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The effect of chondroitin sulfate on bone healing
Research
The effect of chondroitin on bone healing
S
sulfate
timulation of ostcogenesis by a substance present in borer has been the snbject. of a rrurnhe~ of ins-estigations.l-I-’ Levandcrl” deswihed hone or cart,ilage formation following the intrammxwlar injection of a cell-free alcoholic extract of yonng rabbit hone. Goldhahe+ reported on the suhcutaxlrons implantation of homogi-aft bone contained in difCsion chambws in mice preriously immunized to the homograft tissue. Sew hone formation on the host side of the filter suggested the diffusion of an osteogenic inductor substa~we. 110~s’~ placed Gelfoam sponges impregnated with a solution of dwdroitin sulfate, est,racted from calf bone paste. beneath the leptomeningcs of rats: Histologic examination a.fter 13 days rerealed new ectopic bone formation in all cases. Burger, Sherman, and Xobcl” also employed chonduoitin sulfate as an inductor substance. Thy repwt.ed a consistent acceleration of hone healing in rat calraria when the chondroit~in sulfate was added to varions implant materials. Three chondroitin sulfate compounds hax-e been isolated: A and C, which are normall constituents of hone and cartilage, and B. ~vhich is found in the aorta, skin, and some tendons. 1\Iu~ol)olysaccl?arides form 4 per cent of the OF ganic niatris of bone, with collagen comprisin g 95 per cent.. Cliondroitin sulfate is the major mwopolysaccharide of hone.15-‘9 The present experiment. was designed to test the effect of chondroitin sulfate upon osseous repair in extraction sockets in the maxillae of dogs.
Volume Numljer
Eflect of chondroitin
20 6
adfate
0% bone heal&g
797
The connective tissue that filled the socket was of two types. One was a loose, immature tissue containing inflammatory cells (Fig. 1) ; this was never associated with new bone growth. The second type was characterized by its density. It was cellular and appeared to have a great deal of collagen, but this was not arranged in bundles or in any recognizable pattern The collagen and the ground substance stained deeply eosinophilic. The cells that were present were large and basophilic (Fig. 2). In areas where bone was forming, the cells, became larger and more basophilic. They lined up in the shape of trabeculae and gave the impression that a change in the connective tissue cells had taken place, transforming them into osteoblasts. The control and experimental sides varied with respect to the proportions of the different types of connective tissue present. In all animals killed at 2, 4, and 6 weeks, the control side had more of the loose connective tissue and less of the dense cellular tissue than did t,he chondroitin sulfate side. The appearance of the dense cellular connective tissue preceded bone formation. In the early specimens the chondroitin sulfate side had noticeably larger quantities of t,his dense tissue, which corresponded to the larger quantities of new bone that appeared in these sockets. A periosteal layer of mature connective tissue outlining the new contour that the bone was forming made its appearance at the apical end of the 6-week ehondroitin sulfate socket. At 8 weeks the periosteum outlined the apical portion of both sockets, and by 16 weeks it had progressed to cover the entire socket. Bone growth always began along pre-existent bone and proceeded outward into the socket. Bone did not appear in the center of the socket without contacting one of the walls. The formation of new bone or osteofd tissue had begun in all sockets prior to the first sacrifice. It seemed to start simultaneously all along the walls of the socket. The apex usually appeared to have more rapid growth, but this w.as probably because the labial plate was intact in this area. and the diameter of the socket was narrow.
Pig.
I. Loose
connective
tissue
from
2-week
socket,
coritrol
side.
Pig.
2.
connective
I. Quantitative
hubie
tissue
from
Z-vie&
Arm
(weeks)
4
6
8
16
experimental
side.
evaluation of young bone by areas within the sockets
Sacrifice interval
2
soeket,
Ciest
/
Crest-middle
mX~i?z
socket )
afiaak
/
Apex
/
Totals
1 2 2 1
2 2 1 2
2 2 2 2
6 8 6 5
i
2 32
0 3
3 41
6 IO.
4 C.X.
2
3
i
4
1;
5 5 G 6
c. C.8. c. C.X.
