- Email: [email protected]
Growth and transformation of the mandibular joint in the rat. V. The effect of pantothenic acid deficiency from birth
Research __- --..GROWTH
AND TRANSFORMATION OF THE MANDIBULAR IN THE RAT. V. THE EFFECT OF PANTOTHENIC ACID DEFICIENCY FROM BIRTH
EVERAL studies have been reported in recent years the S fraction and pantothenic acid deficiencies on the osseous structures mice, and rats.1, 2, 3. j, C,7, S.9, II on
effects
JOINT
of filtrate
of dogs, The changes seen in the long bones of mice” and rat+ B have been described as impairment of chondrogenesis, osteogenesis, and hematopoiesis accompanied in rat,sb by a rapid resorption of bone trabcculac and early “sealing off” of the epiphyseal disc. Rats dcficicnt in pantot,henic acid from birth” showed severe oral ehang::~s, which consisted of hyperkeratosis and necrosis of the oral mueosa and iutcrdcntal papillae in t,hc complete absence of a leukocytic response. Hcsorption of alveolar bone was observed frequently. Similar changes were induced in wcanling rats on a low pantothenic acid diet by t,osic doses of zinc carbonate” and in dogs 1)s deficiency of the entire filtrate fractiom3 The effects of a pantothenic acid tlclficic~ncy on the mandibular joint of mice deficient from weaning (5 t,o 28 da)-s of age) consisted of a retardation of growth, cartilage hypoplasia, and dccrcased rate of bone formation.” The severe changes reported by K\‘clson and associatrs” in the tibia OF rats maintained on a diet deficient in pantothenic. acid f’rom birth (day 1 ) suggested studies of the mandibular condgle of the same animals. Material
and Methods
Litters of normal rats totaling 330 animals, together with their mothers (Long-Evans strain), werr placed on the pantothenic acid or control diet on the day of birth. The litters were weaned at 21 continued on the same diets.* The average survival for 226 deficient
lactating deficient days and rats was
From tlw Division of Dental Medicine: College of Denistry, The George Williams Hoopel Foundation for Medical Research, the ln-,tltute of Experimental Biology, University of California, San Francisco and Rerkeley, Calif. 13ided by grants fronr thr American Foumlation for Dental Science, the Board of Research and the Department of Agriculture of the University of California, the Rockefeller Foundation, New York City, and the California State Dental Association. Received for publication June 9. 1952. *The pantothenic acid deficient dietaP 9 was CornPosed of 24% alcohol-extracted casein, 64% SUCrOse, SC/O hydrogenated cottonseed oil (Crisco) supplemented with crystalline thiamine, pyridoxin. riboflavin, p-aminobenzoic acid, nicotinic acid, inositol, and choline chloride. A fat-soluble vitamin mixture containing vitamins 4 D, I<, and unsaturated fatty acids was given Once a week. Control animals received the Identical purified diet supplementedwith a high level of synthetic calcium pantothenate.
GROWTH
AND
TRANSFORMATION
OF
MANDIBULAR
JOIKT
IN
RAT.
V
893
35 days and only 13 per cent lived past 60 days of age. Twenty deficient rats (13 males and 7 females) t,hat were most severely affected were sacrificed, together with their controls, between 3 and 16 weeks of age for histological study. The mandibular joints were dissected free, decalcified in 5 per cent nitric acid, embedded in nitrocellulose, sectioned in the mediolateral direction, and stained with hematoxylin and eosin.
