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Replantation of enzymatically treated teeth in monkeys. Part I
Replantation of enzymatically treated teeth in monkeys. Part I Alun .I. Nevins, D.D.S., * Robert F. LuPorta, M.S., ** Bernard G. Borden, D.M.D., Peter Lorenzo, A.A.S.,**** East Meadow, N. Y. NASSAU
COUNTY
MEDICAL
*** and
CENTER
Chemical alterations in cementum of avulsecl teeth may be implied as a causative factor in their eventual resorption when replanted. Root surface decak3icatiin, with subsequent enzymatic deletion of glycoproteins and cross linking of cementum collagen, resutts in liffle or no root resorption for up to 3 months after replantation.
W
hile vast energieshave been expended on replantation techniques, underlying mechanismsof resorption of replanted tooth structure remain poorly understood.’ Integrity of the periodontal ligament has proved to be the single most significant factor for success.2s 3 Unfortunately, this tissue is rarely viable after 20 minutes, when most replantation procedures are attempted. The resulting denuded cementum apparently triggers a rejection phenomenon in which progressive root resorption and ankylosis occur..’ Several attempts to reduce the rate of resorption, including use of sodium fluoride, have yielded inconclusive results.“. fi Since chemical alterations of cementurn have been implied as a possiblecausative factor,’ we hypothesized that replantation would be more successful if potentially antigenic components of cementurn matrix were enzymatically removed subsequentto root surface decalcification. In addition, cross-linking agents would further inactivate remaining antigenic sites.” MATERIALS
AND METHODS
Sixteen maxillary and mandibular incisors of six juvenile cynomologous monkeys were extracted and their crowns were removed. A scalpel was usedto strip periodontal ligaments, denuding cementum of all soft tissue. In addition, pulps were removed with endodontic This
study
*Director Endodontics.
was supported of Dental
in part
Research,
by NlDR
Department
No.
DIG04552.
of Dentistry,
Grant
Division
of
**Ph.D. Candidate, Department of Oral Biology, State University of New
York
***Chairman, ****Executive
at Stony
Brook.
Department Assistant
0030-4220/80/090277+05$00.50~0
of Dentistry. Director. Department
0
1980-h
of Animal C. V. Mosby(‘o.
Research.
files and normal saline irrigation. Five hours after extraction and the subsequentexperimental or control treatments indicated in Table I, all teeth were replanted without root canal fillings. Interproximal sutures were placed, but soft-tissue closure of sockets was not attempted. Monkeys received daily intramuscular injections of 300,000 units of procaine penicillin G plus 300,000 units of benzathine penicillin G for 7 days. Postproceduralradiographs were taken at 1 hour and at 4, 8, and 12 weeks. Bone sections (each containing two teeth) were excised and all animals killed at 12 weeks. Each bone block was fixed in 10 percent neutral buffered formalin and decalcified in 20 percent formic acid in citrate buffer. Serial cross sections were cut at 5 microns and alternately stained with hematoxylin-eosin and Masson’s trichrome stains for histologic examination. RESULTS
Soft-tissue closure of sockets was evident within 4 weeks. At 12 weeks all saline-treatedand Group 2 experimental teeth demonstratedextensive resorption and ankylosis radiographically and on histologic examination (Figs. I to 4 and Fig. 6). Seven of eight Group 1 experimental teeth, in contrast, demonstratedlittle or no resorptionof cementumin the sametime period (Figs. I, 2, 3, and 5). A uniform thickness of cementum and subjacent dentin stained intensely blue with Masson’s trichrome, indicating an area of hypocalcification or increaseddensity of collagen (Figs. 3 and 5). This zone was not present in saline-treatedcontrol teeth, Group 2 experimental teeth, or normal nonprocedural teeth. Connective tissue growth was evident within root canals of most teeth. However, Group 1 experimental 277
278
Nevins et al.
