J. BIOMED. MATER. RES.
VOL. 10, PP. 733-741 (1976)
In vitro Effects of Bone Cement on S. aureus
Growth and Phagocytosis and Glucose Metabolism of Macrophage: A Preliminary Study HIROMU SHOJI, Department of Orthopaedic Surgery, School of Medicine, University of California, Davis, California 96616 , and JOSEPH F. NICASTRO, GEORGE D. ROVERE, and ANTHONY G. GRISTINA, Section of Orthopaedic Surgery, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 271 03 '
Summary A preliminary study of the effects of Surgical Simplex, mixed for various time periods, was performed on S. aureus growth rate, and phagocytosis and glucose metabolism of macrophage in vitro. Quantitatively, no significant effects of the mixture on S. aureus growth rate were observed. The cement mixed for less than 4 min tended to cause adverse effects on phagocytosis rate and hexose monophosphate shunt activity of macrophage. This information may not be directly applicable to in viuo, but in vitro the cement mixture in a certain situation can cause decreased phagocytosis and hexose monophosphate shunt activity of macrophage.
INTRODUCTION Total joint replacement in the treatment of arthritis has resulted in the increased use of methyl methacrylate (MM), and its effects in the biologic tissue have become a growing concern to orthopedic surgeons. Although the systemic effects of MM have been fairly well investigated,3 relatively little information is available regarding its biologic effects at the cellular level. The qualitative evidence of bacterial growth inhibition by MM has recently been shown.2 The so-called bone cement used in orthopedic surgery is provided in powder (polymerized MM and benzoyl peroxide) and liquid (MM monomer and aromatic tertiary amine) forms. Polymerization 733 @ 1976 by John Wiley & Sons, Inc.
734
SHOJI ET AL.
of MM monomer is achieved through the reaction between benzoyl peroxide and aromatic tertiary amine, and Surgical Simplex is one of the bone cements employing this system. Since S. aureus is the most common devastating organism in early postoperative infections in general, and macrophage plays a great role in the defense mechanisms in the tissue in chronic or late infection, in this preliminary study we have attempted to: 1) measure quantitatively the growth rate of S. aureus, 2) investigate the phagocytosis rate of macrophage, and 3) study the glucose metabolism of macrophage in vitro under the influence of MM a t its various curing stages, using Surgical Simplex.
MATERIALS AND METHODS Methyl methacrylate of various curing stages was prepared by mixing the Surgical Simplex. Eight units (1 unit: one pack of powder and one ampoule of liquid) of Surgical Simplex were mixed for 1, 2, 3, 4, 6, 8, 10, and 12 min a t room temperature (18OC). Each of the eight mixtures was placed into an Erlenmeyer flask containing 500 ml of TSB (treated TSB) (Trypticase Soy Broth, obtained from Baltimore Biological Laboratory Company, Cockeysville, Maryland.) The same process was performed on another eight units of Surgical Simplex, and these mixtures were placed into Erlenmeyer flasks containing 250 ml of MEM media (treated MEM) (MEM Eagle w/o Earle's Base obtained from Baltimore Biological Laboratory Company, Cockeysville, Maryland). The Surgical Simplex, which was to be mixed for long periods of time, tended to harden before the mixing time had lapsed. In such a situation, mixing was stopped and the mixture was left alone until the time expired. It was then placed into an Erlenmeyer flask. The top of each Erlenmeyer flask was air-tightly sealed with a rubber stopper and the flask stored for more than a month (1 month-6 weeks) in the refrigerator until used. Immediately before the experiment, the treated media were filtrated through a 0.2 p Millipore filter.
Bacterial Growth Quantitation S. aureus from a blood agar plate was innoculated into 10 ml of TSB and incubated for 24 hr a t 37°C. Six-tenths milliliter of the innoculated TSB was added to 60 ml of each treated TSB, which was then incubated for 24 hr. The sequential turbidity was read a t
I N VITRO EFFECTS OF BONE CEMENT ON MACROPHAGE 735
540 X at 2, 4, 7, and 24 hr incubation. Five experiments were performed for each category (control and each treated TSB group categorized by the time period of mixing).
