rate of skeletal turnover is slowed down to half or less of its normal value in the PTX rats compared with intact animals (Jowsey et al., 1958; Lindgren, 1976). This might be the reason for the difference in the external diameter of the fourth lumbar vertebra between PTX and intact IMO rats. In intact IMO rats, the area of the cross section of the vertebral body including cortical bone was enlarged. On the contrary, there was no difference in cortical bone mass in PTX rats. In the intact group the marrow cavity was also enlarged, and in both groups cortical bone was thinner. The enlargement of the lumbar vertebra of intact rats resembles those seen in human osteoporosis and with increasing age (Lindahl and Lindgren, 1967). Nevertheless, it should be noted again that our experimental animals were still growing. Planimetric analysis was preferred to linear measurement on the cross section of vertebra because the latter is more subject to local variations in cortical thickness than is the former. Due to suppressed skeletal turnover in the absence of the parathyroids, the parathyroid glands are able to modify the rate of reaction of the bone, although they do not play a permissive role in the development of the IMO.
Acknowledgement: We wish to express appreciation to Mrs. Werner for her technical assistence. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, West Germany.
References Dinarello C.A.: lnterleukln 1 and the pathogenesis
of the acute-phase response. N.Engl.J.Med. 311:1413-1418, 1984. Frost H.M.: Bone Modeling and Skeletal Modeling Errors. Charles C. Thomas, Springfield, IL, 1973, pp. 73-74. Gasser A.B.. Morgan D.B.. Fleisch H.A. and Richelle L.J. The influence of
J. F’feilschifter et al.: Inflammation-mediated
osteopenia.
two diphosphonates on calcium metabolism in the rat. C/in..% 43:3145, 1972. Goldner J.: A modrfrcahon of the Masson trichrome technique for routme laboratory purpose. Am J.Pathol. 14:237-243, 1938. Jowsey J., Rowland R.E., Marshall J.H. and MC Lean F.C.: The effect of parathyroidectomy on haversian remodeling of bone. Endocrinology 63:903-908, 1958. Kemm J.R.: The effect of parathyroidectomy and large doses of cholecalciferol on the ability of rats to adapt to changes in dietary intake of calcium. J.Pbysiol. 256:103-l 15, 1976. Kenny A-D.: Survival and serum calcium levels of rats after parathyroidectomy. Endocrinology 70 715-722, 1962. Kimmel D.B. and Jee W.S.S : A quantitative histological analysis of the growing long bone metaphys,rcs. Calcif. Tisue Int. 32:113-122, 1980. Lindahl D. and Lrndgren A.G.M.: Cortical bone in man. Acta OfThop. Stand. 36:133-140, 1967. Lindgren J.U.: Studies of the calcium accretion rate of bone during immobilization in intact and parathyroidectomized rats. Calcif. Tissue Res. 22:41-47. 1976. Merz W.A.: Die Streckenmessung an gerichteten Sirukturen im Mrkroskop und ihre Anwendung zur Bestimmung von Oberflachen-Volumen-Relationen im Knochengewebe. Mikroskopie 22:132-142, 1967. Merz W.A. and Schenk R.K.: Quantitative structural analysis of human cancellous bone. Acta Anaf. 7554-66, 1970.
465
Minne H.W., Pfeilschifter J., Scharla S., Mutschelknauss S., Schwarz A., Krempien B. and Ziegler R.: Inflammation-mediated osteopenia in the rat: A new animal model for pathological loss of bone mass. Endocrinology 115:50-54. 1984. Miller S.C. and Jee W.S.S.: The comparative effects of dichloromethylene diphosphonate (CI,MDP) and ethane-1-hydroxy-I. I-diphosphonate (EHDP) on growth and modekng of the rat tibia. Calcif. Tissue Res. 23:207-214, 1977. Peng T.-C. and Garner SC.: Hypercalcitoninemia assocrated with return of serum calcium ioward normal in chronically parathyroidectomized rats. Endocrinology 104: 1624.1630, 1979. Pfeiischifter J., Scharla S., Bauss F.. Krempien B. and Minne H.W.: Nonspecific inflammation corrects PTX-dependent hypocalcemia and leads to inflammation-mediated osteopenia (IMO). Acta Endocrinol. 105 [Suppl. 264]:16. 1984. Schenk R.K.: Zur histologischen Verarbeitung von unentkalkten Knochen. Acta Anat. 60:3-19, 1965. Schenk R.K.. Merz W.A. and Mijller J.: A quantitative histological study on bone resorption in human cancellous Done. Acta Anaf 74:44-53, 1969.