Oral Surg. September, 1980
Fig. 1. Radiograph taken immediately after replantation of Group 1 experimental (left) and saline-treated control (right) mandibular central incisors. Table
I. Treatment Experimental
of root structure prior to Group
rephntatiOn Control
I (8 teeth)
Hydrochloric acid
Normal saline solution
Hyaluronidase* 0.4 mg/ml. in 0.1 M NaPO, buffer pH 5.5 Normal saline solution
Buffered glutaraldehydet (0.2%, pH 7.4) Normal saline solution
Experimental
Fig. 2. Three-month postprocedural radiograph demonstrating lack of resorption of Group 1 experimental incisor and extensive resorption of control incisor.
group
(4 teeth)
I change 10 minutes
Normal saline solution 10 minutes
24°C.
24°C.
3 changes 45 minutes (total)
Normal saline solution 45 minutes
37°C.
37°C.
2 changes 30 minutes (total)
Normal saline solution 30 minutes
37°C.
37°C.
3 changes 45 minutes (total)
Normal saline solution 45 minutes
37°C.
37°C.
2 changes 30 minutes (total)
Normal saline solution 30 minutes
37°C.
37°C.
3 changes 45 minutes (total)
Normal saline solution 45 minutes
37°C.
37°C.
Group 2 (4 teeth)
Treatment is identical to Experimental Group 1 without hydrochloric acid. *Worthington Biochemical Corporation, Freehold, N.J. tHancock’s solution, Hancock Laboratories, Inc., Anaheim, Calif.
Volume Number
50 3
Replantation
of enzymatically
treated
teeth
in monkeys
279
Figs. 3 aml4. Histologicsectionsof Group 1 experimental(Fig. 3) andcontrol (Fig. 4) teethshownin Figs. 1and 2 demonstratinglack of resorptionof experimentalandextensiveresorptionof control. (Trichromestain. Magnification, X50.) teeth exhibited little or no internal resorption in contrast to other groups. Alveolar and/or periapical bone loss with moderateto severeinflammation occurred in three teeth from experimental Group 1, two teeth from experimental Group 2, and two teeth from the salinetreated control group. DISCUSSION Decalcified cementum contains primarily Type I collagen which is insoluble in both neutral and acid buffers.s Hydrochloric acid (0.6M) was used to decalcify cementum of Group 1 experimental teeth without denaturing the collagen component. The blue peripheral zone present on histologic sections stained with trichrome was probably a result of this initial decalcification procedure and enzymatic deletion of ground substance. Hyaluronic acid and chondroitin sulfate within cementum matrix contain potentially antigenic unsaturated disaccharides.lo The glucose content within cementum glycoprotein (1.5 Gm. / 14 Gm. hydroxyproline) is several times greater than 0.5 Gm.114 Gm. hydroxyproline found in skin or dentin matrix.”
Hyaluronidase was the enzyme of choice to hydrolyze endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin-sulfuric acids A and C,12 facilitating their removal from cementurn. Subsequentto deletion of these potential antigens, 0.2 percent glutaraldehyde buffered to pH 7.4 was used to cross-link the remaining cementumcollagen, thereby reducing its rate of resorption. I3 It was hypothesized that this step would also render any antigenic sites inactive. In a study transplanting bovine carotid arteries, DeFalcoR demonstrated that carotid (enzymatically treated to remove ground substanceand subsequently cross linked) produces no antibody response. Salinewashedcarotid and saline-washedcarotid digestedwith enzyme both evoke antibodies. Glutaraldehyde has been used as a storage medium for collagen cardiovascular bioprosthesis13and was therefore selected as the cross-linking agent in our study. Progressive resorption and ankylosis of salinetreated control teeth was a predictable outcome corresponding to results obtained in other studies.‘4x ” In contrast, Group 1 experimental results appear to be unique in that alveolar bone was found in close adapta-
280 Nevins et al.
Oral Surg. September,
1980
Figs. 5 and 6. High magnification of areas shown in Figs. 3 and 4. Note close approximation of bone to intensely stained peripheral zone (arrow) of experimental tooth (Fig. 5). Inflammatory (r) and replacement(R) resorption is evident in the saline-treated control (Fig. 6). (Magnification, x 100.)
tion to unresorbedcementum in five of eight teeth. This phenomenonis probably due to either an abatementof immunogenicity or simply increaseddensity of cementurn collagen. Future immunofluorescent studieswould indicate if the former is a factor in replantation. From contrasting results of experimental Groups 1 and 2, we have demonstrated that calcium phosphate (hydroxyapatite) must first be removed from cementum for enzymatic hydrolysis of ground substanceto be effective. Past studies indicate that hydrochloric acid or crosslinking agents alone do not inhibit resorption of replanted teeth. ’ SUMMARY
We have conducted a short-term study in which teeth denuded of periodontal ligament and pulp were chemically treated and replanted with minimal resorption of cementum occurring. In similar long-term studiesendodontically filled teeth are being replanted in an attempt to eliminate areas inaccessibleto enzymatic treatment and prevent alveolar bone lossdue to bacterial contamination.
