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The effect of reconstituted collagen gels on the healing of experimental bony defects: A preliminary report
JOURNAL
THE
OF
SURGICAL
12, 318-321
RCSEARCII,
EFFECT OF RECONSTITUTED EXPERIMENTAL BONY ROBERT,
L.
CT’CIN,
M.D.,*
DICRAN 9ND
(1972)
COLLAGEN GELS ON DEFECTS: A PRELIMINARY GOULIAN, ALBERT
JR., L.
BELL [2] HAS DESCRIBED the following properties for an ideal bone graft or bone substitute : (1) it is accepted with little or no inflammatory reaction; (2) it is rapidly revascularized; (3) it is replaced with new host osseous tissue; and (4) it undergoes at least surface resorption by the host. Fresh autogenous bone is the most suitable material for this purpose [l], but has the disadvantage that an additional surgical procedure is necessary to obtain the graft. Collagen has been shown to make up 89.2% of t,he organic matrix of bone [7] and to induce calcification both in vitro [lo, 151 and in viva [9]. This report investigates the healing of experimental bony defects following the implantation of antigenically altered collagen gels.
Collagen was prepared from calf skin by solubilizing with proctase, a protease with pH optimum of 3.0, known to remove telopeptides and noncollagenous proteins. It greatly diminishes the antigenicity of the preparat,ion [II].
Ino.
H.
STENZEL,
M.D.,?
M.D.?
One-centimeter lengths of bone were removed from the right 6th and 8th ribs of 11 2-3 kg. albino rabbits after carefully incising and elevating the periosteum under sterile operating conditions. The reconstituted collagen gels were shaped and inserted int’o the defects of t,he 6th ribs in six animals and the 8th ribs in the other five. In all cases, the other rib defect was left without implant; periosteum and intercostal muscles were closed over both ribs in like fashion with 60 silk. No internal or external splinting was attempted. Animals were killed at weekly intervals from 1 to 6 weeks. Radiographs were t’aken before death and read by a radiologist who 318
Press,
K1;RT
OF
METHOD
Submitted for publication Nov. 19, 1971. The authors thank Dr. George Stassa, Departments of Radiology and Anatomy, The New York Hospital-Cornell Medical Center, for interpreting the radiographs of the postoperative rabbits and Dr. George F. Gray, Department of Pathology, The New York Hospital-Cornell Medical Center, for reviewing the decalcified rib sections. *Department of Surgery, Division of Plastic Surgery, The New York Hospital-Cornell Medical Center, New York. ? Rogosin Laboratories, Departments of Biochemistry and Surgery, The New York Hospital-Cornell Medical Center, New York.
Q 1972 by Academic
M.D.,*
HEALING REPORT
It was purified by repeated precipitation by dialysis against 0.02 1M disodium hydrogen phosphate (Na2HP04) and resolubilization in 0.0570 acetic acid. The preparation of this gel for use as a vitrous implant has been reported previously [5, 131. The collagen was then lyophilized and dissolved in dilute hydrochloric acid to obtain a 5% solution of pH 3.0. This 5% collagen solution was then extruded into glass tubing 5-mm. i.d. and gamma irradiat#ed with 1.5 x lo6 rads in nitrogen atmosphere. Gamma irradiation results in increased cross-bonding and tensile strength as well as sterilization of the gels since the sterilizing dose of lo6 rads is exceeded. These reconstituted gels, now in the solid phase, were then dialyzed against sterile lactated Ringer’s solution to remove acid and complete the preparation of sterile implants.
MATERIALS
Copyright
RTBIX,
THE
CUCIN
ET
AL.:
IZECONSTITUTED
COLLAGEN
319
GELS
of the proximal and d&al segments of resected rib; (2) apposition of the two segments; (3) abundance and density of callus. Evans blue dye (0.5%) (T 1824) was injected in a dose of 5 cc/kg. I.V. l/z hour prior to death in one animal of each age. Uptake was interpreted as demonstrating some degree of revascularization of the implant [6]. The ribs were examined grossly, fixed in 10% neutral formalin, decalcified in 10% nitric acid, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were then reviewed for inflammation, callus and new bone formation, and to establish the fate of the collagen implants.