Received: September 12, 7984 Revised: February 14, 1985 Accepted: May 24, 1985
Copyright
8756-3282185 $3.00 + .OO @ 1985 Pergamon Press Ltd.
Inflammation-Mediated Osteopenia in the Rat: The Effects of Artificial Granuloma and Sham Operation on Cortical and Trabecular Bone J. PFEILSCHIFTER,’
H.W. MINNE,’
E. ENZMANN,’
B. KREMPIEN,’
and R. ZIEGLER’
Qepartment of internal Medicine VI, Endocrinology, University of Heidelberg, D-6900 Heidelberg, 2/nstitufe of Pathology, University of Heidelberg, O-6900 Heidelberg, West Germany. Address for corresoondence and reprints: Dr. H.W. Minne, Department Luisenstr., 5, D-6990 Heidelberg, West Germany.
Recent studies have established that generalized loss of trabecular bone occurs in the growing rat following day-today inflammatory irritation for a period of 3 wk. We can now demonstrate that there are similar effects on bone in milder but more prolonged chronic inflammation (14 wk). Thus, there were significant decreases in trabecular bone mass as well as in cortical bone after weekly subcutaneous injections or implantations of nonspecific irritants. Osteopenia, induced by a single but extensive inflammatory lesion, remained apparent even 14 wk after induction. This indicates that inflammation-mediated osteopenia is at least incompletely reversible. A less pronounced but similar reduction of cortical and trabecular bone was observed in rats following sham operation. This might be of importance in all animal studies on bone metabolism that include surgical procedures. Cortical Bone-Serum
of Internal Medicine VI, Endocrinology,
University of Heidelberg,
investigated the effects on cortical bone in both intact and parathyroidectomized rats. IMO may be due to a generalized host reaction irrespective of the nature of inflammatory substances. As a consequence, even sham-operated animals might be affected. We therefore compared the bone mass of animals after implantation of foreign body granulomas with the bone mass of sham-operated and untreated rats.
Abstract
Key Words: Inflammation-Osteopenia-Trabecular
West Germany.
Material and Methods Female rats (strain Chbb Thorn, Thomae Co., Biberach; FRG) weighing 180 to 200 g at the beginning of the study were used. Some of the animals had been parathyroidectomized 3 to 4 wk prior to the experiments. Parathyroidectomy (PTX) was performed under light ether anesthesia by electrocoagulation. A decrease in total serum calcium below 6.5 mg/dl proved successful PTX. Rats were fed a standard rat chow (Altromin Co., Lage; FRG) and water ad libitum.