Results Histologically the condyles of the control rats fed the purified diet supplemented with calcium pantothenate were consistent with the normal standard series reported by Collins and associates.4 The mandibular condyle of a 38-dayold control rat is illustrated in Fig. 1. The cartilage of the condylar head is crescent-shaped and broad. It is differentiated into the three zones of embryonic, intermediate, and vesicular cells. There is active invasion of the cartilage bv capillaries at the line of erosion. The bone trabeculae are short and delicate and they are densely lined with osteoblasts. At 106 days of age the appearance of t,he normal mandibular condyle has changed considerably (Fig. 2). The cartilage is narrower, the zone of vesicular cells has almost disappeared, and the calcification of the cartilage extends into the intermediate zone. The activity in the zone of erosion has slowed down, with only few capillaries invading cartilage cells. Osteoblasts are scarce; the bone trabeculae are coarse and united with the calcified cartilage. The marked progressive changes in the mandibular condyles of rats fed the diet deficient in pantothenic acid were classified into four stages according to severity of changes. Due to variations in the individual response to the lack of pantothenic acid, the degree of histologic and morphologic change did not necessarily correlate with the duration of the deficiency period.
Stage I.-The shown in Fig. 3. It in the control (Fig. zone. The zone of bone trabeculae are are seen occasionally
condyle of a 38-day-old pantothenic acid deficient rat is can be noted that the broad zone of vesicular cells as seen 1) is absent, as well as the major part of the intermediate erosion is narrow and osteoblasts seem to be scarce. The coarse and blunt. Slightly below the erosion zone giant cells near the surface of the bony trabeculae.
Stage IL-The condyle of a 107-day-old pantothenic acid deficient rat (Fig. 4) shows a greater decrease in the width of the cartilage than the normal control (Fig. 2). The zone of vesicular cells is absent.. The embryonic and intermediate zones are considerably narrower and the latter almost fully calcified. Osteogenesis seems to have ceased, as hardly any osteoblasts are seen and only a few scattered trabeculae remain. Stage III.-In this stage (Fig. 5, the condyle of a 109-day-old deficient rat) the changes as seen in Stage II are still more pronounced. The contours of the condylar head are fading away. The vesicular and intermediate zones of the cartilage have disappeared, leaving a narrow zone of embryonic cells. The bone trabeculae are scarce and the lamina compacta on the medial side
=Z c~ouspiciious nw formation of immature, of the condylar head is atwllt. spongy bone is uotcd gwnving o(lt from t Ire Iattral likikiiua vompacta. l’llc rnctlial inferior aspect of the ayticular disc is undergoin g resorption; histiocytes WC seen infiltrating tlw tissue’.
7,909. X,(i34. 8,580. 8,577.
Fig. Fig. Fir. Pl. Fig. PI. Fig. PI. Fig. PI.
I.-Mandibular 2.-Mandibular 3.-Mandibular 9,900.) I.-Mandibular 9.910.) 5.-Mandibular 9,912.) F.-Mandibulav 9,636.)
condylc of RB-clay--ol~l rat. (X90. Spec. 7,920. PI. 13 387.) rondyl~ of 1 (IC-
of Ill:-day-old
pantothenic
acid
deficient
rat.
cvndyle
of 109-clay-old
pantothenic
acid
deficient
rat.
(X90.
rnndyle
of 97.(lay-old
deficient
rat.
( X 90. Spec.
pantothenic
acid
( x 90. Spec. Spec.
GROWTH
AND
TRANSFORMATION
OF MANDIBULAR
,JOlNT
IN
RAT.
V
895
Stage IV.-This stage represents the severest changes encountered (Fig. 6). The illustration is t,aken from the condyle of a 97-day-old deficient rat. Morphologically the condyle has only little resemblance to that of a normal rat.
Fig. 7.-Same rat as in Fig. 6. Survey of condyle and ascending ramus A, Osteophytes arising from the ramus; osteophytes arising from the B, (X40. Pl. B 391.) Fig. K-Higher magnification of area A in Fig. 7. (X90. Pl. 9,637.) Pl. R 390.) .Fig. I).-Higher magnification of area B in Fig. 7. (X90.
of the mandible. squamosal bone.