had no prior knowledge of which rib had rccei\-cd the collngcn implant. The following characteristics were evaluated: (1) linearity
RESULTS (1) Chest radiographs showed significantly superior linearity of the rib segments in 64% (VII) and significantly superior apposition of the segmentsin 82% (s 1) of the collagen-implanted ribs. Denser and more abundant callus formation was found in 89% (yg) of the collagen-implanted ribs taken of rabbits killed 2 weeks and longer postoperatively (Fig. 1). (2) Gross examination of the ribs upon death showed surface resorption and decrease in mass by 30-40s of the collagen implant at 1 week. Uptake of Evans blue dye was visible
Fit/. 1. The radiograph depicts resected ribs at 6 nt~~l;s ~)(,~tol’rra~ivc,l~. .Irrows designate the resected ribs ; (* ticnolw tlrr lib wilb the collagen implant.)
Fig.
2. I’hotornicrograph
(II-I<:,
125~)
depicts
osteoid
which
has rrplared
the collagen
implalrt
320
JOURNAL
OF
SURGICAL
RESEARCH,
at 2 w-eeks and beyond by granulation tissue which was later microscopically shown to hare incorporated the collagen implant. The collngen implants could not be grossly distinguished beyond 3 weeks. There were no wound infections. (3) Histologic review demonstrated that the collagen implant was populated mit’h fibroblasts, osteoblasts and osteoclasts, incorporated into the callus and replaced with osteoid (Fig. 2). It evoked no greater inflammatory response than was present in nonimplantcd ribs. DISCUSSION Mergenhagen et al. [9] demonstrated that’ reconstituted collagen gels can be induced to calcify in nonorthotopic locations in Sprague-Dawley rats. Burger, Sherman and Sobel [3] found a moderately accelerated rate of repair of bone defects in the calvaria of albino rats into which acid-soluble collagen was implanted. Pepsin-treated citrate extracts of ox bone have been shown to be a powerful potentiator of new bone formation in rat maxillae as judged by tetracycline uptake [12] and canine maxillae as judged by histologic study [18]. However, the collagen preparation itself greatly influences the tissue handling of the collagen implant so that other preparat’ions have been unsuccessful because of problems of antigenicity, foreign body reactions and infectious complications [4, 141. Two important differences between our study and those of others are: (1) the use of an orthotopic location with scrupulous preservation of the periosteum; and (2) the use of a virtually nonantigenic collagen preparation. This study demonstrated that antigenically altered collagen gels fulfill the criteria for a bone substitute outlined in the introduction, namely, they are noninflammat,ory, revascularized, replaced with host osseous tissue, and resorbed. The tensile strength of the collagen implant could possibly have offered a splinting effect which itself contributed in part to the beneficial effect, on healing. Further experiments which eliminate this effect by choice of implantation site and control implants are in preparation by the authors.
VOL.
12,
NO.
4,
APRIL
1972
SUMRI4RY Sterile ln+octase-treated collagen gels were implanted into surgically created defects in the ribs of rabbits. Ribs were studied radiographically, grossly and microscopically, and by Evans blue dye studies. (1) Inflammatory response to the collagen implant was absent; (2) the collngcn implant aided in the healing of resected ribs by radiographic criteria; (3) the collagen implant was shown microscopically to be included into the callus and granulation tissue and to bc cellularly repopulated. (4) By uptake of Evans blue dye, the collagen implant. was shown to be revascularized. (5) The results indicate that nntigenically nltcrcd collagen aids bone rcconst)itution and furCher studies are warranted. REFERENCES 1. -4ndcrson. K. J. Behavior of 9u1ogcnous and Homogenous Bone Transplants. J. Boric Joi?lt Swg. 43Li :980. 1961. 2. Brll. W. H. Rcsorl)tion Clr:tr:wteristics of Bone and Bone Substitutes. Ot~l Stcty. 17:640. 1964. 3. Burgrr. M.. Shcrmnn. B. S., Sobrl. -A. .\cwlrmtion of Bonp Rrl~air by Chondroitin Sulfate Trentmrnt. of Implants. Report 61-63. School of Aerospace Medicine, USAF, Aerospace Medical Center (ATC). Brooks Air Force Base, Texas, July 1961. 4. Colago, V. Hollenberg, C. Penn, I. Ross. H. M. Singg. W. Fcrguson. C. C. An Evaluation of the Effect, of Powdered Collagen on the Healing of Experimental Bone Dcfrcts. Cnwd. J. Swg. S:412. 1965. 5. Dunn, M. W. Stenzrl. K. H. Rubin. A. 1,. Miynta. T. Collngrn Implants in the Vitrous. An-h. ophthnl. 82 :840. 1969. 6. Goulian. D.. ar. Early Diffrrcntintion Bet,accn Nwrotic and Viable Tissue in Burns. Plnst. Reconstr. Xwq. 27:359, 1961. 7. Herring. G. M. Chclmistry of lhe Boric Matrix. ch. Ortho,. 36:1169, 1964. 5. Hiatt. W. H. Solomons, C. C. Butler, E. D. The Induction of Nrw Bone and Cementum Formnlion 2: Utilizing a Collagen Extract of Ox Bonr. J. Periodonl, 41:274, 1970. 9. Mergenhngm. S. E. Martin. G. R. Risso. rl. :I. Wright, D. N. Scott. D. B. Calcification in Jriz)o of Implantrd Collnpcn. Biochiwz. Xiophys. Acta 43 :563, 19600. 10. Sontanam, M. S. Calcification of Collagen. J. Nolec. Biol. 155, 1959. 11. Schmitt. F. 0. Levine, L. Drake, M. P. Rubin, A. I,. Pfahl. D. Davison. P. F. The Antigcnirit.y of Trophocollngc~n. PJ.OC. Not. Acrid. Rci. (T:SA) 51 :493. 1964.