Bone-
Calcium Experimental
design
Fifteen PTX and fifteen intact rats were subcutaneously injected with a 1 ml saline suspension of 400 mg magnesium silicate weekly for a period of 14 wk. Thirty PTX and fifteen intact control rats received weekly injections of the same volume of saline. Pellets of sterile compressed cotton (diameter 0.8 cm) were subcutaneously implanted in PTX rats. In one group of 10 animals, 8 pellets were implanted simultaneously, and the rats were then kept for 14 wk without further treatment. In another group of 14 animals, implantation of one pellet was performed weekly for a period of 8 wk. The skin of the back was shaved and washed, and the pellets were inserted into the subcutaneous fascial space via a 1 cm incision after creation of a subcutaneous pocket by blunt dissection. The incision was closed with two metal clips. In a third group of PTX rats the same surgical procedure was performed but no pellets were implanted. Nine PTX rats without further treatment were used as controls. Blood samples were obtained from the orbital plexus under light ether anesthesia at different intervals. Serum calcium was determined by atomic absorption spectrophotometry. The rats were fasted overnight prior to the collection of the blood samples. After sacrifice the right femur, the left tibia, and fourth lumbar
Introduction Previous studies have demonstrated the occurrence of bone loss in rats following Inonspecific inflammation (Minne et al., 1984; Pfeilschifter et al., 1984). This inflammation-mediated osteopenia (IMO) developed independent of PTH and vitamin D metabolites. Elone rarefaction was generalized and appeared as a decrease of trabecular bone volume and bone ash weight. In parathyroidectomized rats bone loss was associated with significant increase in serum calcium. The effect of inflammation on bone metabolism by daily injections of magnesium silicate had been examined for a period of 2 to 3 wk. The present study was performed to determine whether more prolonged inflammatory irritation would lead to similar changes in bone mass and if there is any restitution of bol-te mass after inflammation-mediated bone loss in the rat. Previously, only the effects of inflammation on trabecular bone had been studied. We have now 461
J. Pfeilschifter et al.: Inflammation-mediated
462
vertebra were removed and cleaned from adherent soft tissue. The femur was rehydrated in distilled water in vacua for 24 h, weighed without removal from water (WUW) and then again in air (WIA) after blotting. The difference between WUW and WIA measured the bone volume according to Archimedes’ principle. The bone was then ashed at 600°C for 24 h and the ash weighed. In prior experiments concerning IMO, correlations between bone density and bone calcium content had been made. Both parameters correlated positively with a correlation coefficient of r = 0.92. Bone density was calculated as ash weight/bone volume. The proximalmost 1 cm of the tibia was cut off the shaft with a razor blade and embedded in methylmethacrylate without prior decalcification (Schenk, 1965). A longitudinal section was cut to the midsagittal plane with a precision bone saw, and from there sections of 3 pm thickness were prepared on a Jung Model K microtome (Jung Co., Heidelberg; FRG). The body of the embedded fourth lumbar vertebra was cut in half by a transverse section, and 3 pm sections were prepared. Sections were stained according to a modified Goldner method (Goldner, 1938; Schenk et al., 1969) and then subjected to quantitative histologic analysis. A method reliant on a Merz grid was employed for quantitative estimation of the trabecular bone (Merz, 1967; Merz and Schenk. 1970; Kimmel and Jee, 1980). A 1 mm band 1.75 mm distal to the epiphyseal cartilage was analyzed from cortical to cortical bone by point counting. Using a x 400 magnification, this area comprised about 90 to 100 fields. The percentage of trabecular bone was calculated as: Number of points over trabecular bone x 100 Total number of points The area of trabecular bone in the fourth lumbar vertebra was defined by two parallels tangential to the vertebral canal and perpendicular to the ventral bordering of the vertebral canal. Using a x 400 magnification, this area comprised about 60 to 80 fields. The cortical area of the fourth lumbar vertebra was determined either by point counting or by planimetry using a half-automated image analysis system (Videoplan; Kontron Elektronik, Eching; FRG). Cortical bone was expressed as percent of the total vetebral body. The area of the cross section of each vertebral body was measured and expressed as percent area of total vertebral area of control animals. Statistical analysis was performed using the Wilcoxon rank sum test, Q was determined to be 0.05 before the study. All data were collected using the single blind technique to reduce bias of interpretation. The data are expressed as the mean + the standard deviation (f SD).
osteopenia.
Results All groups
of animals weight steadily.