The cartilage is destroyed except for a few isolated groups of cells. Osteoclasts are noted on the surface of the few remaining trabeculae. Fibrous tissue has taken the place of most of the trabeculae filling the large marrow spaces. The avascular, fibrous tissue of the glenoid fossa and the condylar head, the synovial
membrane, and the articular disc are necrotic. Immature, spongy boric is extending from the medial side of ilte bone in the neck region. The same condyle as seen in Fig. 6, together wit,11 the ramux and the cranial portion of the .joint,, is shown in a survey photomicrograph in %‘ig. 7. The cxtensivc destruction of bono and cartilage and the process of osteoclasia advancing in inferolateral direction Ostcophytes are arising from both sides from the condylar head may be noted. of the ramus (Fig. 7, A) and from the superolateral surface of the squamosal bone (Fig. 7, B). The trabeculae of the osteophytic bone from the ramus arc’ directed obliquely downward following the orientatidn of the connective tissue’. The osteophytes arising from the lateral surface of the ramus arc s(lcn at a higher magnification in Fig. 8. The new bone is extrcrnely cellular and the apices of the trabeculae consist of osteoid tissue. This, in connrction with the presence of a pronounced zone of cellular differentiation around the tips of the trabeculae, indicates rapid bone formation. The formation of new spongy bone on the superolateral surface of the squamosal bone is illustrated in Fig. 9. The trabeculae are shorter and more anastomosed than those proliferating from the ramus. IIere the muscle tissue. which under normal conditions lies closet t,o the bone surface, is pushed away 1,~ the new growth. Summary and Conclusions The histologic changes observed in the condyles of 20 rats maintained on a pantothenic acid deficient diet from birth are characterized by marked impairment of chondrogenesis and osteogenesis and in the late stages by necrosis of the articular capsule, the articular disc, and the fibrous tissue in the glenoid fossa resulting in an almost complete destruction of the mandibular joint. The severity of the changes seems to depend more upon the individual response to the deficiency than upon the duration of the deficiency. Fibrous tissue replaces the boric marrow and t,he disintegrated bone trabeculae. The inhibition of chondrogcnesis and osteogenesis is more severe than that reported for pantothenic acid deficiency in mict?.F This might be anticipated in view of the acute syndrome produced by instituting the deficiency at birth in the rat. In the late morphologic stages seen in this stud>- the bone resorption led to an almost complete absencxc of trabcculae in the condylar head and to parGal disappearance of the lamina compacta of the ramus. At the same time ostcophytic growth arose from the pcriosteal surface of the ramus and squamosal bone. The common feature in all instances of osteophytic growth was a disintegration of the lamina compacta and loss of trabeculac. The growth may be esplaimd as a compensation for the rapid destruction of the condylar head. Despite the pronounced morphologic changes produced by t,he lilck of pantothenic acid and the prevention of all changes by administration of calcium pantothenate, it should not be in-l’rrrcd that, all of these changes wcrc~ due specifically to the lack of this vitamin alone. Daft and associate9 indicated that the blood and boric marrow changes which t,hep observed were not wholly mani‘festations of uncomplicated deficienc..v of t,his vitamin. Moreover, it, has been lo that the proper utilization of pantothenie suggested by Wright and Welch
GROWTH
AND
TRANSFORMATION
OF MANDIBULAR
JOINT
IN
RAT.