We thank N. Levycky for photographic assistance, J. Behrendt and I. B. Bender for constructive criticism of the manuscript, S. Soliman, D.V.M., for care of the animals, and S. Slezak for typing the manuscript. REFERENCES
1. Natiella, J. R., Armitage, J. E., and Green, G. W.: The Replantation and Transplantation of Teeth, ORAL SURG. 29: 397-415, 1970. 2. Loe, H., and Waerhang, J.: Experimental Replantation of Teeth in Dogs and Monkeys, Arch. Oral Biol. 3: 176-1961. 3. Nasjleti, C. E., Caffesse, R. G., Castelli, W. A., and Hoke, J. A.: Healing After Tooth Reimplantation in Monkeys: A Radioautographic Study, ORAL SURG. 39: 361-375, 1975. 4. Andreasen, J. 0.: The Effect of Splinting Upon Periodontal Healing After Replantation of Permanent Incisors in Monkeys, Acta Odontol. Stand. 33: 313-323, 1975. 5. Shulman, L. B., Gedalia, I., and Feingold, R. M.: Fluoride Concentration in Root Surfaces and Alveolar Bone of FluorideImmersed Monkey Incisors Three Weeks After Replantation, J. Dent. Res. 52: 1314-1316, 1973. 6. Barbakow, F. H., Dent, H. D., Austin, J. C., and CleatonJones, P. E.: Histologic Response of Replanted Teeth Pretreated With Acidulated Sodium Fluoride, ORAL SURG. 45: 621-628, 1978. 7. Herbert, W E.: A Case of Complete Dislocation of a Tooth, Br. Dent. J. 105: 137-138, 1958.
Volume Number
50 3
Replantation
8. DeFalco, R. J.: Immunologic Studies of Untreated and Chemically Modified Bovine Carotid Arteries, J. Surg. Res. 10: 95100, 1970. 9. Birkedal-Hansen, H., Butler, W. T., and Taylor, R. E.: Proteins of the Periodontium; Characterization of the Insoluble Collagens of Bovine Dental Cementum, Calcif. Tiss. Res. 23: 39-44, 1977. 10. Boume, G. H.: The Biochemistry and Physiology of Bone, ed. 2, New York, 1972, Academic Press, Inc., p. 158. 11. Chovelon, A., Carmichael, D. J., and Pearson, C. H.: The Composition of the Organic Matrix of Bovine Cementum, Arch. Oral Biol. 20: 537-541, 1975. 12. Ludowieg, J., Vennesland, B., and Dorfman, A.: The Mechanism of Action of Hyaluronidase, J. Biol. Chem. 236: 333-339, 1961. 13. Hall, D. A., and Jackson, D. S.: International Review of Con-
of enzymatically treated teeth in monkeys
281
nective Tissue Research, New York, 1973, Academic Press, Inc., vol. 6, pp. 8, 35. 14. Andreasen, J. O., and HjBrting-Hansen, E.: Replantation of Teeth. I. Radiographic and Clinical Study of 110 Human Teeth Replanted After Accidental Loss, Acta Odontol. Stand. 24:
263-286, 1966. 15. Andreasen, J. O., and Hjerting-Hansen, E.: Replantation of Teeth. II. Histological Study of 22 Replanted Anterior Teeth in Humans, Acta Odontol. Stand. 24: 287-306, 1966. Reprint requests to: Dr. Alan J. Nevins Department of Dentistry Nassau County Medical Center 2201 Hempstead Turnpike East Meadow, N. Y. 11554
INFORMATION FOR AUTHORS Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership ofthe manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. I sign for and accept responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.