appeared
healthy
and
active
and
At the end of the experiment there was no significant difference in body weight between treated and untreated rats. Body weight of the magnesium silicateinjected rats was corrected by subtracting the weight of the injected material. Parathyroidectomy caused a slight retardation in growth of the rats. Liver and kidney appeared normal in both histology and fresh weight per body weight, but spleen was enlarged and showed activation of the lymphatic nodules in all animals treated with irritants once a week. The bone marrow of these animals was markedly hypercellular with an increase in mature neutrophils. gained
Trabecular bone and ash weight A significant decrease in mean femur ash weight per volume of bone occurred in PTX (7%) as well as in intact animals (9%) treated with magnesium silicate. Trabecular bone volume of the tibia and of the fourth lumbar vertebra was decreased in the same animals (Table I). Similar changes were found in PTX rats that underwent implantation of one pressed cotton pellet once a week. The amount of ash per volume of femur was reduced by 6%, the trabecular bone volume of the tibia by 29%, and trabecular bone volume of the fourth lumbar vertebra by 17%, as compared with these same parameters in the PTX controls. Similar changes were also found in rats that underwent the same surgical procedure but actually did not receive cotton pellets, i.e., in sham-operated animals. Again the trabecular bone volumes of the tibia and of the fourth lumbar vertebra were reduced, though to a lesser extent, whereas the amount of ash per volume of femur was not significantly changed (Table II). Bone rarefaction produced by single implantation of eight cotton pellets was seen even 14 wk after implantation. We found the trabecular bone volume in the tibia metaphysis decreased by 40% and a 10% less ash weight of the right femur. However, ash weight per volume of femur, as well as vertebral trabecular bone, failed to show any significant differences.
Table I. Effect of weeklv iniections of maonesium silicate or of single implantation of cotton pellets on bone parameters after 14 wk
Groups PTX (n=30) PTX-magnesium
Tibia Trabecular Bone (%)
Vertebra Trabecular Bone (%)
Vertebra Cortical Bone 1%)
639 (i-30)
18.8 (k5.8)
15.1 (f3.2)
25.9 (1t3.6)
102.3 (+9.9)
99.9 (+9.5)
Femur Ash Weight/ Volume (pg/mm?
Vertebra Area of Cross Section (Int.= 100%)
Vertebra Marrow Cavity (Int. = iOO%p
593 (f30)”
10.9 (f4.1)’
11.4 (f2.3)”
21.8 (k3.3)”
101.3 (*3.4)
104.5 (k9.5)
silicate (n = 15) PTX-cotton
618 (f27)
10.2 (f4.0)”
12.7 (f2.3)
23.7 (f 1.3)'
100.5 (f9.7)
101.3 (+10.4)
pellets (n = 10) Intact
637 (+40)
20.8 (f4.9)
17.9 (f3.8)
24.3 (k3.6)
100.0 (i9.0)
100.0 (19.7)
582 (f27)b
14.5 (k3.8)
13.4 (+_2.5)b
21.3 (f2.1)b
111.4 (+12.4)b
115.2 (+14.9)b
(n=l5) Intact magnesium silicate (n = 15)
Data expressed as mean + SD. aSignificantly different from PTX control. bSignificantly different from intact control (P Clnt. = Intact (control) animals’ values.
< 0.05).
b
J. Pfeilschifter et al.: Inflammation-mediated
Table II. Effect of weekly implantation
of pressed cotton pellets or of sham operation on bone parameters
Femur Ash weight/ Volume Groups PTX (n=9) PTX sham op. (n= 14) PTX cotton pellets (n = 14)
463
osteopenia.
Tibia Trabecular Bone (%)
(I*g/mm?
Vertebra Trabecular Bone (%)
Vertebra Cortical Bone (%)
after 8 wk.
