- Email: [email protected]
Collagen gel in osseous defects
Collagen gel in osseous defects A preliminary
study
A. M. DelBalso, Captain, DC, USA,* and J. C. Adrian, Colonel, DC, UXA’* UNITED MEDICAL
STATES CENTER,
ARMY
INSTITUTE
WASHINGTON,
OF DENTAL
RESEARCH,
WALTER
REED
ARMY
D. C.
In a preliminary study, the effects of a biodegradable collagen gel on the healing of osseous defects were examined. Histologic and biochemical observations made on the seventh day after placement revealed that the collagen gel is well tolerated by the recipient and might be an effective stimulator of the formation of reparative bone. However, between the seventh and fourteenth days after placement, the deposition of reparative osseous tissues at the site of implant was significantly retarded. The possible implication of these findings is discussed.
T
he efficacy of using various reconstituted collagen heterografts in the treatment and management of osseous injuries has been reported by several investigat0rs.l’ 2 In many instances, these collagens appear to be effective stimulators of osteogenesis. However, since species differences may exist between donors and recipients, these materials may trigger immunologic reactions. Various “nonantigenic” biodegradable collagens have been developed which offer much promise since they are nonantigenic in nature and can be manufactuced in a variety of shapes and physical states. One form that would be of definite value to the dental profession is a biodegradable gel that could readily be injected into surgical defects, bony pockets, and periapical lesions. Such a gel might also be an effective vehicle for delivery of sustained-release drugs.3 Recently, we obtained a xenogeneic collagen gel and attempted to evaluate its biochemical and histologic effects on osseous injuries.t In this preliminary study, the activities of alkaline and acid phosphatases, as well as those of N-acetyl-/3-D-glucosaminidase *Division **Division tsupplied
562
of Basic Sciences. of Pathology. by Nippi, Inc., Tokyo,
Japan.
Volume Number
12 5
C~ollugcl~
gel
iw osseous
defects
563
and /3-glucuronidase, were determined in ‘I-day and 14-day control and implant sites. Alkaline phosphatase activity is believed to be an index of the population of osteogenic cells, whereas increases in the activities of the other enzymes have been noted during bone formation.4-7 The present article describes the results of these preliminary studies. MATERIALS
AND
METHODS
Sixteen adult male Hartley guinea pigs, weighing 350 to 500 grams, were used in this study. With the animal under ether anesthesia, the tibiae were exposed, and two 11/s-mm. holes were drilled in the shaft of each bone. The holes were approximately 5 mm. apart, and extended 3 mm. into the bone. One hole in each tibia was then filled with the biodegradable collagen gel, and the other wound was left void in order to serve as a control. On the seventh and fourteenth days after insult, eight animals were killed and both tibiae were immediately removed. One tibia was subjected to histologic study, and the other tibia was used in biochemical studies. The samples used in the histologic studies were placed in a 10 per cent buffered formalin solution and later decalcified. These specimens were then cut at 6 pm and stained with hematoxylin and eosin. The tibiae used in the biochemical studies were frozen in liquid nitrogen immediately after removal, and were subsequently lyopholized. Materials present in the experimental wounds at 7 and 14 days, and in the control wounds at 14 days were removed and used in the preparation of tissue homogenates. The repair process at the 7-day control site had not progressed to the point at which sufficient material was available for quantitative analysis. Samples of tissue weighing 0.75 to 1.5 mg. were homogenized in 1 ml. of deionized distilled water at O0 C. for use in the respective assays. Alkaline and acid phosphatase activities were determined by means of the methods of Lowry and associates,8 with the use of 7- to lo-pg tissue samples. Activities of N-acetyl+-n-glucosaminidase and /3-glucuronidase were determined by means of the method of Robins and associates,g with the use of 7- to lo-pg tissue samples. Preliminary enzyme studies with the use of 10 ,ug of dry tissue revealed linearity with respect to time from 0 to 60 minutes. Protein was determined according to the method of Lowry and associates,” with the use of bovine serum albumin as a standard. RESULTS
Gross examination of the tibiae on the seventh postoperative day revealed the presence of a firm, fibrous plug in the experimental wound, whereas the control wound was essentially void of any material. Histologic examination of the implant site at one week revealed the presence of young, highly vascularized connective tissue that contained noticeable amounts of young spindle-shaped and stellate fibroblasts (Fig. 1). Focal areas of osteoid and woven bone were also present in the defect area ; these areas were always observed to be in immediate contact with either vital or nonvital osseous fragments (Fig. 2). The inflammatory response elicited by the collagen was minimal,
564
LlelBnlso
Fig. 1. and stellate Fig. 1. position to
and Adrim
Oral November,
Surg. 1976
Implant site, 7 days. (Magnification, x250.) Note the presence of spindle-shaped fibroblasts and numerous small vascular channels. Implant site, 7 days. (Magnification, x250.) Young proliferating osteoid in apthe endosteal surface of the remaining cortical bone in the area of the defect.