COUNTY
MEDICAL
*** and
CENTER
Chemical alterations in cementum of avulsecl teeth may be implied as a causative factor in their eventual resorption when replanted. Root surface decak3icatiin, with subsequent enzymatic deletion of glycoproteins and cross linking of cementum collagen, resutts in liffle or no root resorption for up to 3 months after replantation.
W
hile vast energieshave been expended on replantation techniques, underlying mechanismsof resorption of replanted tooth structure remain poorly understood.’ Integrity of the periodontal ligament has proved to be the single most significant factor for success.2s 3 Unfortunately, this tissue is rarely viable after 20 minutes, when most replantation procedures are attempted. The resulting denuded cementum apparently triggers a rejection phenomenon in which progressive root resorption and ankylosis occur..’ Several attempts to reduce the rate of resorption, including use of sodium fluoride, have yielded inconclusive results.“. fi Since chemical alterations of cementurn have been implied as a possiblecausative factor,’ we hypothesized that replantation would be more successful if potentially antigenic components of cementurn matrix were enzymatically removed subsequentto root surface decalcification. In addition, cross-linking agents would further inactivate remaining antigenic sites.” MATERIALS
AND METHODS
Sixteen maxillary and mandibular incisors of six juvenile cynomologous monkeys were extracted and their crowns were removed. A scalpel was usedto strip periodontal ligaments, denuding cementum of all soft tissue. In addition, pulps were removed with endodontic This
study
*Director Endodontics.
was supported of Dental
in part
Research,
by NlDR
Department
No.
DIG04552.
of Dentistry,
Grant
Division
of
**Ph.D. Candidate, Department of Oral Biology, State University of New
York
***Chairman, ****Executive
at Stony
Brook.
Department Assistant
0030-4220/80/090277+05$00.50~0
of Dentistry. Director. Department
0
1980-h
of Animal C. V. Mosby(‘o.
Research.
files and normal saline irrigation. Five hours after extraction and the subsequentexperimental or control treatments indicated in Table I, all teeth were replanted without root canal fillings. Interproximal sutures were placed, but soft-tissue closure of sockets was not attempted. Monkeys received daily intramuscular injections of 300,000 units of procaine penicillin G plus 300,000 units of benzathine penicillin G for 7 days. Postproceduralradiographs were taken at 1 hour and at 4, 8, and 12 weeks. Bone sections (each containing two teeth) were excised and all animals killed at 12 weeks. Each bone block was fixed in 10 percent neutral buffered formalin and decalcified in 20 percent formic acid in citrate buffer. Serial cross sections were cut at 5 microns and alternately stained with hematoxylin-eosin and Masson’s trichrome stains for histologic examination. RESULTS
Soft-tissue closure of sockets was evident within 4 weeks. At 12 weeks all saline-treatedand Group 2 experimental teeth demonstratedextensive resorption and ankylosis radiographically and on histologic examination (Figs. I to 4 and Fig. 6). Seven of eight Group 1 experimental teeth, in contrast, demonstratedlittle or no resorptionof cementumin the sametime period (Figs. I, 2, 3, and 5). A uniform thickness of cementum and subjacent dentin stained intensely blue with Masson’s trichrome, indicating an area of hypocalcification or increaseddensity of collagen (Figs. 3 and 5). This zone was not present in saline-treatedcontrol teeth, Group 2 experimental teeth, or normal nonprocedural teeth. Connective tissue growth was evident within root canals of most teeth. However, Group 1 experimental 277
278
Nevins et al.
Oral Surg. September, 1980
Fig. 1. Radiograph taken immediately after replantation of Group 1 experimental (left) and saline-treated control (right) mandibular central incisors. Table
I. Treatment Experimental
of root structure prior to Group
rephntatiOn Control
I (8 teeth)
Hydrochloric acid
Normal saline solution
Hyaluronidase* 0.4 mg/ml. in 0.1 M NaPO, buffer pH 5.5 Normal saline solution
Buffered glutaraldehydet (0.2%, pH 7.4) Normal saline solution
Experimental
Fig. 2. Three-month postprocedural radiograph demonstrating lack of resorption of Group 1 experimental incisor and extensive resorption of control incisor.
group
(4 teeth)
I change 10 minutes
Normal saline solution 10 minutes
24°C.