Vertebra Area of Cross Section (PTX= 100%)
Vertebra Marrow Cavity (PTX= 100%)
634 (+39)
30.3 (k6.2)
18.3 (k2.2)
26.4 (f3.9)
100.0 (k7.5)
100.0 (*9.4)
607 (+30)
24.3 (f4.8)a
16.4 (f2.8)a
23.1 (&4.4)a
100.0 (k7.4)
106.0 (k6.2)
595 (&38)a
21.4 (k6.5)”
15.1 (k2.8)”
21.3 (+3.2)a
105.7 (f7.5)
115.6 (k9.4)”
Data expressed as mean * SD. “Significantly different from PTX control (P ~0.05)
Cortical bone Measurements of the cortical area of cross sections from the fourth lumbar vertebra clearly demonstrate the differ-
ences in cortical bone between untreated and IMO rats at the end of both experiments. The data are presented as percent cortical bone area of the vertebral body in relation to the total area of vertebral body (Tables I and II). In animals that were subcutaneously injected with magnesium silicate, the cortical area decreased by 16% in PTX and 13% in intact animals when compared with PTX or intact controls. PTX rats that received one cotton pellet once a week showed a similar decrease of 19%. Sham-operated animals at least had a 12% less cortical area at the end of the study. The cortical bone of those PTX animals that underwent single implantation of eight pellets was significantly decreased by 9% even after a period of 14 wk. Marrow cavity and total bone area Whereas the marrow cavity was found enlarged in IMO PTX rats and in IMO intact rats, only the latter had an enlarged total area of the vertebral body. In all PTX groups no significant changes in total area of body were found as compared with untreated controls. Serum calcium One month after parathyroidectomy concentration was 5 1:; 6 mg/dl.
athyroidectomized
table
the mean serum calcium
in animals that were parand then subcutaneously injected with
magnesium silicate once a week, serum calcium began to rise after two injections and thereafter was only slightly elevated as compared with PTX controls. However serum calcium of PTX controls did rise from 5 to 8 mg/dl during the experiment. By 14 wk the serum calcium levels of IMO rats could not be distinguished statistically from those found in untreated PTX animals (Table III). In those animals that were parathyroidectomized and then received a single subcutaneous implantation of cotton pellets, an initial rise of serum calcium to 7.6 mg/dl occurred within the first 2 wk. However, by 4 wk the serum calcium values of these rats were low again and remained so for the remainder of the experiment. In intact rats treated with magnesium silicate, serum calcium concentration was slightly decreased throughout the experiment.
Discussion Cortical and trabecular bone of the growing rat undergoes extensive modeling by continued bone formation and bone resorption. Thus, the growing rat bone offers an excellent opportunity for the short-term study of various conditions thought to influence bone formation or bone resorption. For the same reasons it may be difficult to draw conclusions about long-term effects from short-term experiments, especially since bone formation and bone resorption may be
Ill. Effect of weekly injections of magnesium silicate or of single implantation of cotton pellets on serum calcium concentration. Groups
Serum Calcium in mg/dl Day 0
PTX (n=30) PTX magnesium silicate (n=15) PTX cotton pellets (n=lO) Intact (n = 15) Intact magnesium silicate (n = 15)
14
28
55
100
4.8 (kO.9) 4.6 (10.6)
6.2 (kO.7) 6.7 (*I 0)”
6.1 (k1.0) 6.8 (*1.2)
7.1 (fO.9) 8.5 (f0.7)a
7.9 (f0.8) 8.0 (kO.9)
4.5 (L-0.8)
7.6 (f0.9)a
6.4 (kO.7)
6.7 (k1.0)
7.9 (*0.7)
9.6 (f0.4) 9.6 (f0.3)
9.6 (L-0.7) 9.6 (f0.2)
10.0 (+0.3) 9.6 (+0.2)b
10.0 (f0.5) 9.8 (kO.6)
10.1 (kO.4) 9.7 (&0.3)b
Data expressed as mean + SD. aSignificantly different from PTX control. bSignificantly different from intact control (P < 0.05).