and consisted chiefly of round cells and a few neutrophils (Fig. 3). In contrast, the control wound at one week was characterized by a dense inflammatory infiltrate and/or a partial void at the site (Fig. 4). On the fourteenth day after insult, tissue plugs were evident in both the experimental and control wounds. Histologically, the implant site at 2 weeks was characterized by a marked inflammatory infiltrate of both acute and chronic cells (Fig. 5). The fibroblasts were less numerous and also were not so spindle-shaped or stellate as in the one-week specimen. In addition, because of the inflammatory overlay, it was not possible to determine the presence of residual implant. Filling of the defect with woven bone varied from none to a moderate amount (Fig. 6).
XTolume 42 iTumber 5
Fig. implant. focus of view of kling of
Table
Collagen
gel
i9~ osseous
defects
565
J. A, Implant site, 7 days. (Magnification, x40.) Y, Marrow cavity. CG, Collagen + Areas of inflammation. Minimal inflammatory infiltrate present with only a rare cellular accumulation. li, Implant site, 7 days. (Magnification, x100.) Higher power implant material (CG) being infiltrated by proliferating fibroblasts, with a sprininflammatory cells (c). Jf, Marrow cavity.
I. Enzyme activities in control an d implant sites p MOlx
Alkaline phosphatase Acid phosphatase N-acetyl-p-D-glucosaminidase &&xuronidase
Control* -
?-day ( Implant 1.090 f 0.340 .014 f 0.020 3.195 f 0.414 .305 f 0.063
mg. protein-’
( 6) (6) (6) (6)
x min-’
1I-day Control 1 Implant 1.023 f 0.196 (6) 0.047 f 0.201 (6) .O130f 0.0 10 (6) 0.069 31 0.0 19 (6) 1.561 f 0.509 (5j 1.072 f 0..345 i5i ,162 f 0.034 (6) 0.145 f 0.081 i6j
“Seven-day control wounds did not present sufficient material for the performance of quantitative assays. Values listed are means + S.E.M. of tissue samples taken from five or six animals. Sample sizes are listed in parentheses. Each determination was carried out in triplicate. Details concerning analytical procedures are presented in the Materials and Methods section.
In no instance did this deposition of bone occur when not in direct apposition to existing bone. Inflammation in the 2-week controls was minimal to absent. The formation of woven bone varied from moderate to exuberant (Fig. 7). Biochemical data obtained in the present investigation are presented in Table I. Seven-day control wounds did not contain sufficient material on which to perform quantitative studies.
566
DelBalso
and
Adrian
Oral November,
Surg. 1976
Pig. 4. A, Control site, 7 days. (Magnification, x40.) Low-power photomicrograph demonstrating the partial void at the control site and a marked inflammatory infiltrate. B, Control site, 7 days. (Magnification, x400.) High-power photomicrograph of the site of the dense inflammatory infiltrate.