V
897
acid by the tissues of rats requires the availability of folic acid and biotin. Neither of these vitamins was added to the purified diet used in this study. Finally, while the marked inanition which accompanied this deficiency may explain retardation of chondrogenesis and osteogenesis, it is not likely to account for the striking changes seen in the later stages. References 1. Ashburn, L. L.: The Effect of Administration of Pantothenic Acid on the Histopathology of the Filtrate Factor Deficiency State in Rats, Pub. Health Rep. 55: 1337, 1940. The Effect of Deficiencies of the Filtrate Fraction of 2. Becks, H., and Morgan, A. F.: J. the Vitamin B Comnlex and of Nicotinic Acid on Teeth and Oral Structures, Periodontol, 13: 18, i942. 3. Becks, H., Wainwright, W. W., and Morgan, A. F.: Comparative Study of Oral Changes in Dogs Due to Deficiencies of Pantothenic Acid, Nicotinic Acid, and Unknowns of the B Vitamin Complex, Am. J. Orthodontics and Oral Surg. 29: 153, 1943. 4. Collins, D. A., Becks, H., Simpson, M. E., and Evans, H. M.: Growth and Transformation of the Mandibular Joint in the Rat. I. Normal Female Rats, Am. J. Orthodontics and Oral Surg. 32: 431, 1946. 5. Daft, F. S., Kornberg, A., Ashburn, T. L., and Sebrell, W. H.: Anemia and Granulocytopenia in Rats Fed a Diet Low in Pantothenic Acid, Pub. Health Rep. 60: 1201. 1945. 6. Levy, B.‘M.: Effect of Pantothenic Acid Deficiency on the Mandibular Joints and Periodontal Structures of Mice, J. Am. Dent. A. 38: 215, 1949. 7. Levy, B. M., and Silberberg, M.: Inhibition of Endochondral Ossification in Pantothenic Acid Deficiency, Proe. Sot. Exper. Biol. & Med. 63: 380, 1946. 8. Nelson, M. M., Sulon, E., Becks, H., Wainwright, W. W., and Evans, H. M.: Changes in Endochondral Ossification of the Tibia Accompanying Acute Pantothenic Acid Deficiency in Young Rats, Proc. Sot. Exper. Biol. & Med. 73: 31, 1950. 9. Wainwright, W. W., and Nelson, M. M.: Changes in the Oral Mucosa Accompanying Acute Pantothenic Acid Deficiency in Young Rats, Am. J. Orthodontics and Oral Burg. (Oral Surg. Sect.) 31: 406, 1945. 10. Wright, L. D., Welch, A. D.: Role of “Folic Acid” and Biotin in the Utilization of Pantothenic Acid bv the Rat. Sc’ience 97: 426.1943. 11. Ziskin, D. E., Stein, G:, Gross, ‘P., and Runne, ‘E.: Oral, Gingival, and Periodontal Pathology Induced in Rats on a Low Pantothenic Acid Diet by Toxic Doses of Zinc Carbonate, Am. J. Orthodontics and Oral Surg. (Oral Surg. Sect.) 33: 407, 1947.
AND TRANSFORMATION OF THE MANDIBULAR IN THE RAT. V. THE EFFECT OF PANTOTHENIC ACID DEFICIENCY FROM BIRTH
EVERAL studies have been reported in recent years the S fraction and pantothenic acid deficiencies on the osseous structures mice, and rats.1, 2, 3. j, C,7, S.9, II on
effects
JOINT
of filtrate
of dogs, The changes seen in the long bones of mice” and rat+ B have been described as impairment of chondrogenesis, osteogenesis, and hematopoiesis accompanied in rat,sb by a rapid resorption of bone trabcculac and early “sealing off” of the epiphyseal disc. Rats dcficicnt in pantot,henic acid from birth” showed severe oral ehang::~s, which consisted of hyperkeratosis and necrosis of the oral mueosa and iutcrdcntal papillae in t,hc complete absence of a leukocytic response. Hcsorption of alveolar bone was observed frequently. Similar changes were induced in wcanling rats on a low pantothenic acid diet by t,osic doses of zinc carbonate” and in dogs 1)s deficiency of the entire filtrate fractiom3 The effects of a pantothenic acid tlclficic~ncy on the mandibular joint of mice deficient from weaning (5 t,o 28 da)-s of age) consisted of a retardation of growth, cartilage hypoplasia, and dccrcased rate of bone formation.” The severe changes reported by K\‘clson and associatrs” in the tibia OF rats maintained on a diet deficient in pantothenic. acid f’rom birth (day 1 ) suggested studies of the mandibular condgle of the same animals. Material
and Methods
Litters of normal rats totaling 330 animals, together with their mothers (Long-Evans strain), werr placed on the pantothenic acid or control diet on the day of birth. The litters were weaned at 21 continued on the same diets.* The average survival for 226 deficient
lactating deficient days and rats was
From tlw Division of Dental Medicine: College of Denistry, The George Williams Hoopel Foundation for Medical Research, the ln-,tltute of Experimental Biology, University of California, San Francisco and Rerkeley, Calif. 13ided by grants fronr thr American Foumlation for Dental Science, the Board of Research and the Department of Agriculture of the University of California, the Rockefeller Foundation, New York City, and the California State Dental Association. Received for publication June 9. 1952. *The pantothenic acid deficient dietaP 9 was CornPosed of 24% alcohol-extracted casein, 64% SUCrOse, SC/O hydrogenated cottonseed oil (Crisco) supplemented with crystalline thiamine, pyridoxin. riboflavin, p-aminobenzoic acid, nicotinic acid, inositol, and choline chloride. A fat-soluble vitamin mixture containing vitamins 4 D, I<, and unsaturated fatty acids was given Once a week. Control animals received the Identical purified diet supplementedwith a high level of synthetic calcium pantothenate.