464
mechanistically linked in the growing skeleton (Gasser et al., 1972; Miller and Jee, 1977). Previous studies have demonstrated a dramatic loss of trabecular bone in growing rats following daily injections of nonspecific irritants for a period of 3 wk. One aim of this study was to find out whether the growing rat is able to compensate for bone loss in milder, more prolonged inflammation In addition, we examined whether bone loss after 3 wk of daily inflammatory stimulus is completely reversible. Our data suggest that prolonged inflammation is a powerful stimulus of bone loss and that bone rarefaction induced by a single inflammatory lesion remains apparent even after 14 wk. If there is any reversibility in the volume of cortical and trabecular bone, it is only minor. The bone of the growing rat is quite different from the bone of an adult human (Frost, 1973). Therefore, our results may not be representative of the effects of inflammation on the remodeling of human adult bone. Nevertheless, the experiments described may serve as a new model for long-term or even lasting development of bone loss in a biologic system. Rats that were injected weekly with magnesium silicate for a period of 14 wk showed bone loss similar to the rats that received daily injections for a period of 3 wk. This indicates that each inflammatory stimulus might have a cumulative effect on bone. Up to now, we have little information about the minimal inflammatory intensity of a single stimulus needed to influence bone volume. The effects appeared to be relatively independent of whether the changes were produced by magnesium silicate or subcutaneous implantation of cotton pellets. Animals that underwent the same surgical procedure with no implantation of pellets had milder but similar changes in cortical and trabecular bone. We conclude therefore that the foreign body material is not the main stimulus necessary to induce IMO. There were no visible signs of local wound infection, and all operated animals appeared healthy. However, bone loss was associated with enlargement of the spleen and a marked proliferation of bone marrow. Therefore, surgical trauma as well as bacterial contamination may have contributed to the oberved loss of bone in sham-operated animals. Irrespective of the nature of the inciting stimulus, nearly all infections, injuries, and inflammatory processes stimulate mononuclear cell phagocytes to synthesize and release interleukin 1 (Dinarello, 1984). Interleukin 1, therefore, may play an important role in bone loss associated with inflammation. In animal experiments on bone metabolism, particular attention should be paid to all surgical procedures, since the operation itself may have some effect on calcium and bone parameters. Daily injections of magnesium silicate in PTX rats were followed by a return of serum calcium toward subnormal levels (Minne et al., 1984). This study demonstrates that a powerful single stimulus of inflammatory irritation can raise serum calcium concentration in PTX rats toward a subnormal level 2 wk after the onset of the inflammation. However, 2 wk later there is a return of serum calcium toward preinflammatory levels. Thereafter, serum calcium remained unchanged. Weekly injections of magnesium silicate resulted in slightly but significant changes of serum calcium levels as compared with controls. In PTX animals, serum calcium increased to the upper normal range or slightly above the serum calcium of untreated PTX rats. However, we also
J. Pfeilschifier
et al.: Inflammation-mediated
osteopenia.
observed a continuous increase in serum calcium of untreated PTX rats. Calcium had increased from 4.8 to 7.9 mg/dl 14 wk after parathyroidectomy. It is unlikely that the return of serum calcium toward normal levels was due to compensatory function of parathyroid remnants after incomplete parathyroidectomy. Our results are consistent with those of other authors (Kenny, 1962; Kemm, 1976; Peng and Garner, 1979), who also reported a return of serum calcium toward normal in PTX rats. According to Peng and Garner (1979), this phenomenon is associated with an increase of serum calcitonin. Rats with intact parathyroid glands tended to have slightly but significantly lower serum calcium levels after weekly injections of magnesium silicate. These surprising data indicate that the elevated calcium levels of IMO PTX rats are not necessarily due to increased calcium mobilization from bone. Recent studies of the biphosphonate APD (Pfeilschifter, unpublished data) demonstrated that APD is completely able to inhibit inflammation-mediated bone loss without inhibiting the rise of serum calcium in PTX rats. Inflammation may, at least to some extent, raise serum calcium concentration by decreasing urinary excretion of calcium or by increasing the gastrointestinal absorption of calcium. Further work is needed to define the mode of action of inflammation on calcium homeostasis. The present results provide further confirmation that IMO occurs to the same degree in PTX and intact rats. The rate of skeletal turnover is slowed down to half or less of its normal value in the PTX rats compared with intact animals (Jowsey et al., 1958; Lindgren, 1976). This might be the reason for the difference in the external diameter of the fourth lumbar vertebra between PTX and intact IMO rats. In intact IMO rats, the area of the cross section of the vertebral body including cortical bone was enlarged. On the contrary, there was no difference in cortical bone mass in PTX rats. In the intact group the marrow cavity was also enlarged, and in both groups cortical bone was thinner. The enlargement of the lumbar vertebra of intact rats resembles those seen in human osteoporosis and with increasing age (Lindahl and Lindgren, 1967). Nevertheless, it should be noted again that our experimental animals were still growing. Planimetric analysis was preferred to linear measurement on the cross section of vertebra because the latter is more subject to local variations in cortical thickness than is the former. Due to suppressed skeletal turnover in the absence of the parathyroids, the parathyroid glands are able to modify the rate of reaction of the bone, although they do not play a permissive role in the development of the IMO.