Alkaline phosphatase activities were found to be equal in material taken from the ‘I-day implant and in that from 14-day control sites. However, the activity of this enzyme was significantly lower in material taken from 14-day implant sites (P < 0.05). The equal alkaline phosphatase activities observed in 7-day implant and 14-day control material may reflect an equal population of osteogenic cells. The acid phosphatase activity observed in the 14-day implant site was twice that observed in 7-day implant material. N-Acetyl-P-n-glucosaminidase and /3-glucuronidase activities were found to be at control levels in material taken from the implant site on the fourteenth day. The respective activities of N-acetyl-P-n-gbmosaminidase and ,&glucuronidase observed in both 14-day control and implant material were one half those observed in material taken from the 7-day implant site.
Volume Sumber
-f2 5
Collagex gel in osseo~~sdefects
567
Pig. 5. Implant site, 14 days. (Magnification x30.) Low-power view of the implant site. Note the Iack of woven-bone formation and the dense inflammatory infiltrate. Fig. 6. Implant site, 14 days. (Magnification, x40.) This lowpower photomicrograph demonstrates the greatest amount of bone formation seen at an implant site. There is, in addition, an obvious arca of foreign-body type of inflammation (PB).
DISCUSSION
The histologic and biochemical observations made on Day ‘7 suggest the occurrence of active reparative processes at the implant site. The granulation tissue and areas of osteoid deposition observed in the T-day experimental wound, as well as the high alkaline phosphatase, are in accord with the proposal. Alkaline phosphatase activity is a commonly accepted indicator of the differentiation of osteoblasts3, * In the present investigation, the activity of alkaline phosphatase in ‘7-day experimental sites and that in 14-day controls were equal, which suggests an equal distribution of osteogenic cells. Although specific roles in osseous reparative processes have not been assigned to K-acetyl-/.?-n-glucosaminidase and ,&glucuronidase, increases in their activities have been noted during the development of osseous tissues.“, 5 In these studies, positive correlations were found
568
DelBalso
and Adrian
Oral Sovember,
Surg. 19i6
Fig. 7. Control site, 14 days. (Magnification, x40.) Woven-bone formation seen in this photomicrograph demonstrates an exuberant response to the insult. Also note the lack of inflammation. This specimen was examined under polarized light, and early remodeling was evident. In no instance WM this seen in the implant sites.
between the histologic differentiation of osteogenic cells and the activities of alkaline phosphatases and acid hydrolases. The high hydrolase activities observed in the ‘I-day site may reflect the differentiation of preosteogenic mesenchymal cells into osteogenic cells. The deposition of osseous tissue at the implant sites was significantly impaired between Day 7 and Day 14. This observation correlated with a marked decrease in the activities of all enzymes, except acid phosphatase, and the dearth of histologic evidence of woven-bone formation. The factors underlying these events are unknown; however, a number of speculative proposals may be raised. These include a recognition of the “foreign-ness” of the collagen matrix by differentiating osteogenic cells, the possible release of an inhibitor of osteogenesis from the reconstituted gel, and the destruction of newly synthesized osteoid by cells involved in the degradation of the collagen implant. These hypotheses are being currently investigated in our laboratory. SUMMARY
The effect of reconstituted collagen gel on osseous defects was studied histologically and biochemically. Early follow up (7 days) indicates that the collagen gel stimulates or actively supports the repair processes in the defect. However, later examination (14 days )suggests that the opposite is true. The control defects have abundant bone formation, whereas bone formation in the implant site appears to be retarded. Three possible explanations for this phenomenon are the following: (1) recognition by the host of the “foreign” collagen matrix; (2) release of an inhibitor of osteogenesis from the reconstit,uted gel ; and (3) destruction of the osteoid by cells involved in the degradation of the implant.