1 2 0 0
2 ii
3
3 3 3 3
9 1.1 3 6
7 7 8 8
c. C.S. c. C.S.
2 2 2 2
2 3 2 2
3 -3. 3 2
10 11 9 8
9 9 10 10
c. C.S. c. C.S.
2 2 2 3
2
3
10 9 9 9
1 c. 1 C.S.
1 2
2 c.
1
2 C.R.
0
3 c. 3 (2.x. 4 6.
“6. = Control socket. C.S. = Chondroitin sulfate
1
1
3’ 2
2 0~ 2 3
; i 33 2 2
i: 2
socketd
At 2 weeks the quantity of bsr;e or osteoid tissue,that, had formed Tniassimilar on both sides in each animal, but at 4 reeks, the &m&&iii sulfate socket forged far ahead of the control socket (Fig. 3). At 6 ~&ks, the- chondroitin sulfate socket contained more bpne than the control s+&t, but th8 di&rence in tbe’cjuantity of bond ppreswas less marked. There was no sjgnific&,nt difference ent in the sockets of the dogs killed 8 &I& 16 weeks postoperatively.
Volume Number
20 6
Effect
of chond~oitin
adfate
on bone keaihg
799
A
B
FOU
Fig. 3 Corn1 jarison r-wee :k’s ocket, control
of new side.
bone
formation.
A, Four-week
socket,
expf:rim
enta ,l side.
B,
In an attempt to make a direct comparison of the quantity and rate of osseous. repair, we examined ea.ch socket in turn. A socket was divided into four portions: crest, crest-middle, middle, and apex. Sn arbitrary rating scale of 0 to 4 was used to compare the quantity of new or young bone present in each portion. A rating of 0 indicated that there was no new bone (or that the quantity present was minute), and a 4 rating indicated that this was the greatest quantity of bone seen. These ratings a,re compiled in Table I. The arithmetic means for each sacrifice interval were calculated and are presen&ed graphically in Fig. 4. Tests for statistical significance were not employed because of the small nutiber of samplesin each sacrifice interval. Resorption of the older bone occurred primarily in two places-the crest
1. A series of mongre! dogs were suhjeeted to hilatexil snrgicai 2xiz3ction of masillar~ cuspids. A gelatin sponge imprcgnat.ed with a solntion of choridr9itill sulfate was implanted in one socket9 and a sal ine-iaipre~rMed sponge vas implmtecl in t,lie control socket. The animais were killed at post.operat.~.l-e intervals of 1 t.o 16 weeks. 2. Within 2 weeks all sockets were cofilpletely filied wit,h cwnnectii-e .tissne. The esperimental sockets contained a greater amount of dense c.ellulsr t,issue: usuall;v associated w&h osteogenesis, than tile control sockets. 3. After 4 and 6 weeks the experimental sockets had m.nre young bon.egro\TtiI than ihe eonwols. This was most,pro:~on;uxc~ at 4 weeks. 4. After 8 weeks no difference ii: the bc~ay l*eljair v-as evident ‘i;et\reen ihe experimental and control sockets. 5. Chond~oit.in sulfate appeared to accelerate the rate of hncy xpair but did not affect the ultimate quantity or quality of bone produced.