GROWTH
AND
TRANSFORMATION
OF
MANDIBULAR
JOIKT
IN
RAT.
V
893
35 days and only 13 per cent lived past 60 days of age. Twenty deficient rats (13 males and 7 females) t,hat were most severely affected were sacrificed, together with their controls, between 3 and 16 weeks of age for histological study. The mandibular joints were dissected free, decalcified in 5 per cent nitric acid, embedded in nitrocellulose, sectioned in the mediolateral direction, and stained with hematoxylin and eosin.
Results Histologically the condyles of the control rats fed the purified diet supplemented with calcium pantothenate were consistent with the normal standard series reported by Collins and associates.4 The mandibular condyle of a 38-dayold control rat is illustrated in Fig. 1. The cartilage of the condylar head is crescent-shaped and broad. It is differentiated into the three zones of embryonic, intermediate, and vesicular cells. There is active invasion of the cartilage bv capillaries at the line of erosion. The bone trabeculae are short and delicate and they are densely lined with osteoblasts. At 106 days of age the appearance of t,he normal mandibular condyle has changed considerably (Fig. 2). The cartilage is narrower, the zone of vesicular cells has almost disappeared, and the calcification of the cartilage extends into the intermediate zone. The activity in the zone of erosion has slowed down, with only few capillaries invading cartilage cells. Osteoblasts are scarce; the bone trabeculae are coarse and united with the calcified cartilage. The marked progressive changes in the mandibular condyles of rats fed the diet deficient in pantothenic acid were classified into four stages according to severity of changes. Due to variations in the individual response to the lack of pantothenic acid, the degree of histologic and morphologic change did not necessarily correlate with the duration of the deficiency period.
Stage I.-The shown in Fig. 3. It in the control (Fig. zone. The zone of bone trabeculae are are seen occasionally
condyle of a 38-day-old pantothenic acid deficient rat is can be noted that the broad zone of vesicular cells as seen 1) is absent, as well as the major part of the intermediate erosion is narrow and osteoblasts seem to be scarce. The coarse and blunt. Slightly below the erosion zone giant cells near the surface of the bony trabeculae.
Stage IL-The condyle of a 107-day-old pantothenic acid deficient rat (Fig. 4) shows a greater decrease in the width of the cartilage than the normal control (Fig. 2). The zone of vesicular cells is absent.. The embryonic and intermediate zones are considerably narrower and the latter almost fully calcified. Osteogenesis seems to have ceased, as hardly any osteoblasts are seen and only a few scattered trabeculae remain. Stage III.-In this stage (Fig. 5, the condyle of a 109-day-old deficient rat) the changes as seen in Stage II are still more pronounced. The contours of the condylar head are fading away. The vesicular and intermediate zones of the cartilage have disappeared, leaving a narrow zone of embryonic cells. The bone trabeculae are scarce and the lamina compacta on the medial side
=Z c~ouspiciious nw formation of immature, of the condylar head is atwllt. spongy bone is uotcd gwnving o(lt from t Ire Iattral likikiiua vompacta. l’llc rnctlial inferior aspect of the ayticular disc is undergoin g resorption; histiocytes WC seen infiltrating tlw tissue’.