Volume Number
-l2 5
Collngw
gel in osseous defects
569
REFERENCES
1. Cucin, R. L., Goulian, D., Stenzel, K. H., and Rubin, A. L.: The Effects of Reconstituted Collagen Gels on the Healing of Experimental Bony Defects: A Preliminary _ Report, J. _ Surg.-Res. 12: 318-321, 1972._ 2. DeVore, D. T.: Collagen Hetcrografts for Bone Replacement, ORAL SURG. 35: 609-615, 1972. par les substances 3. Horakova, Z., Krajicek, M., Chvapil, M., and Boissier, J.: Prolongation collagenes de quelques actions pharmacologiques, Therapie 22: 1455-1460, 1967. 4. Jibril. A. J.: Phosnhatase and Phosahates in Preosseous Tissues. Biochim. Bionhvs. I Y Acta I 141: 805613, 1967.L 5. Yoshiki, S. : Histochemistry of Various Enzymes in Developing Bone, Cartilage and Tooth of Rat, Bull. Tokvo Dent. Coll. 3: 14-28. 1962. 6. Vates, h., and Jaiques, F.: The Assay ‘of Acid Hydrolases and Other Enzymes in Bone, Biochrm. J. 97: 380-387, 1965. 7. DrlBalso, A. M., and Kauffman, F. C.: Acid Hydrolases in Developing Oral-Facial Structures of the Rat, Arch. Oral Biol. 20: 247-249, 1975. 8. Lowry, 0. H., Roberts, N. R., Wu, M. L., Hixon, TV. S., and Crawford, E. J.: The Quantitative Histochemistry of the Brain. II. Enzyme Measurements, J. Biol. Chem. 207: 19.37, 1954. 9. Robins, E., Hirsch, H. S., and Emmons, S. J.: Glycosidases in the Nervous System, J. Biol. Chem. 243: 4246-4252, 1968. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein Measurements of the Folin Phenol Reagent, J. Biol. Chem. 193: 265-275, 1951. Reprint requests to: Colonel James C. Adrian U. S. Army Institute of Dental Research Walter Reed Army Medical Center Washington, D. C. 20012
study
A. M. DelBalso, Captain, DC, USA,* and J. C. Adrian, Colonel, DC, UXA’* UNITED MEDICAL
STATES CENTER,
ARMY
INSTITUTE
WASHINGTON,
OF DENTAL
RESEARCH,
WALTER
REED
ARMY
D. C.
In a preliminary study, the effects of a biodegradable collagen gel on the healing of osseous defects were examined. Histologic and biochemical observations made on the seventh day after placement revealed that the collagen gel is well tolerated by the recipient and might be an effective stimulator of the formation of reparative bone. However, between the seventh and fourteenth days after placement, the deposition of reparative osseous tissues at the site of implant was significantly retarded. The possible implication of these findings is discussed.
T
he efficacy of using various reconstituted collagen heterografts in the treatment and management of osseous injuries has been reported by several investigat0rs.l’ 2 In many instances, these collagens appear to be effective stimulators of osteogenesis. However, since species differences may exist between donors and recipients, these materials may trigger immunologic reactions. Various “nonantigenic” biodegradable collagens have been developed which offer much promise since they are nonantigenic in nature and can be manufactuced in a variety of shapes and physical states. One form that would be of definite value to the dental profession is a biodegradable gel that could readily be injected into surgical defects, bony pockets, and periapical lesions. Such a gel might also be an effective vehicle for delivery of sustained-release drugs.3 Recently, we obtained a xenogeneic collagen gel and attempted to evaluate its biochemical and histologic effects on osseous injuries.t In this preliminary study, the activities of alkaline and acid phosphatases, as well as those of N-acetyl-/3-D-glucosaminidase *Division **Division tsupplied
562
of Basic Sciences. of Pathology. by Nippi, Inc., Tokyo,
Japan.