Acknowledgement: We wish to express appreciation to Mrs. Werner for her technical assistence. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, West Germany.
References Dinarello C.A.: lnterleukln 1 and the pathogenesis
of the acute-phase response. N.Engl.J.Med. 311:1413-1418, 1984. Frost H.M.: Bone Modeling and Skeletal Modeling Errors. Charles C. Thomas, Springfield, IL, 1973, pp. 73-74. Gasser A.B.. Morgan D.B.. Fleisch H.A. and Richelle L.J. The influence of
J. F’feilschifter et al.: Inflammation-mediated
osteopenia.
two diphosphonates on calcium metabolism in the rat. C/in..% 43:3145, 1972. Goldner J.: A modrfrcahon of the Masson trichrome technique for routme laboratory purpose. Am J.Pathol. 14:237-243, 1938. Jowsey J., Rowland R.E., Marshall J.H. and MC Lean F.C.: The effect of parathyroidectomy on haversian remodeling of bone. Endocrinology 63:903-908, 1958. Kemm J.R.: The effect of parathyroidectomy and large doses of cholecalciferol on the ability of rats to adapt to changes in dietary intake of calcium. J.Pbysiol. 256:103-l 15, 1976. Kenny A-D.: Survival and serum calcium levels of rats after parathyroidectomy. Endocrinology 70 715-722, 1962. Kimmel D.B. and Jee W.S.S : A quantitative histological analysis of the growing long bone metaphys,rcs. Calcif. Tisue Int. 32:113-122, 1980. Lindahl D. and Lrndgren A.G.M.: Cortical bone in man. Acta OfThop. Stand. 36:133-140, 1967. Lindgren J.U.: Studies of the calcium accretion rate of bone during immobilization in intact and parathyroidectomized rats. Calcif. Tissue Res. 22:41-47. 1976. Merz W.A.: Die Streckenmessung an gerichteten Sirukturen im Mrkroskop und ihre Anwendung zur Bestimmung von Oberflachen-Volumen-Relationen im Knochengewebe. Mikroskopie 22:132-142, 1967. Merz W.A. and Schenk R.K.: Quantitative structural analysis of human cancellous bone. Acta Anaf. 7554-66, 1970.
465
Minne H.W., Pfeilschifter J., Scharla S., Mutschelknauss S., Schwarz A., Krempien B. and Ziegler R.: Inflammation-mediated osteopenia in the rat: A new animal model for pathological loss of bone mass. Endocrinology 115:50-54. 1984. Miller S.C. and Jee W.S.S.: The comparative effects of dichloromethylene diphosphonate (CI,MDP) and ethane-1-hydroxy-I. I-diphosphonate (EHDP) on growth and modekng of the rat tibia. Calcif. Tissue Res. 23:207-214, 1977. Peng T.-C. and Garner SC.: Hypercalcitoninemia assocrated with return of serum calcium ioward normal in chronically parathyroidectomized rats. Endocrinology 104: 1624.1630, 1979. Pfeiischifter J., Scharla S., Bauss F.. Krempien B. and Minne H.W.: Nonspecific inflammation corrects PTX-dependent hypocalcemia and leads to inflammation-mediated osteopenia (IMO). Acta Endocrinol. 105 [Suppl. 264]:16. 1984. Schenk R.K.: Zur histologischen Verarbeitung von unentkalkten Knochen. Acta Anat. 60:3-19, 1965. Schenk R.K.. Merz W.A. and Mijller J.: A quantitative histological study on bone resorption in human cancellous Done. Acta Anaf 74:44-53, 1969.
Received: September 12, 7984 Revised: February 14, 1985 Accepted: May 24, 1985