CUCIK
ET
AL.:
RECO~STITCTEI~
12. Solomons. C. C. Gregory, G. W. An Evaluation of the Effects of Collagen Implants on New Bone Formation in Biro. Tetracycline Uptake by New Bone Mineral. J. Periodont. Res. 1:218, 1966. 13. Stenzel, I<. H. Dunn, M. W. Rubin, A. L. Miyata, T. Collagen Gels: Design for Vitrous Replacenlent. Science 164:1282, 1969.
COLLAGEN
GELS
321
14. Thilander, B. L. Stenstrom. S. J. Bone Healing after Implantation of Some Heteroplnstic and -Al1oplasIic Materials: an Experimental Study on the Guinea Pig. Cleft PnInte J. 7:540, 19iO. 15. Wadkins, C. L. Experimental Factors that Influrnce Collagen Calcification i?L T;itj,o. Cnlcij. 2’bsIce &es 2:214. 196s.
THE
OF
SURGICAL
12, 318-321
RCSEARCII,
EFFECT OF RECONSTITUTED EXPERIMENTAL BONY ROBERT,
L.
CT’CIN,
M.D.,*
DICRAN 9ND
(1972)
COLLAGEN GELS ON DEFECTS: A PRELIMINARY GOULIAN, ALBERT
JR., L.
BELL [2] HAS DESCRIBED the following properties for an ideal bone graft or bone substitute : (1) it is accepted with little or no inflammatory reaction; (2) it is rapidly revascularized; (3) it is replaced with new host osseous tissue; and (4) it undergoes at least surface resorption by the host. Fresh autogenous bone is the most suitable material for this purpose [l], but has the disadvantage that an additional surgical procedure is necessary to obtain the graft. Collagen has been shown to make up 89.2% of t,he organic matrix of bone [7] and to induce calcification both in vitro [lo, 151 and in viva [9]. This report investigates the healing of experimental bony defects following the implantation of antigenically altered collagen gels.
Collagen was prepared from calf skin by solubilizing with proctase, a protease with pH optimum of 3.0, known to remove telopeptides and noncollagenous proteins. It greatly diminishes the antigenicity of the preparat,ion [II].
Ino.
H.
STENZEL,
M.D.,?
M.D.?
One-centimeter lengths of bone were removed from the right 6th and 8th ribs of 11 2-3 kg. albino rabbits after carefully incising and elevating the periosteum under sterile operating conditions. The reconstituted collagen gels were shaped and inserted int’o the defects of t,he 6th ribs in six animals and the 8th ribs in the other five. In all cases, the other rib defect was left without implant; periosteum and intercostal muscles were closed over both ribs in like fashion with 60 silk. No internal or external splinting was attempted. Animals were killed at weekly intervals from 1 to 6 weeks. Radiographs were t’aken before death and read by a radiologist who 318
Press,
K1;RT
OF
METHOD
Submitted for publication Nov. 19, 1971. The authors thank Dr. George Stassa, Departments of Radiology and Anatomy, The New York Hospital-Cornell Medical Center, for interpreting the radiographs of the postoperative rabbits and Dr. George F. Gray, Department of Pathology, The New York Hospital-Cornell Medical Center, for reviewing the decalcified rib sections. *Department of Surgery, Division of Plastic Surgery, The New York Hospital-Cornell Medical Center, New York. ? Rogosin Laboratories, Departments of Biochemistry and Surgery, The New York Hospital-Cornell Medical Center, New York.