Macrophage Phagocytosis Rate Macrophage was obtained from rabbit’s lung.6 Macrophage was placed in 5 ml of treated MEM containing 10% calf serum, and a cell count of macrophage was performed. Lyophilized BCG was weighed (7 X lo9 BCG/mg), placed in treated MEM, and sonicated for 10 min. Macrophage and BCG aliquots were mixed. The macrophage: BCG ratio was made 1: 12. The mixture was then incubated for 1 hr at 37°C on a rotating wheel. After Wright and Ziehl-Neelsen stains, the phagocytosis rate.was calculated by microscopy. Viability of macrophage was also tested by using tripan blue, and the results were corrected. Five experiments were performed for each category.
Macrophage Glucose Metabolism Macrophage was placed in treated MEM containing 10% calf serum. Cell count and viability tests were performed. Consumption rate of glucose, labeled a t either 1- or 6-position (G-1-CI4 or G-6-C14), was determined by a modified method of Holmes.6 Twotenths microcurie of G-l-Cl4 and G-6-C14 was used for 1.5 X lo6 cells. The incubation was performed for 1 hr at 37°C. The released 14C02 was collected in 0.5 ml of hyamine hydroxide and counted in a liquidscintillation spectrometer. Zymosan, suspended t o an optical density of 1.00 in phosphate buffer saline at 525 X, was used to determine the glucose metabolism a t the phagocytic state. Five experiments were performed for each category. 14C02measurement was done in duplicate in each sample. In all the aforementioned experiments, untreated MEM or TSB served as a control. Statistical analysis was done using Student’s t-test, comparing the control with each experimental group categorized by the time period of mixing.
RESULTS S. aureus Growth Quantitation The results of sequential turbidity read at 540 X were as follows. In the control, the results of the reading were 0.02 f 0.006 (average
736
SHOJI ET AL.
f l S.D.), 0.25 f 0.07, 2.4 f 0.97, and 11.3 f 3.11 a t the incubation period of 2, 4, 7, and 24 hr, respectively. In the experimental samples (innoculated TSBs treated with Surgical Simplex of various times of mixing), the average value of each experimental group categorized by the time of mixing were in the range of 0.01-0.03 a t 2 hr incubation, 0.24-0.28 a t 4 hr incubation, and 10.9-13.5 at 24 hr incubation. The statistical comparison by Student’s l-test between each experimental group at these time intervals of incubation and the control at corresponding time intervals did not yield any significance.
Macrophage Phagocytosis Rate The results of the macrophage phagocytosis rate on lyophilized BCG are shown in Figure 1. The slight decrease in the macrophage phagocytosis rate was noted in the MEM samples treated with the cement which had been mixed for less than 4 min, although statistical differences were not noted except for in the MEM treated with the cement mixed for 3 min (p < 0.05).
Macrophage Glucose Metabolism The significant decrease of G-1-CI4 consumption rate of macrophage was noted in the MEM samples treated with the cement mixed for less than 4 min, both in resting and phagocytic states (p < 0.05) (Figs. 2a and 2b). There was no significant change noted in the G-6-CI4 consumption rate in either state (Figs. 3a and 3b) In the phagocytic state, the consumption of both G-1-CI4 and G-6-C14 was increased. The G-1-CI4/G-6-Cl4consumption rate was
Fig. 1. Macrophage phagocytosis rate on lyophiliaed BCG under the influence of cement mixed for various time intervals. ( 0 )control; ( 0 )samples.
4
(a)
6
8
10
12 minutes
-
~
I
-
4
-: :qbl
600-
700
800
I,
900-
(b)
6
8
10
12 minutes
I S.D
-}
Fig. 2 . (resting state of macrophage): (0) control; ( 0 )samples. (b) G-1-Cu consumption rate of macrophage under the influence control; ( 0 )samples. of cement mixed for various time intervals (phagocytic state of macrophage): (0)
(a) G-l-Cu consumption rate of macrophage under the influence of cement mixed for various time intervals
I
“11
5001
cpm/l 5x106eelk
*
-4
-J W
8
*
8x
P
Q
SHOJI ET AL.