24°C.
3 changes 45 minutes (total)
Normal saline solution 45 minutes
37°C.
37°C.
2 changes 30 minutes (total)
Normal saline solution 30 minutes
37°C.
37°C.
3 changes 45 minutes (total)
Normal saline solution 45 minutes
37°C.
37°C.
2 changes 30 minutes (total)
Normal saline solution 30 minutes
37°C.
37°C.
3 changes 45 minutes (total)
Normal saline solution 45 minutes
37°C.
37°C.
Group 2 (4 teeth)
Treatment is identical to Experimental Group 1 without hydrochloric acid. *Worthington Biochemical Corporation, Freehold, N.J. tHancock’s solution, Hancock Laboratories, Inc., Anaheim, Calif.
Volume Number
50 3
Replantation
of enzymatically
treated
teeth
in monkeys
279
Figs. 3 aml4. Histologicsectionsof Group 1 experimental(Fig. 3) andcontrol (Fig. 4) teethshownin Figs. 1and 2 demonstratinglack of resorptionof experimentalandextensiveresorptionof control. (Trichromestain. Magnification, X50.) teeth exhibited little or no internal resorption in contrast to other groups. Alveolar and/or periapical bone loss with moderateto severeinflammation occurred in three teeth from experimental Group 1, two teeth from experimental Group 2, and two teeth from the salinetreated control group. DISCUSSION Decalcified cementum contains primarily Type I collagen which is insoluble in both neutral and acid buffers.s Hydrochloric acid (0.6M) was used to decalcify cementum of Group 1 experimental teeth without denaturing the collagen component. The blue peripheral zone present on histologic sections stained with trichrome was probably a result of this initial decalcification procedure and enzymatic deletion of ground substance. Hyaluronic acid and chondroitin sulfate within cementum matrix contain potentially antigenic unsaturated disaccharides.lo The glucose content within cementum glycoprotein (1.5 Gm. / 14 Gm. hydroxyproline) is several times greater than 0.5 Gm.114 Gm. hydroxyproline found in skin or dentin matrix.”
Hyaluronidase was the enzyme of choice to hydrolyze endo-N-acetylhexosaminic bonds of hyaluronic acid and chondroitin-sulfuric acids A and C,12 facilitating their removal from cementurn. Subsequentto deletion of these potential antigens, 0.2 percent glutaraldehyde buffered to pH 7.4 was used to cross-link the remaining cementumcollagen, thereby reducing its rate of resorption. I3 It was hypothesized that this step would also render any antigenic sites inactive. In a study transplanting bovine carotid arteries, DeFalcoR demonstrated that carotid (enzymatically treated to remove ground substanceand subsequently cross linked) produces no antibody response. Salinewashedcarotid and saline-washedcarotid digestedwith enzyme both evoke antibodies. Glutaraldehyde has been used as a storage medium for collagen cardiovascular bioprosthesis13and was therefore selected as the cross-linking agent in our study. Progressive resorption and ankylosis of salinetreated control teeth was a predictable outcome corresponding to results obtained in other studies.‘4x ” In contrast, Group 1 experimental results appear to be unique in that alveolar bone was found in close adapta-
280 Nevins et al.
Oral Surg. September,
1980
Figs. 5 and 6. High magnification of areas shown in Figs. 3 and 4. Note close approximation of bone to intensely stained peripheral zone (arrow) of experimental tooth (Fig. 5). Inflammatory (r) and replacement(R) resorption is evident in the saline-treated control (Fig. 6). (Magnification, x 100.)
tion to unresorbedcementum in five of eight teeth. This phenomenonis probably due to either an abatementof immunogenicity or simply increaseddensity of cementurn collagen. Future immunofluorescent studieswould indicate if the former is a factor in replantation. From contrasting results of experimental Groups 1 and 2, we have demonstrated that calcium phosphate (hydroxyapatite) must first be removed from cementum for enzymatic hydrolysis of ground substanceto be effective. Past studies indicate that hydrochloric acid or crosslinking agents alone do not inhibit resorption of replanted teeth. ’ SUMMARY
We have conducted a short-term study in which teeth denuded of periodontal ligament and pulp were chemically treated and replanted with minimal resorption of cementum occurring. In similar long-term studiesendodontically filled teeth are being replanted in an attempt to eliminate areas inaccessibleto enzymatic treatment and prevent alveolar bone lossdue to bacterial contamination.