Q 1972 by Academic
M.D.,*
HEALING REPORT
It was purified by repeated precipitation by dialysis against 0.02 1M disodium hydrogen phosphate (Na2HP04) and resolubilization in 0.0570 acetic acid. The preparation of this gel for use as a vitrous implant has been reported previously [5, 131. The collagen was then lyophilized and dissolved in dilute hydrochloric acid to obtain a 5% solution of pH 3.0. This 5% collagen solution was then extruded into glass tubing 5-mm. i.d. and gamma irradiat#ed with 1.5 x lo6 rads in nitrogen atmosphere. Gamma irradiation results in increased cross-bonding and tensile strength as well as sterilization of the gels since the sterilizing dose of lo6 rads is exceeded. These reconstituted gels, now in the solid phase, were then dialyzed against sterile lactated Ringer’s solution to remove acid and complete the preparation of sterile implants.
MATERIALS
Copyright
RTBIX,
THE
CUCIN
ET
AL.:
IZECONSTITUTED
COLLAGEN
319
GELS
of the proximal and d&al segments of resected rib; (2) apposition of the two segments; (3) abundance and density of callus. Evans blue dye (0.5%) (T 1824) was injected in a dose of 5 cc/kg. I.V. l/z hour prior to death in one animal of each age. Uptake was interpreted as demonstrating some degree of revascularization of the implant [6]. The ribs were examined grossly, fixed in 10% neutral formalin, decalcified in 10% nitric acid, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Slides were then reviewed for inflammation, callus and new bone formation, and to establish the fate of the collagen implants.
had no prior knowledge of which rib had rccei\-cd the collngcn implant. The following characteristics were evaluated: (1) linearity
RESULTS (1) Chest radiographs showed significantly superior linearity of the rib segments in 64% (VII) and significantly superior apposition of the segmentsin 82% (s 1) of the collagen-implanted ribs. Denser and more abundant callus formation was found in 89% (yg) of the collagen-implanted ribs taken of rabbits killed 2 weeks and longer postoperatively (Fig. 1). (2) Gross examination of the ribs upon death showed surface resorption and decrease in mass by 30-40s of the collagen implant at 1 week. Uptake of Evans blue dye was visible
Fit/. 1. The radiograph depicts resected ribs at 6 nt~~l;s ~)(,~tol’rra~ivc,l~. .Irrows designate the resected ribs ; (* ticnolw tlrr lib wilb the collagen implant.)
Fig.
2. I’hotornicrograph
(II-I<:,
125~)
depicts
osteoid
which
has rrplared
the collagen
implalrt
320
JOURNAL
OF
SURGICAL
RESEARCH,
at 2 w-eeks and beyond by granulation tissue which was later microscopically shown to hare incorporated the collagen implant. The collngen implants could not be grossly distinguished beyond 3 weeks. There were no wound infections. (3) Histologic review demonstrated that the collagen implant was populated mit’h fibroblasts, osteoblasts and osteoclasts, incorporated into the callus and replaced with osteoid (Fig. 2). It evoked no greater inflammatory response than was present in nonimplantcd ribs. DISCUSSION Mergenhagen et al. [9] demonstrated that’ reconstituted collagen gels can be induced to calcify in nonorthotopic locations in Sprague-Dawley rats. Burger, Sherman and Sobel [3] found a moderately accelerated rate of repair of bone defects in the calvaria of albino rats into which acid-soluble collagen was implanted. Pepsin-treated citrate extracts of ox bone have been shown to be a powerful potentiator of new bone formation in rat maxillae as judged by tetracycline uptake [12] and canine maxillae as judged by histologic study [18]. However, the collagen preparation itself greatly influences the tissue handling of the collagen implant so that other preparat’ions have been unsuccessful because of problems of antigenicity, foreign body reactions and infectious complications [4, 141. Two important differences between our study and those of others are: (1) the use of an orthotopic location with scrupulous preservation of the periosteum; and (2) the use of a virtually nonantigenic collagen preparation. This study demonstrated that antigenically altered collagen gels fulfill the criteria for a bone substitute outlined in the introduction, namely, they are noninflammat,ory, revascularized, replaced with host osseous tissue, and resorbed. The tensile strength of the collagen implant could possibly have offered a splinting effect which itself contributed in part to the beneficial effect, on healing. Further experiments which eliminate this effect by choice of implantation site and control implants are in preparation by the authors.