738 cpm/l.5x106cells
t
300
200 -
I
4
6
8
10
12 minutes
8
10
12 minutes
(a)
cpm/i 5~106ce11s
t
300
L
A
I
4
6
(b)
Fig. 3. (a) G-6-C1, consumption rate of macrophage under the influence of cement mixed for various time intervals (resting state of macrophage): (0)control; ( 0 )samples. (b) G - 6 - G consumption rate of macrophage under the influence of cement mixed for various time intervals (phagocytic state of macrophage); (0) control; ( 0 )samples.
I N VITRO EFFECTS O F BONE CEMENT ON MACROPHAGE 739
3.2 in the resting state and 4.2 in the phagocytic state in the control (Fig. 4). In the MEM samples treated with the cement of various curing stages, the rate tended to remain lower in both resting and phagocytic states, and its decrease was prominent in those treated with the cement mixed for less than 4 min.
DISCUSSION The results of the present study showed that 1) Surgical Simplex mixture did not inhibit or stimulate the growth rate of S. aweus quantitatively; 2) Surgical Simplex mixed for less than 4 min tended to induce a slight decrease of phagocytosis rate of macrophage on BCG; 3) Surgical Simplex mixed for less than 4 min caused a significant decrease of G-l-Cl4 consumption in macrophages both in the resting and phagocytic phase, whereas G-6-C14 consumption rate was not affected by Surgical Simplex mixture; and 4) The increased consumption rate of G-1-G and G-6-G in macrophages in the phagocytic phase was noted, when compared with resting phase. Before commenting on the significance of the results of the present study, however, it is important to mention some of the drawbacks attached to this experimental design which may pose some limitation on the validity of the results. The systemic toxic effects of MM monomer is fairly well known, but the side effects of the cement additives have also been reported.3 The liquid part of Surgical
-
2-
I-
minutes
Fig. 4. The G-1-C14/G-6-C14consumption rates. Resting state: (--) control; samples; phagocytic state: (-) control; (.-.) samples. (.--a)
740
SHOJI ET AL.
Simplex does not readily mix with culture media, and a significant amount of MM monomer is dissipated into the air during the time of mixing the cement. Although it has been said that most of the MM monomer applied in vivo is released into the system and is dissipated from the lungs before the mixed mass hardens13there is no study indicating the exact amount of MM monomer remaining in the local tissue where the cement was applied. In view of these, in this preliminary study we adopted the system in which the in vitro effects of Surgical Simplex, mixed for various periods of time, was investigated, rather than those of individual ingredients of the cement, since we also felt that as an initial study it may also bear practical significance to study the effects of whole cement mixture. The cement, mixed for various time intervals, was air-tightly stored in the culture media at 4°C for a minimum arbitrary period of a month. Although we assume that in this system MM monomer concentration would be different in the culture media depending on various times of mixing performed at 18"C, it cannot be certain. Despite a demonstration of inhibition zone by the cement on S. aureus on the blood agar plate,2 we have failed to show inhibitory effects of the cement on S. aureus growth rate quantitatively. The exact reason for this discrepancy is unexplainable, but the inhibitory effects of cement on S. aureus growth do not appear so significant to be detectable quantitatively. Although, in general, the most common devastating organism in early postoperative infections is S. aureus, in late infections of total joint replacement, organisms with low-grade virulence leading chronic infection are the most c ~ m m o n . Late ~ infection frequently is a cause of loosening of the prosthesis. In view of this, in part of the leucocyte study, macrophage was selected rather than neutrophil, which is predominant in acute infection. BCG was used because its staining character simplifies the counting of phagocytosis rate and because the tuberculous nature induces chronic granulomatous lesion, in which macrophage plays a significant role. Under the influence of cement mixed for less than 4 min, the phagocytosis rate of macrophage tended to be decreased. As noted in the change of G-1-C14 and G-6-C14 consumption rates, hexose monophosphate shunt activity of macrophage was decreased under the influence of the cement mixture of less than 4 min in both resting and phagocytic states. The increased hexose monophosphate shunt activity during the phagocytic state? com-
I N VITRO EFFECTS OF BONE CEMENT ON MACROPHAGE 741
pared with the resting state was observed, regardless of influence by the cement mixture. Whether the increased hexose monophosphate shunt activity causes' or results from4 phagocytosis is still undetermined. As mentioned previously in the experimental system of this preliminary study, it is not known whether the adverse effects of phagocytosis rate and hexose monophosphate shunt activity of macrophage are due to MM monomer, MM polymer, additives, or combinations of these. It may seem reasonable to assume that MM monomer is likely to be most responsible, but further study is needed for a definite conclusion. However, we can state that Surgical Simplex mixture, in a certain situation, can cause decreased phagocytosis and decreased hexose monophosphate shunt activity of macrophage in vitro.