Volume Number
12 5
C~ollugcl~
gel
iw osseous
defects
563
and /3-glucuronidase, were determined in ‘I-day and 14-day control and implant sites. Alkaline phosphatase activity is believed to be an index of the population of osteogenic cells, whereas increases in the activities of the other enzymes have been noted during bone formation.4-7 The present article describes the results of these preliminary studies. MATERIALS
AND
METHODS
Sixteen adult male Hartley guinea pigs, weighing 350 to 500 grams, were used in this study. With the animal under ether anesthesia, the tibiae were exposed, and two 11/s-mm. holes were drilled in the shaft of each bone. The holes were approximately 5 mm. apart, and extended 3 mm. into the bone. One hole in each tibia was then filled with the biodegradable collagen gel, and the other wound was left void in order to serve as a control. On the seventh and fourteenth days after insult, eight animals were killed and both tibiae were immediately removed. One tibia was subjected to histologic study, and the other tibia was used in biochemical studies. The samples used in the histologic studies were placed in a 10 per cent buffered formalin solution and later decalcified. These specimens were then cut at 6 pm and stained with hematoxylin and eosin. The tibiae used in the biochemical studies were frozen in liquid nitrogen immediately after removal, and were subsequently lyopholized. Materials present in the experimental wounds at 7 and 14 days, and in the control wounds at 14 days were removed and used in the preparation of tissue homogenates. The repair process at the 7-day control site had not progressed to the point at which sufficient material was available for quantitative analysis. Samples of tissue weighing 0.75 to 1.5 mg. were homogenized in 1 ml. of deionized distilled water at O0 C. for use in the respective assays. Alkaline and acid phosphatase activities were determined by means of the methods of Lowry and associates,8 with the use of 7- to lo-pg tissue samples. Activities of N-acetyl+-n-glucosaminidase and /3-glucuronidase were determined by means of the method of Robins and associates,g with the use of 7- to lo-pg tissue samples. Preliminary enzyme studies with the use of 10 ,ug of dry tissue revealed linearity with respect to time from 0 to 60 minutes. Protein was determined according to the method of Lowry and associates,” with the use of bovine serum albumin as a standard. RESULTS
Gross examination of the tibiae on the seventh postoperative day revealed the presence of a firm, fibrous plug in the experimental wound, whereas the control wound was essentially void of any material. Histologic examination of the implant site at one week revealed the presence of young, highly vascularized connective tissue that contained noticeable amounts of young spindle-shaped and stellate fibroblasts (Fig. 1). Focal areas of osteoid and woven bone were also present in the defect area ; these areas were always observed to be in immediate contact with either vital or nonvital osseous fragments (Fig. 2). The inflammatory response elicited by the collagen was minimal,
564
LlelBnlso
Fig. 1. and stellate Fig. 1. position to
and Adrim
Oral November,
Surg. 1976
Implant site, 7 days. (Magnification, x250.) Note the presence of spindle-shaped fibroblasts and numerous small vascular channels. Implant site, 7 days. (Magnification, x250.) Young proliferating osteoid in apthe endosteal surface of the remaining cortical bone in the area of the defect.
and consisted chiefly of round cells and a few neutrophils (Fig. 3). In contrast, the control wound at one week was characterized by a dense inflammatory infiltrate and/or a partial void at the site (Fig. 4). On the fourteenth day after insult, tissue plugs were evident in both the experimental and control wounds. Histologically, the implant site at 2 weeks was characterized by a marked inflammatory infiltrate of both acute and chronic cells (Fig. 5). The fibroblasts were less numerous and also were not so spindle-shaped or stellate as in the one-week specimen. In addition, because of the inflammatory overlay, it was not possible to determine the presence of residual implant. Filling of the defect with woven bone varied from none to a moderate amount (Fig. 6).
XTolume 42 iTumber 5
Fig. implant. focus of view of kling of
Table
Collagen
gel
i9~ osseous
defects
565
J. A, Implant site, 7 days. (Magnification, x40.) Y, Marrow cavity. CG, Collagen + Areas of inflammation. Minimal inflammatory infiltrate present with only a rare cellular accumulation. li, Implant site, 7 days. (Magnification, x100.) Higher power implant material (CG) being infiltrated by proliferating fibroblasts, with a sprininflammatory cells (c). Jf, Marrow cavity.