Around Autoplastic Ban)Transplants in Hellsta.dius, A. : Studies on Osteqgenesis Bon)- Defects, Acta orthop. scandiuar. 29: 278. 1955. Koenig. H.. and Heller, J. 13.: Affecting the Dynamics of Bone Healing by Administration’of a Bone Extract, Surg. Gynec. Lt Olmt,. 111: 203. 19617. Lacrois! P.: The Organization of Bones? New York, I&&l. The Blakistou Companp. Lacrois, P.: Recent investigations un the Growth of Bone, .Yature, London 156: 576, 1945. Levander, G.: Study of Bone Regeneration, Surg. C;-net. & Obst. 67: 705. 19.33. Rul~stance From Bone Levander, Q.. and Willstaedt, H. : hlc.01~01 Soluble Osteogenic Xarrow, Nature, London 157: 587, I946. Moss, 1%. I,.: Extraction of an Osteogenic Inductor Factor From Bone, Science 127: 7.55, 1.95s. &Ioss, M. I,.: E:xperiment,al Induction of Oxteogenesis. In Sognnaes. R. F. (editor) : CaIcification in Biological Sgsteme, Xashington, 1960, American Associatiou for the Advancement of Science. Willstaedt. H.. Lerander. Ci., and Hult, I,.: Ytudiw in Osteoprnesis, dcta orthol;. I scandinar. 19: i-19, 19.30. Curran, R. C.: The Hisf.ological Demonstration of Connective Tissue Muc.opol-saccharides. III, Clark, 1;‘. (editor) : The Biocbrmistrp of ~~ncopol~sac~harides of Connectire Tissue, London, 1961, Cambridge Unix-ersity Press. Kent, P.. W.: Some Biochemica.1 Asperts of Sulphated Dl~~cosul;stnnccs. Ill, Clark, F. (editor) : The Biochemistry of ~~u~opol~sacchal-isles 0-f Connective Tissue, London;, 1961, Cambridge University Press. Meyer, l<. : The ~Iucopol~saccharides of Bone. 1n SYolstenholme, G. E. TV.: and 0 ?Connor, C. AI. (edit.ors) : Bone Structure and 1ietaholixn. Boston. 1956, Little, Brow1 6r Company. Stacep, AI., and Barker, 9. A.: Carbohpcl~ates of Living Tissues, London, 1.962, D. Van Nostrand Company, Ltd. Suzuki, H. Ii.: Some Studies on the ?&ole of ~,IucopolvsaocIla.rides iu Ossification, J. Xrkansas hf. sot. 59: 576, 1963. Schram: X1’. R.: d Histologic )Study of Repair in the I\lauillary Bones Follorving Surgery, J. Am. Dent. A. 16: 1987> 1929.
The effect of chondroitin on bone healing
S
sulfate
timulation of ostcogenesis by a substance present in borer has been the snbject. of a rrurnhe~ of ins-estigations.l-I-’ Levandcrl” deswihed hone or cart,ilage formation following the intrammxwlar injection of a cell-free alcoholic extract of yonng rabbit hone. Goldhahe+ reported on the suhcutaxlrons implantation of homogi-aft bone contained in difCsion chambws in mice preriously immunized to the homograft tissue. Sew hone formation on the host side of the filter suggested the diffusion of an osteogenic inductor substa~we. 110~s’~ placed Gelfoam sponges impregnated with a solution of dwdroitin sulfate, est,racted from calf bone paste. beneath the leptomeningcs of rats: Histologic examination a.fter 13 days rerealed new ectopic bone formation in all cases. Burger, Sherman, and Xobcl” also employed chonduoitin sulfate as an inductor substance. Thy repwt.ed a consistent acceleration of hone healing in rat calraria when the chondroit~in sulfate was added to varions implant materials. Three chondroitin sulfate compounds hax-e been isolated: A and C, which are normall constituents of hone and cartilage, and B. ~vhich is found in the aorta, skin, and some tendons. 1\Iu~ol)olysaccl?arides form 4 per cent of the OF ganic niatris of bone, with collagen comprisin g 95 per cent.. Cliondroitin sulfate is the major mwopolysaccharide of hone.15-‘9 The present experiment. was designed to test the effect of chondroitin sulfate upon osseous repair in extraction sockets in the maxillae of dogs.