We thank N. Levycky for photographic assistance, J. Behrendt and I. B. Bender for constructive criticism of the manuscript, S. Soliman, D.V.M., for care of the animals, and S. Slezak for typing the manuscript. REFERENCES
1. Natiella, J. R., Armitage, J. E., and Green, G. W.: The Replantation and Transplantation of Teeth, ORAL SURG. 29: 397-415, 1970. 2. Loe, H., and Waerhang, J.: Experimental Replantation of Teeth in Dogs and Monkeys, Arch. Oral Biol. 3: 176-1961. 3. Nasjleti, C. E., Caffesse, R. G., Castelli, W. A., and Hoke, J. A.: Healing After Tooth Reimplantation in Monkeys: A Radioautographic Study, ORAL SURG. 39: 361-375, 1975. 4. Andreasen, J. 0.: The Effect of Splinting Upon Periodontal Healing After Replantation of Permanent Incisors in Monkeys, Acta Odontol. Stand. 33: 313-323, 1975. 5. Shulman, L. B., Gedalia, I., and Feingold, R. M.: Fluoride Concentration in Root Surfaces and Alveolar Bone of FluorideImmersed Monkey Incisors Three Weeks After Replantation, J. Dent. Res. 52: 1314-1316, 1973. 6. Barbakow, F. H., Dent, H. D., Austin, J. C., and CleatonJones, P. E.: Histologic Response of Replanted Teeth Pretreated With Acidulated Sodium Fluoride, ORAL SURG. 45: 621-628, 1978. 7. Herbert, W E.: A Case of Complete Dislocation of a Tooth, Br. Dent. J. 105: 137-138, 1958.
Volume Number
50 3
Replantation
8. DeFalco, R. J.: Immunologic Studies of Untreated and Chemically Modified Bovine Carotid Arteries, J. Surg. Res. 10: 95100, 1970. 9. Birkedal-Hansen, H., Butler, W. T., and Taylor, R. E.: Proteins of the Periodontium; Characterization of the Insoluble Collagens of Bovine Dental Cementum, Calcif. Tiss. Res. 23: 39-44, 1977. 10. Boume, G. H.: The Biochemistry and Physiology of Bone, ed. 2, New York, 1972, Academic Press, Inc., p. 158. 11. Chovelon, A., Carmichael, D. J., and Pearson, C. H.: The Composition of the Organic Matrix of Bovine Cementum, Arch. Oral Biol. 20: 537-541, 1975. 12. Ludowieg, J., Vennesland, B., and Dorfman, A.: The Mechanism of Action of Hyaluronidase, J. Biol. Chem. 236: 333-339, 1961. 13. Hall, D. A., and Jackson, D. S.: International Review of Con-
of enzymatically treated teeth in monkeys
281
nective Tissue Research, New York, 1973, Academic Press, Inc., vol. 6, pp. 8, 35. 14. Andreasen, J. O., and HjBrting-Hansen, E.: Replantation of Teeth. I. Radiographic and Clinical Study of 110 Human Teeth Replanted After Accidental Loss, Acta Odontol. Stand. 24:
263-286, 1966. 15. Andreasen, J. O., and Hjerting-Hansen, E.: Replantation of Teeth. II. Histological Study of 22 Replanted Anterior Teeth in Humans, Acta Odontol. Stand. 24: 287-306, 1966. Reprint requests to: Dr. Alan J. Nevins Department of Dentistry Nassau County Medical Center 2201 Hempstead Turnpike East Meadow, N. Y. 11554
INFORMATION FOR AUTHORS Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following written statement, signed by one author: “The undersigned author transfers all copyright ownership ofthe manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The undersigned author warrants that the article is original, is not under consideration by another journal, and has not been previously published. I sign for and accept responsibility for releasing this material on behalf of any and all co-authors.” Authors will be consulted, when possible, regarding republication of their material.