References 1. R. L. Bachner and M. L. Karnovsky, Science, 162, 1277 (1968). 2. M. W. Chapman and W. K. Hadley, J . Bone Joint Surg., 56A,846 (1974). 3. J. Charnley, in Acrylic Cement in Orthopaedic Surgery, J. Charnley, Ed., E. and S. Livingston, Ltd., Edinburgh and London, 1970, p. 72. 4. L. R. DeChatelet, R. M. Cooper, and C. E. McCall, Inf. Immun., 3,66 (1971). 5 . R. H. Fitzgerald, Jr., L. F. A. Peterson, J. A. Washington, 11, R. E. Van Scoy, and M. B. Coventry, J . Bone Joint Surg., 55A, 1242, (1973). 6. B. Holmes, P. G. Quie, D. B. Windhorst, and R. A. Good, Lancet, 1, 1225 (1966). 7. Q. N. Myrvik and D. G. Evans, Arch. Environ. Health, 14, 92 (1967).
Received October 1, 1975 Revised November 18, 1975
VOL. 10, PP. 733-741 (1976)
In vitro Effects of Bone Cement on S. aureus
Growth and Phagocytosis and Glucose Metabolism of Macrophage: A Preliminary Study HIROMU SHOJI, Department of Orthopaedic Surgery, School of Medicine, University of California, Davis, California 96616 , and JOSEPH F. NICASTRO, GEORGE D. ROVERE, and ANTHONY G. GRISTINA, Section of Orthopaedic Surgery, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 271 03 '
Summary A preliminary study of the effects of Surgical Simplex, mixed for various time periods, was performed on S. aureus growth rate, and phagocytosis and glucose metabolism of macrophage in vitro. Quantitatively, no significant effects of the mixture on S. aureus growth rate were observed. The cement mixed for less than 4 min tended to cause adverse effects on phagocytosis rate and hexose monophosphate shunt activity of macrophage. This information may not be directly applicable to in viuo, but in vitro the cement mixture in a certain situation can cause decreased phagocytosis and hexose monophosphate shunt activity of macrophage.
INTRODUCTION Total joint replacement in the treatment of arthritis has resulted in the increased use of methyl methacrylate (MM), and its effects in the biologic tissue have become a growing concern to orthopedic surgeons. Although the systemic effects of MM have been fairly well investigated,3 relatively little information is available regarding its biologic effects at the cellular level. The qualitative evidence of bacterial growth inhibition by MM has recently been shown.2 The so-called bone cement used in orthopedic surgery is provided in powder (polymerized MM and benzoyl peroxide) and liquid (MM monomer and aromatic tertiary amine) forms. Polymerization 733 @ 1976 by John Wiley & Sons, Inc.
734
SHOJI ET AL.
of MM monomer is achieved through the reaction between benzoyl peroxide and aromatic tertiary amine, and Surgical Simplex is one of the bone cements employing this system. Since S. aureus is the most common devastating organism in early postoperative infections in general, and macrophage plays a great role in the defense mechanisms in the tissue in chronic or late infection, in this preliminary study we have attempted to: 1) measure quantitatively the growth rate of S. aureus, 2) investigate the phagocytosis rate of macrophage, and 3) study the glucose metabolism of macrophage in vitro under the influence of MM a t its various curing stages, using Surgical Simplex.