VOL.
12,
NO.
4,
APRIL
1972
SUMRI4RY Sterile ln+octase-treated collagen gels were implanted into surgically created defects in the ribs of rabbits. Ribs were studied radiographically, grossly and microscopically, and by Evans blue dye studies. (1) Inflammatory response to the collagen implant was absent; (2) the collngcn implant aided in the healing of resected ribs by radiographic criteria; (3) the collagen implant was shown microscopically to be included into the callus and granulation tissue and to bc cellularly repopulated. (4) By uptake of Evans blue dye, the collagen implant. was shown to be revascularized. (5) The results indicate that nntigenically nltcrcd collagen aids bone rcconst)itution and furCher studies are warranted. REFERENCES 1. -4ndcrson. K. J. Behavior of 9u1ogcnous and Homogenous Bone Transplants. J. Boric Joi?lt Swg. 43Li :980. 1961. 2. Brll. W. H. Rcsorl)tion Clr:tr:wteristics of Bone and Bone Substitutes. Ot~l Stcty. 17:640. 1964. 3. Burgrr. M.. Shcrmnn. B. S., Sobrl. -A. .\cwlrmtion of Bonp Rrl~air by Chondroitin Sulfate Trentmrnt. of Implants. Report 61-63. School of Aerospace Medicine, USAF, Aerospace Medical Center (ATC). Brooks Air Force Base, Texas, July 1961. 4. Colago, V. Hollenberg, C. Penn, I. Ross. H. M. Singg. W. Fcrguson. C. C. An Evaluation of the Effect, of Powdered Collagen on the Healing of Experimental Bone Dcfrcts. Cnwd. J. Swg. S:412. 1965. 5. Dunn, M. W. Stenzrl. K. H. Rubin. A. 1,. Miynta. T. Collngrn Implants in the Vitrous. An-h. ophthnl. 82 :840. 1969. 6. Goulian. D.. ar. Early Diffrrcntintion Bet,accn Nwrotic and Viable Tissue in Burns. Plnst. Reconstr. Xwq. 27:359, 1961. 7. Herring. G. M. Chclmistry of lhe Boric Matrix. ch. Ortho,. 36:1169, 1964. 5. Hiatt. W. H. Solomons, C. C. Butler, E. D. The Induction of Nrw Bone and Cementum Formnlion 2: Utilizing a Collagen Extract of Ox Bonr. J. Periodonl, 41:274, 1970. 9. Mergenhngm. S. E. Martin. G. R. Risso. rl. :I. Wright, D. N. Scott. D. B. Calcification in Jriz)o of Implantrd Collnpcn. Biochiwz. Xiophys. Acta 43 :563, 19600. 10. Sontanam, M. S. Calcification of Collagen. J. Nolec. Biol. 155, 1959. 11. Schmitt. F. 0. Levine, L. Drake, M. P. Rubin, A. I,. Pfahl. D. Davison. P. F. The Antigcnirit.y of Trophocollngc~n. PJ.OC. Not. Acrid. Rci. (T:SA) 51 :493. 1964.
CUCIK
ET
AL.:
RECO~STITCTEI~
12. Solomons. C. C. Gregory, G. W. An Evaluation of the Effects of Collagen Implants on New Bone Formation in Biro. Tetracycline Uptake by New Bone Mineral. J. Periodont. Res. 1:218, 1966. 13. Stenzel, I<. H. Dunn, M. W. Rubin, A. L. Miyata, T. Collagen Gels: Design for Vitrous Replacenlent. Science 164:1282, 1969.
COLLAGEN
GELS
321
14. Thilander, B. L. Stenstrom. S. J. Bone Healing after Implantation of Some Heteroplnstic and -Al1oplasIic Materials: an Experimental Study on the Guinea Pig. Cleft PnInte J. 7:540, 19iO. 15. Wadkins, C. L. Experimental Factors that Influrnce Collagen Calcification i?L T;itj,o. Cnlcij. 2’bsIce &es 2:214. 196s.