7,909. X,(i34. 8,580. 8,577.
Fig. Fig. Fir. Pl. Fig. PI. Fig. PI. Fig. PI.
I.-Mandibular 2.-Mandibular 3.-Mandibular 9,900.) I.-Mandibular 9.910.) 5.-Mandibular 9,912.) F.-Mandibulav 9,636.)
condylc of RB-clay--ol~l rat. (X90. Spec. 7,920. PI. 13 387.) rondyl~ of 1 (IC-
of Ill:-day-old
pantothenic
acid
deficient
rat.
cvndyle
of 109-clay-old
pantothenic
acid
deficient
rat.
(X90.
rnndyle
of 97.(lay-old
deficient
rat.
( X 90. Spec.
pantothenic
acid
( x 90. Spec. Spec.
GROWTH
AND
TRANSFORMATION
OF MANDIBULAR
,JOlNT
IN
RAT.
V
895
Stage IV.-This stage represents the severest changes encountered (Fig. 6). The illustration is t,aken from the condyle of a 97-day-old deficient rat. Morphologically the condyle has only little resemblance to that of a normal rat.
Fig. 7.-Same rat as in Fig. 6. Survey of condyle and ascending ramus A, Osteophytes arising from the ramus; osteophytes arising from the B, (X40. Pl. B 391.) Fig. K-Higher magnification of area A in Fig. 7. (X90. Pl. 9,637.) Pl. R 390.) .Fig. I).-Higher magnification of area B in Fig. 7. (X90.
of the mandible. squamosal bone.
The cartilage is destroyed except for a few isolated groups of cells. Osteoclasts are noted on the surface of the few remaining trabeculae. Fibrous tissue has taken the place of most of the trabeculae filling the large marrow spaces. The avascular, fibrous tissue of the glenoid fossa and the condylar head, the synovial
membrane, and the articular disc are necrotic. Immature, spongy boric is extending from the medial side of ilte bone in the neck region. The same condyle as seen in Fig. 6, together wit,11 the ramux and the cranial portion of the .joint,, is shown in a survey photomicrograph in %‘ig. 7. The cxtensivc destruction of bono and cartilage and the process of osteoclasia advancing in inferolateral direction Ostcophytes are arising from both sides from the condylar head may be noted. of the ramus (Fig. 7, A) and from the superolateral surface of the squamosal bone (Fig. 7, B). The trabeculae of the osteophytic bone from the ramus arc’ directed obliquely downward following the orientatidn of the connective tissue’. The osteophytes arising from the lateral surface of the ramus arc s(lcn at a higher magnification in Fig. 8. The new bone is extrcrnely cellular and the apices of the trabeculae consist of osteoid tissue. This, in connrction with the presence of a pronounced zone of cellular differentiation around the tips of the trabeculae, indicates rapid bone formation. The formation of new spongy bone on the superolateral surface of the squamosal bone is illustrated in Fig. 9. The trabeculae are shorter and more anastomosed than those proliferating from the ramus. IIere the muscle tissue. which under normal conditions lies closet t,o the bone surface, is pushed away 1,~ the new growth. Summary and Conclusions The histologic changes observed in the condyles of 20 rats maintained on a pantothenic acid deficient diet from birth are characterized by marked impairment of chondrogenesis and osteogenesis and in the late stages by necrosis of the articular capsule, the articular disc, and the fibrous tissue in the glenoid fossa resulting in an almost complete destruction of the mandibular joint. The severity of the changes seems to depend more upon the individual response to the deficiency than upon the duration of the deficiency. Fibrous tissue replaces the boric marrow and t,he disintegrated bone trabeculae. The inhibition of chondrogcnesis and osteogenesis is more severe than that reported for pantothenic acid deficiency in mict?.F This might be anticipated in view of the acute syndrome produced by instituting the deficiency at birth in the rat. In the late morphologic stages seen in this stud>- the bone resorption led to an almost complete absencxc of trabcculae in the condylar head and to parGal disappearance of the lamina compacta of the ramus. At the same time ostcophytic growth arose from the pcriosteal surface of the ramus and squamosal bone. The common feature in all instances of osteophytic growth was a disintegration of the lamina compacta and loss of trabeculac. The growth may be esplaimd as a compensation for the rapid destruction of the condylar head. Despite the pronounced morphologic changes produced by t,he lilck of pantothenic acid and the prevention of all changes by administration of calcium pantothenate, it should not be in-l’rrrcd that, all of these changes wcrc~ due specifically to the lack of this vitamin alone. Daft and associate9 indicated that the blood and boric marrow changes which t,hep observed were not wholly mani‘festations of uncomplicated deficienc..v of t,his vitamin. Moreover, it, has been lo that the proper utilization of pantothenie suggested by Wright and Welch
GROWTH
AND
TRANSFORMATION
OF MANDIBULAR
JOINT
IN
RAT.