Volume Numljer
Eflect of chondroitin
20 6
adfate
0% bone heal&g
797
The connective tissue that filled the socket was of two types. One was a loose, immature tissue containing inflammatory cells (Fig. 1) ; this was never associated with new bone growth. The second type was characterized by its density. It was cellular and appeared to have a great deal of collagen, but this was not arranged in bundles or in any recognizable pattern The collagen and the ground substance stained deeply eosinophilic. The cells that were present were large and basophilic (Fig. 2). In areas where bone was forming, the cells, became larger and more basophilic. They lined up in the shape of trabeculae and gave the impression that a change in the connective tissue cells had taken place, transforming them into osteoblasts. The control and experimental sides varied with respect to the proportions of the different types of connective tissue present. In all animals killed at 2, 4, and 6 weeks, the control side had more of the loose connective tissue and less of the dense cellular tissue than did t,he chondroitin sulfate side. The appearance of the dense cellular connective tissue preceded bone formation. In the early specimens the chondroitin sulfate side had noticeably larger quantities of t,his dense tissue, which corresponded to the larger quantities of new bone that appeared in these sockets. A periosteal layer of mature connective tissue outlining the new contour that the bone was forming made its appearance at the apical end of the 6-week ehondroitin sulfate socket. At 8 weeks the periosteum outlined the apical portion of both sockets, and by 16 weeks it had progressed to cover the entire socket. Bone growth always began along pre-existent bone and proceeded outward into the socket. Bone did not appear in the center of the socket without contacting one of the walls. The formation of new bone or osteofd tissue had begun in all sockets prior to the first sacrifice. It seemed to start simultaneously all along the walls of the socket. The apex usually appeared to have more rapid growth, but this w.as probably because the labial plate was intact in this area. and the diameter of the socket was narrow.
Pig.
I. Loose
connective
tissue
from
2-week
socket,
coritrol
side.
Pig.
2.
connective
I. Quantitative
hubie
tissue
from
Z-vie&
Arm
(weeks)
4
6
8
16
experimental
side.
evaluation of young bone by areas within the sockets
Sacrifice interval
2
soeket,
Ciest
/
Crest-middle
mX~i?z
socket )
afiaak
/
Apex
/
Totals
1 2 2 1
2 2 1 2
2 2 2 2
6 8 6 5
i
2 32
0 3
3 41
6 IO.
4 C.X.
2
3
i
4
1;
5 5 G 6
c. C.8. c. C.X.
1 2 0 0
2 ii
3
3 3 3 3
9 1.1 3 6
7 7 8 8
c. C.S. c. C.S.
2 2 2 2
2 3 2 2
3 -3. 3 2
10 11 9 8
9 9 10 10
c. C.S. c. C.S.
2 2 2 3
2
3
10 9 9 9
1 c. 1 C.S.
1 2
2 c.
1
2 C.R.
0
3 c. 3 (2.x. 4 6.
“6. = Control socket. C.S. = Chondroitin sulfate
1
1
3’ 2
2 0~ 2 3
; i 33 2 2
i: 2
socketd
At 2 weeks the quantity of bsr;e or osteoid tissue,that, had formed Tniassimilar on both sides in each animal, but at 4 reeks, the &m&&iii sulfate socket forged far ahead of the control socket (Fig. 3). At 6 ~&ks, the- chondroitin sulfate socket contained more bpne than the control s+&t, but th8 di&rence in tbe’cjuantity of bond ppreswas less marked. There was no sjgnific&,nt difference ent in the sockets of the dogs killed 8 &I& 16 weeks postoperatively.
Volume Number
20 6
Effect
of chond~oitin
adfate
on bone keaihg
799
A
B
FOU
Fig. 3 Corn1 jarison r-wee :k’s ocket, control
of new side.
bone
formation.
A, Four-week
socket,
expf:rim
enta ,l side.