I. Enzyme activities in control an d implant sites p MOlx
Alkaline phosphatase Acid phosphatase N-acetyl-p-D-glucosaminidase &&xuronidase
Control* -
?-day ( Implant 1.090 f 0.340 .014 f 0.020 3.195 f 0.414 .305 f 0.063
mg. protein-’
( 6) (6) (6) (6)
x min-’
1I-day Control 1 Implant 1.023 f 0.196 (6) 0.047 f 0.201 (6) .O130f 0.0 10 (6) 0.069 31 0.0 19 (6) 1.561 f 0.509 (5j 1.072 f 0..345 i5i ,162 f 0.034 (6) 0.145 f 0.081 i6j
“Seven-day control wounds did not present sufficient material for the performance of quantitative assays. Values listed are means + S.E.M. of tissue samples taken from five or six animals. Sample sizes are listed in parentheses. Each determination was carried out in triplicate. Details concerning analytical procedures are presented in the Materials and Methods section.
In no instance did this deposition of bone occur when not in direct apposition to existing bone. Inflammation in the 2-week controls was minimal to absent. The formation of woven bone varied from moderate to exuberant (Fig. 7). Biochemical data obtained in the present investigation are presented in Table I. Seven-day control wounds did not contain sufficient material on which to perform quantitative studies.
566
DelBalso
and
Adrian
Oral November,
Surg. 1976
Pig. 4. A, Control site, 7 days. (Magnification, x40.) Low-power photomicrograph demonstrating the partial void at the control site and a marked inflammatory infiltrate. B, Control site, 7 days. (Magnification, x400.) High-power photomicrograph of the site of the dense inflammatory infiltrate.
Alkaline phosphatase activities were found to be equal in material taken from the ‘I-day implant and in that from 14-day control sites. However, the activity of this enzyme was significantly lower in material taken from 14-day implant sites (P < 0.05). The equal alkaline phosphatase activities observed in 7-day implant and 14-day control material may reflect an equal population of osteogenic cells. The acid phosphatase activity observed in the 14-day implant site was twice that observed in 7-day implant material. N-Acetyl-P-n-glucosaminidase and /3-glucuronidase activities were found to be at control levels in material taken from the implant site on the fourteenth day. The respective activities of N-acetyl-P-n-gbmosaminidase and ,&glucuronidase observed in both 14-day control and implant material were one half those observed in material taken from the 7-day implant site.
Volume Sumber
-f2 5
Collagex gel in osseo~~sdefects
567
Pig. 5. Implant site, 14 days. (Magnification x30.) Low-power view of the implant site. Note the Iack of woven-bone formation and the dense inflammatory infiltrate. Fig. 6. Implant site, 14 days. (Magnification, x40.) This lowpower photomicrograph demonstrates the greatest amount of bone formation seen at an implant site. There is, in addition, an obvious arca of foreign-body type of inflammation (PB).
DISCUSSION
The histologic and biochemical observations made on Day ‘7 suggest the occurrence of active reparative processes at the implant site. The granulation tissue and areas of osteoid deposition observed in the T-day experimental wound, as well as the high alkaline phosphatase, are in accord with the proposal. Alkaline phosphatase activity is a commonly accepted indicator of the differentiation of osteoblasts3, * In the present investigation, the activity of alkaline phosphatase in ‘7-day experimental sites and that in 14-day controls were equal, which suggests an equal distribution of osteogenic cells. Although specific roles in osseous reparative processes have not been assigned to K-acetyl-/.?-n-glucosaminidase and ,&glucuronidase, increases in their activities have been noted during the development of osseous tissues.“, 5 In these studies, positive correlations were found
568
DelBalso
and Adrian
Oral Sovember,
Surg. 19i6
Fig. 7. Control site, 14 days. (Magnification, x40.) Woven-bone formation seen in this photomicrograph demonstrates an exuberant response to the insult. Also note the lack of inflammation. This specimen was examined under polarized light, and early remodeling was evident. In no instance WM this seen in the implant sites.