MATERIALS AND METHODS Methyl methacrylate of various curing stages was prepared by mixing the Surgical Simplex. Eight units (1 unit: one pack of powder and one ampoule of liquid) of Surgical Simplex were mixed for 1, 2, 3, 4, 6, 8, 10, and 12 min a t room temperature (18OC). Each of the eight mixtures was placed into an Erlenmeyer flask containing 500 ml of TSB (treated TSB) (Trypticase Soy Broth, obtained from Baltimore Biological Laboratory Company, Cockeysville, Maryland.) The same process was performed on another eight units of Surgical Simplex, and these mixtures were placed into Erlenmeyer flasks containing 250 ml of MEM media (treated MEM) (MEM Eagle w/o Earle's Base obtained from Baltimore Biological Laboratory Company, Cockeysville, Maryland). The Surgical Simplex, which was to be mixed for long periods of time, tended to harden before the mixing time had lapsed. In such a situation, mixing was stopped and the mixture was left alone until the time expired. It was then placed into an Erlenmeyer flask. The top of each Erlenmeyer flask was air-tightly sealed with a rubber stopper and the flask stored for more than a month (1 month-6 weeks) in the refrigerator until used. Immediately before the experiment, the treated media were filtrated through a 0.2 p Millipore filter.
Bacterial Growth Quantitation S. aureus from a blood agar plate was innoculated into 10 ml of TSB and incubated for 24 hr a t 37°C. Six-tenths milliliter of the innoculated TSB was added to 60 ml of each treated TSB, which was then incubated for 24 hr. The sequential turbidity was read a t
I N VITRO EFFECTS OF BONE CEMENT ON MACROPHAGE 735
540 X at 2, 4, 7, and 24 hr incubation. Five experiments were performed for each category (control and each treated TSB group categorized by the time period of mixing).
Macrophage Phagocytosis Rate Macrophage was obtained from rabbit’s lung.6 Macrophage was placed in 5 ml of treated MEM containing 10% calf serum, and a cell count of macrophage was performed. Lyophilized BCG was weighed (7 X lo9 BCG/mg), placed in treated MEM, and sonicated for 10 min. Macrophage and BCG aliquots were mixed. The macrophage: BCG ratio was made 1: 12. The mixture was then incubated for 1 hr at 37°C on a rotating wheel. After Wright and Ziehl-Neelsen stains, the phagocytosis rate.was calculated by microscopy. Viability of macrophage was also tested by using tripan blue, and the results were corrected. Five experiments were performed for each category.
Macrophage Glucose Metabolism Macrophage was placed in treated MEM containing 10% calf serum. Cell count and viability tests were performed. Consumption rate of glucose, labeled a t either 1- or 6-position (G-1-CI4 or G-6-C14), was determined by a modified method of Holmes.6 Twotenths microcurie of G-l-Cl4 and G-6-C14 was used for 1.5 X lo6 cells. The incubation was performed for 1 hr at 37°C. The released 14C02 was collected in 0.5 ml of hyamine hydroxide and counted in a liquidscintillation spectrometer. Zymosan, suspended t o an optical density of 1.00 in phosphate buffer saline at 525 X, was used to determine the glucose metabolism a t the phagocytic state. Five experiments were performed for each category. 14C02measurement was done in duplicate in each sample. In all the aforementioned experiments, untreated MEM or TSB served as a control. Statistical analysis was done using Student’s t-test, comparing the control with each experimental group categorized by the time period of mixing.