B,
In an attempt to make a direct comparison of the quantity and rate of osseous. repair, we examined ea.ch socket in turn. A socket was divided into four portions: crest, crest-middle, middle, and apex. Sn arbitrary rating scale of 0 to 4 was used to compare the quantity of new or young bone present in each portion. A rating of 0 indicated that there was no new bone (or that the quantity present was minute), and a 4 rating indicated that this was the greatest quantity of bone seen. These ratings a,re compiled in Table I. The arithmetic means for each sacrifice interval were calculated and are presen&ed graphically in Fig. 4. Tests for statistical significance were not employed because of the small nutiber of samplesin each sacrifice interval. Resorption of the older bone occurred primarily in two places-the crest
1. A series of mongre! dogs were suhjeeted to hilatexil snrgicai 2xiz3ction of masillar~ cuspids. A gelatin sponge imprcgnat.ed with a solntion of choridr9itill sulfate was implanted in one socket9 and a sal ine-iaipre~rMed sponge vas implmtecl in t,lie control socket. The animais were killed at post.operat.~.l-e intervals of 1 t.o 16 weeks. 2. Within 2 weeks all sockets were cofilpletely filied wit,h cwnnectii-e .tissne. The esperimental sockets contained a greater amount of dense c.ellulsr t,issue: usuall;v associated w&h osteogenesis, than tile control sockets. 3. After 4 and 6 weeks the experimental sockets had m.nre young bon.egro\TtiI than ihe eonwols. This was most,pro:~on;uxc~ at 4 weeks. 4. After 8 weeks no difference ii: the bc~ay l*eljair v-as evident ‘i;et\reen ihe experimental and control sockets. 5. Chond~oit.in sulfate appeared to accelerate the rate of hncy xpair but did not affect the ultimate quantity or quality of bone produced.
Around Autoplastic Ban)Transplants in Hellsta.dius, A. : Studies on Osteqgenesis Bon)- Defects, Acta orthop. scandiuar. 29: 278. 1955. Koenig. H.. and Heller, J. 13.: Affecting the Dynamics of Bone Healing by Administration’of a Bone Extract, Surg. Gynec. Lt Olmt,. 111: 203. 19617. Lacrois! P.: The Organization of Bones? New York, I&&l. The Blakistou Companp. Lacrois, P.: Recent investigations un the Growth of Bone, .Yature, London 156: 576, 1945. Levander, G.: Study of Bone Regeneration, Surg. C;-net. & Obst. 67: 705. 19.33. Rul~stance From Bone Levander, Q.. and Willstaedt, H. : hlc.01~01 Soluble Osteogenic Xarrow, Nature, London 157: 587, I946. Moss, 1%. I,.: Extraction of an Osteogenic Inductor Factor From Bone, Science 127: 7.55, 1.95s. &Ioss, M. I,.: E:xperiment,al Induction of Oxteogenesis. In Sognnaes. R. F. (editor) : CaIcification in Biological Sgsteme, Xashington, 1960, American Associatiou for the Advancement of Science. Willstaedt. H.. Lerander. Ci., and Hult, I,.: Ytudiw in Osteoprnesis, dcta orthol;. I scandinar. 19: i-19, 19.30. Curran, R. C.: The Hisf.ological Demonstration of Connective Tissue Muc.opol-saccharides. III, Clark, 1;‘. (editor) : The Biocbrmistrp of ~~ncopol~sac~harides of Connectire Tissue, London, 1961, Cambridge Unix-ersity Press. Kent, P.. W.: Some Biochemica.1 Asperts of Sulphated Dl~~cosul;stnnccs. Ill, Clark, F. (editor) : The Biochemistry of ~~u~opol~sacchal-isles 0-f Connective Tissue, London;, 1961, Cambridge University Press. Meyer, l<. : The ~Iucopol~saccharides of Bone. 1n SYolstenholme, G. E. TV.: and 0 ?Connor, C. AI. (edit.ors) : Bone Structure and 1ietaholixn. Boston. 1956, Little, Brow1 6r Company. Stacep, AI., and Barker, 9. A.: Carbohpcl~ates of Living Tissues, London, 1.962, D. Van Nostrand Company, Ltd. Suzuki, H. Ii.: Some Studies on the ?&ole of ~,IucopolvsaocIla.rides iu Ossification, J. Xrkansas hf. sot. 59: 576, 1963. Schram: X1’. R.: d Histologic )Study of Repair in the I\lauillary Bones Follorving Surgery, J. Am. Dent. A. 16: 1987> 1929.