between the histologic differentiation of osteogenic cells and the activities of alkaline phosphatases and acid hydrolases. The high hydrolase activities observed in the ‘I-day site may reflect the differentiation of preosteogenic mesenchymal cells into osteogenic cells. The deposition of osseous tissue at the implant sites was significantly impaired between Day 7 and Day 14. This observation correlated with a marked decrease in the activities of all enzymes, except acid phosphatase, and the dearth of histologic evidence of woven-bone formation. The factors underlying these events are unknown; however, a number of speculative proposals may be raised. These include a recognition of the “foreign-ness” of the collagen matrix by differentiating osteogenic cells, the possible release of an inhibitor of osteogenesis from the reconstituted gel, and the destruction of newly synthesized osteoid by cells involved in the degradation of the collagen implant. These hypotheses are being currently investigated in our laboratory. SUMMARY
The effect of reconstituted collagen gel on osseous defects was studied histologically and biochemically. Early follow up (7 days) indicates that the collagen gel stimulates or actively supports the repair processes in the defect. However, later examination (14 days )suggests that the opposite is true. The control defects have abundant bone formation, whereas bone formation in the implant site appears to be retarded. Three possible explanations for this phenomenon are the following: (1) recognition by the host of the “foreign” collagen matrix; (2) release of an inhibitor of osteogenesis from the reconstit,uted gel ; and (3) destruction of the osteoid by cells involved in the degradation of the implant.
Volume Number
-l2 5
Collngw
gel in osseous defects
569
REFERENCES
1. Cucin, R. L., Goulian, D., Stenzel, K. H., and Rubin, A. L.: The Effects of Reconstituted Collagen Gels on the Healing of Experimental Bony Defects: A Preliminary _ Report, J. _ Surg.-Res. 12: 318-321, 1972._ 2. DeVore, D. T.: Collagen Hetcrografts for Bone Replacement, ORAL SURG. 35: 609-615, 1972. par les substances 3. Horakova, Z., Krajicek, M., Chvapil, M., and Boissier, J.: Prolongation collagenes de quelques actions pharmacologiques, Therapie 22: 1455-1460, 1967. 4. Jibril. A. J.: Phosnhatase and Phosahates in Preosseous Tissues. Biochim. Bionhvs. I Y Acta I 141: 805613, 1967.L 5. Yoshiki, S. : Histochemistry of Various Enzymes in Developing Bone, Cartilage and Tooth of Rat, Bull. Tokvo Dent. Coll. 3: 14-28. 1962. 6. Vates, h., and Jaiques, F.: The Assay ‘of Acid Hydrolases and Other Enzymes in Bone, Biochrm. J. 97: 380-387, 1965. 7. DrlBalso, A. M., and Kauffman, F. C.: Acid Hydrolases in Developing Oral-Facial Structures of the Rat, Arch. Oral Biol. 20: 247-249, 1975. 8. Lowry, 0. H., Roberts, N. R., Wu, M. L., Hixon, TV. S., and Crawford, E. J.: The Quantitative Histochemistry of the Brain. II. Enzyme Measurements, J. Biol. Chem. 207: 19.37, 1954. 9. Robins, E., Hirsch, H. S., and Emmons, S. J.: Glycosidases in the Nervous System, J. Biol. Chem. 243: 4246-4252, 1968. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.: Protein Measurements of the Folin Phenol Reagent, J. Biol. Chem. 193: 265-275, 1951. Reprint requests to: Colonel James C. Adrian U. S. Army Institute of Dental Research Walter Reed Army Medical Center Washington, D. C. 20012