RESULTS S. aureus Growth Quantitation The results of sequential turbidity read at 540 X were as follows. In the control, the results of the reading were 0.02 f 0.006 (average
736
SHOJI ET AL.
f l S.D.), 0.25 f 0.07, 2.4 f 0.97, and 11.3 f 3.11 a t the incubation period of 2, 4, 7, and 24 hr, respectively. In the experimental samples (innoculated TSBs treated with Surgical Simplex of various times of mixing), the average value of each experimental group categorized by the time of mixing were in the range of 0.01-0.03 a t 2 hr incubation, 0.24-0.28 a t 4 hr incubation, and 10.9-13.5 at 24 hr incubation. The statistical comparison by Student’s l-test between each experimental group at these time intervals of incubation and the control at corresponding time intervals did not yield any significance.
Macrophage Phagocytosis Rate The results of the macrophage phagocytosis rate on lyophilized BCG are shown in Figure 1. The slight decrease in the macrophage phagocytosis rate was noted in the MEM samples treated with the cement which had been mixed for less than 4 min, although statistical differences were not noted except for in the MEM treated with the cement mixed for 3 min (p < 0.05).
Macrophage Glucose Metabolism The significant decrease of G-1-CI4 consumption rate of macrophage was noted in the MEM samples treated with the cement mixed for less than 4 min, both in resting and phagocytic states (p < 0.05) (Figs. 2a and 2b). There was no significant change noted in the G-6-CI4 consumption rate in either state (Figs. 3a and 3b) In the phagocytic state, the consumption of both G-1-CI4 and G-6-C14 was increased. The G-1-CI4/G-6-Cl4consumption rate was
Fig. 1. Macrophage phagocytosis rate on lyophiliaed BCG under the influence of cement mixed for various time intervals. ( 0 )control; ( 0 )samples.
4
(a)
6
8
10
12 minutes
-
~
I
-
4
-: :qbl
600-
700
800
I,
900-
(b)
6
8
10
12 minutes
I S.D
-}
Fig. 2 . (resting state of macrophage): (0) control; ( 0 )samples. (b) G-1-Cu consumption rate of macrophage under the influence control; ( 0 )samples. of cement mixed for various time intervals (phagocytic state of macrophage): (0)
(a) G-l-Cu consumption rate of macrophage under the influence of cement mixed for various time intervals
I
“11
5001
cpm/l 5x106eelk
*
-4
-J W
8
*
8x
P
Q
SHOJI ET AL.
738 cpm/l.5x106cells
t
300
200 -
I
4
6
8
10
12 minutes
8
10
12 minutes
(a)
cpm/i 5~106ce11s
t
300
L
A
I
4
6
(b)
Fig. 3. (a) G-6-C1, consumption rate of macrophage under the influence of cement mixed for various time intervals (resting state of macrophage): (0)control; ( 0 )samples. (b) G - 6 - G consumption rate of macrophage under the influence of cement mixed for various time intervals (phagocytic state of macrophage); (0) control; ( 0 )samples.
I N VITRO EFFECTS O F BONE CEMENT ON MACROPHAGE 739
3.2 in the resting state and 4.2 in the phagocytic state in the control (Fig. 4). In the MEM samples treated with the cement of various curing stages, the rate tended to remain lower in both resting and phagocytic states, and its decrease was prominent in those treated with the cement mixed for less than 4 min.
DISCUSSION The results of the present study showed that 1) Surgical Simplex mixture did not inhibit or stimulate the growth rate of S. aweus quantitatively; 2) Surgical Simplex mixed for less than 4 min tended to induce a slight decrease of phagocytosis rate of macrophage on BCG; 3) Surgical Simplex mixed for less than 4 min caused a significant decrease of G-l-Cl4 consumption in macrophages both in the resting and phagocytic phase, whereas G-6-C14 consumption rate was not affected by Surgical Simplex mixture; and 4) The increased consumption rate of G-1-G and G-6-G in macrophages in the phagocytic phase was noted, when compared with resting phase. Before commenting on the significance of the results of the present study, however, it is important to mention some of the drawbacks attached to this experimental design which may pose some limitation on the validity of the results. The systemic toxic effects of MM monomer is fairly well known, but the side effects of the cement additives have also been reported.3 The liquid part of Surgical
-
2-
I-
minutes
Fig. 4. The G-1-C14/G-6-C14consumption rates. Resting state: (--) control; samples; phagocytic state: (-) control; (.-.) samples. (.--a)
740
SHOJI ET AL.