V
897
acid by the tissues of rats requires the availability of folic acid and biotin. Neither of these vitamins was added to the purified diet used in this study. Finally, while the marked inanition which accompanied this deficiency may explain retardation of chondrogenesis and osteogenesis, it is not likely to account for the striking changes seen in the later stages. References 1. Ashburn, L. L.: The Effect of Administration of Pantothenic Acid on the Histopathology of the Filtrate Factor Deficiency State in Rats, Pub. Health Rep. 55: 1337, 1940. The Effect of Deficiencies of the Filtrate Fraction of 2. Becks, H., and Morgan, A. F.: J. the Vitamin B Comnlex and of Nicotinic Acid on Teeth and Oral Structures, Periodontol, 13: 18, i942. 3. Becks, H., Wainwright, W. W., and Morgan, A. F.: Comparative Study of Oral Changes in Dogs Due to Deficiencies of Pantothenic Acid, Nicotinic Acid, and Unknowns of the B Vitamin Complex, Am. J. Orthodontics and Oral Surg. 29: 153, 1943. 4. Collins, D. A., Becks, H., Simpson, M. E., and Evans, H. M.: Growth and Transformation of the Mandibular Joint in the Rat. I. Normal Female Rats, Am. J. Orthodontics and Oral Surg. 32: 431, 1946. 5. Daft, F. S., Kornberg, A., Ashburn, T. L., and Sebrell, W. H.: Anemia and Granulocytopenia in Rats Fed a Diet Low in Pantothenic Acid, Pub. Health Rep. 60: 1201. 1945. 6. Levy, B.‘M.: Effect of Pantothenic Acid Deficiency on the Mandibular Joints and Periodontal Structures of Mice, J. Am. Dent. A. 38: 215, 1949. 7. Levy, B. M., and Silberberg, M.: Inhibition of Endochondral Ossification in Pantothenic Acid Deficiency, Proe. Sot. Exper. Biol. & Med. 63: 380, 1946. 8. Nelson, M. M., Sulon, E., Becks, H., Wainwright, W. W., and Evans, H. M.: Changes in Endochondral Ossification of the Tibia Accompanying Acute Pantothenic Acid Deficiency in Young Rats, Proc. Sot. Exper. Biol. & Med. 73: 31, 1950. 9. Wainwright, W. W., and Nelson, M. M.: Changes in the Oral Mucosa Accompanying Acute Pantothenic Acid Deficiency in Young Rats, Am. J. Orthodontics and Oral Burg. (Oral Surg. Sect.) 31: 406, 1945. 10. Wright, L. D., Welch, A. D.: Role of “Folic Acid” and Biotin in the Utilization of Pantothenic Acid bv the Rat. Sc’ience 97: 426.1943. 11. Ziskin, D. E., Stein, G:, Gross, ‘P., and Runne, ‘E.: Oral, Gingival, and Periodontal Pathology Induced in Rats on a Low Pantothenic Acid Diet by Toxic Doses of Zinc Carbonate, Am. J. Orthodontics and Oral Surg. (Oral Surg. Sect.) 33: 407, 1947.