Simplex does not readily mix with culture media, and a significant amount of MM monomer is dissipated into the air during the time of mixing the cement. Although it has been said that most of the MM monomer applied in vivo is released into the system and is dissipated from the lungs before the mixed mass hardens13there is no study indicating the exact amount of MM monomer remaining in the local tissue where the cement was applied. In view of these, in this preliminary study we adopted the system in which the in vitro effects of Surgical Simplex, mixed for various periods of time, was investigated, rather than those of individual ingredients of the cement, since we also felt that as an initial study it may also bear practical significance to study the effects of whole cement mixture. The cement, mixed for various time intervals, was air-tightly stored in the culture media at 4°C for a minimum arbitrary period of a month. Although we assume that in this system MM monomer concentration would be different in the culture media depending on various times of mixing performed at 18"C, it cannot be certain. Despite a demonstration of inhibition zone by the cement on S. aureus on the blood agar plate,2 we have failed to show inhibitory effects of the cement on S. aureus growth rate quantitatively. The exact reason for this discrepancy is unexplainable, but the inhibitory effects of cement on S. aureus growth do not appear so significant to be detectable quantitatively. Although, in general, the most common devastating organism in early postoperative infections is S. aureus, in late infections of total joint replacement, organisms with low-grade virulence leading chronic infection are the most c ~ m m o n . Late ~ infection frequently is a cause of loosening of the prosthesis. In view of this, in part of the leucocyte study, macrophage was selected rather than neutrophil, which is predominant in acute infection. BCG was used because its staining character simplifies the counting of phagocytosis rate and because the tuberculous nature induces chronic granulomatous lesion, in which macrophage plays a significant role. Under the influence of cement mixed for less than 4 min, the phagocytosis rate of macrophage tended to be decreased. As noted in the change of G-1-C14 and G-6-C14 consumption rates, hexose monophosphate shunt activity of macrophage was decreased under the influence of the cement mixture of less than 4 min in both resting and phagocytic states. The increased hexose monophosphate shunt activity during the phagocytic state? com-
I N VITRO EFFECTS OF BONE CEMENT ON MACROPHAGE 741
pared with the resting state was observed, regardless of influence by the cement mixture. Whether the increased hexose monophosphate shunt activity causes' or results from4 phagocytosis is still undetermined. As mentioned previously in the experimental system of this preliminary study, it is not known whether the adverse effects of phagocytosis rate and hexose monophosphate shunt activity of macrophage are due to MM monomer, MM polymer, additives, or combinations of these. It may seem reasonable to assume that MM monomer is likely to be most responsible, but further study is needed for a definite conclusion. However, we can state that Surgical Simplex mixture, in a certain situation, can cause decreased phagocytosis and decreased hexose monophosphate shunt activity of macrophage in vitro.
References 1. R. L. Bachner and M. L. Karnovsky, Science, 162, 1277 (1968). 2. M. W. Chapman and W. K. Hadley, J . Bone Joint Surg., 56A,846 (1974). 3. J. Charnley, in Acrylic Cement in Orthopaedic Surgery, J. Charnley, Ed., E. and S. Livingston, Ltd., Edinburgh and London, 1970, p. 72. 4. L. R. DeChatelet, R. M. Cooper, and C. E. McCall, Inf. Immun., 3,66 (1971). 5 . R. H. Fitzgerald, Jr., L. F. A. Peterson, J. A. Washington, 11, R. E. Van Scoy, and M. B. Coventry, J . Bone Joint Surg., 55A, 1242, (1973). 6. B. Holmes, P. G. Quie, D. B. Windhorst, and R. A. Good, Lancet, 1, 1225 (1966). 7. Q. N. Myrvik and D. G. Evans, Arch. Environ. Health, 14, 92 (1967).
Received October 1, 1975 Revised November 18, 1975
