Upsala J Med Sci 79: 18-20, 1974 Effect of an Enzyme-Resistant Phosphopeptide on Calcification of Embryo Chicken Bone in Vitro CONNY EDENO Department of Pathology I and Department of Medical Chemistry, University of Goteborg, Sweden ABSTRACT Cultivation of embryonic chicken bone in vitro enables substances to be added to the culture medium in order to ascertain how they affect the histological development of the bone. This method has been adopted for studying an enzyme-resistant phospbopeptide extracted by Mel- lander from casein. By cultivating paired bones that from one side can be used as a control for the contra- lateral bone. The test group was given calcium complexly bound to the phosphopeptide and the control group calcium as CaCI,. Studies of bones from embryos of different incubation ages after cultivation for various periods in media containing different concentration of calcium revealed that similar degress of ossification and rates of osteoid tissue formation were achieved when the phospbopeptide was the source of calcium as when it was CaCI,. These experiments have demonstrated that calcium bound to a phosphopeptide can be utilized in the ossifcation process just as well as readily soluble inorganic calcium. It has long been known that phosphoproteins are present in such products a s milk-there in the form of casein-and that considerable amounts of phosphorylated peptides are formed when these proteins a r e hydrolyzed in the human intestinal tract (1). Using experimental animals with rickets fed special diets, Mellander discovered an anti- rachitic effect of such phosphorylated peptides ( 2 ) . This effect has been interpreted a s the result of increased intestinal absorption of calcium com- plexly bound to phosphorylated peptides ( 3 ) . We have in the present investigation studied their direct action on the ossification of embryonic chicken bone in an in v i t r o system. Most previous trials with bone cultivation have been done with chicken bone. T h e y have revealed that bone from 6-day chick embryos incubated a t 36°C c a n be explanted, that histologically they continue t o differentiate normally, and that they Upsala J Med Sci 79 exhibit considerable longitudinal growth (4). Bone tissue from 6-day chick embryos have also been used for studying the effects in v i t r o of various hormones on longitudinal growth and ossification (5, 6 , 7, 8, 9, 10, 11, 12, 13). Such experiments, all using a mixture of plasma and chicken embryo extract a s culture medium, have disclosed that both longitudinal growth and ossification are significantly affected by insulin, thyroxin, cortisone, vitamin A and parathyroid hormone. Embryo chicken bone, incubated at 36", de- velop in v i v o from commencing differentiation a t 6 days t o a bone with well developed marrow cavity at 12 to 16 days of age (14). Pilot studies in this laboratory o n embryonic chick tibiae aged 6 t o 17 d a y s and cultivated for 1 to 15 days demonstraeted that 10 to 14 days was a suitable age for studying ossification and the formation of osteoid tissue. T E C H N I Q U E S Dissection was carried out with a cataract knife and tweezers under a dissection microscope. All bones were measured before and after cultivation by means of Trowell's grid technique (15). According to Franks, the ambient gas phase may well be air for cultivation of embryonic organs, so we chose air (16). The medium was .Eagle's minimum essential medium for Spinner cul- tures>> with 10% calf serum (17). To avoid cells attaching themselves to the grid, we placed the bones on milli- pore paper which accompanied them to fixation. The specimens were fixed i n 10% formalin neutralized with calcium, dehydrated, embedded in paraffine, sectioned longitudinally in 5 to 10 slices, and stained with haema- toxylin-eosine according to van Gieson and von Kossa. First the tibia was dissected out from 6 embryos in each age group of 10, 12 and 14 days. Thus each embryo yielded 2 bones, one of which was cultivated in medium plus calcium chloride and the other in medium plus phosphopeptide. Caseinphosphopeptide-effect on chicken bone in vitro I9 The amounts of CaCl, and phosphopeptide were such as to yield concentrations of calcium of 100 mg/ litre. Two of the 6 embryos in each age group were for 5 , 10 and 15 days respectively. RESULTS m e specimens were examined after staining and judged on the basis of the degree of ossification and the amount of osteoid tissue. Both variables were classified according to a scale from 0 to 3 , where 0 implies no ossification or osteoid tissue and 3 denotes marked ossification and plenty of osteoid tissue respectively. With respect to both controls and experimental bones it appeared that: (i) both ossification and osteoid tissue were equal to 3 in the group incubated for 14 days regardless of how long they were cultivated; (ii) both ossification and osteoid tissue were rated 0.5 in the group incubated for 10 days, regardless of how long they were cultivated; and (iii) osteoid tissue rated 2.5 in the experimental group and 2.7 in the controls while ossification was 3 in the experimental group and 2 in the controls among embryos incubated for 12 days. The differences observed between experimental and control groups were confined to those embryos incubated for 10 days and cultivated for 10 days and to those incubated for 12 and cultivated for 5 days. Embryos 10 to 12 days old are evidently in a critical state with respect to ossification and formation of osteoid tissue. Hence we examined 20 additional embryos incubated for 10 days and cultivated for 6 and 8 days, as described above. The results were as follows: It will be seen that no differences in amount of osteoid tissue and degree of ossification could be demonstrated between embryonic chicken bones cultivated in a medium containing 100 mg calcium per litre in the form of CaCl, and similar cul- tures in a medium containing calcium complexly bound to a phosphopeptide. Incub. Cultiv No. of time, time Ca source bones days days CaCl, 10 10 6 Phospho-peptide 10 10 6 CaCl, 10 10 8 i Phospho-peptide 10 10 8 To exclude the possibility that a concentration of 100 mg of calcium per litre was too high to allow any differences to appear, we carried out another experiment in which the calcium con- centration was reduced to 25 mg per litre. For this latter experiment we chose embryos incubated for 9 days and cultivated them for 8 days in the same way as before. It turned out that longitudinal growth in the experimental group was 1 . 1 - t O . 5 mm and 1.35 0.6 mm in the control group. The amount of osteoid tissue in the two groups was 2.2 and 2.3 respec- tively. No bone exhibited any calcification. Consequently not even a calcium concentration which is extremely low for tissue cultivation pur- poses could expose any differences between the phosphopeptide and calcium chloride in either ossification or the rate of osteoid tissue formation in embryonic chicken bone. REFERENCES 1. 2 . 3. 4 . 5 . 6 . 7. Mellander, 0. & Folsch, G.: Enzyme resistance and metal binding of phosphorylated peptides. I. E. F. N., vol. 1 1 , p. 569 (ed. E. J . Bigwood). Pergamon Press, Oxford and New York, 1972. Mellander, I. & Olsson, N.: The influence of pep- tide bound calcium and phosphorous on bone cal- cification in rickets. Numero extraordinario del Bolitin Medico del Hospital Infantil, Vol. XIII, Mellander, 0.: Nutritional factors (other than vita- min D) influencing the intestinal absorption of calcium and strontium. Transfer of Calcium and Strontium across biological membranes, Section V. Academic Press, New York, 1963. Fell, H . B. & Robisson, R.: The growth, development and phosphatase activity of embryonic avian femora and limb-buds cultivated in vitro. Biochem J 23: 765, 1929. Chen, J . M.: The effect of insulin on embryonic limb bones cultivated in vitro. J Physioll25: 148, 1954. Hay, M. F.: The effect of growth hormone and insulin on limb-bone rudiments of the embryonic chick cultivated in vitro, J Physiol 144: 490, 1958. Zwilling, E.: Micrornelia as a direct effect of insulin. Evidence from in vitro and in vivo experiments. J Morph 104: 159, 1959. 243-246, 1956. Growth, Osteoid Ossifi- mm tissue cation 1.0k0.4 2.9 2.9 1.420.4 2.9 2.9 0.9k0.4 2.9 2.9 1.0k0.5 2.9 2.9 Upsala J Med Sci 79 20 Conny Edeno 8. Buno, W. & Goyena, H.: Effect of cortisone on growth in vitro of femur of chick embryo. Proc SOC Exp Biol Med89: 622, 1955. 9. Sobel. H . & Freund, 0.: The action ofcortisone on the embryonic cartilage and muscle in vorro. Experi- entia14:421, 1958. 10. Fell, H. B. & Mellanby, R.: The biological action of thyroxine on embryonic bones grown in tissue culture. J Physiol127: 427, 1955. 11. Fell. H. B. & Thomas, L.: The influence of hydro- cortisone on the action of excess vitamin A on limb bone rudiments in culture. J E x p Med: 114: 343 1961. 12. Lawson, K.: 11. Growth rate. J Embryo1 Morph 9: 42, 1961. 13. Gaillard, P. J . : Parathyroid gland tissue and bone in v i f r o . 11. Koninkl Nederl Akad Wetenschap Proc C 58: 579, 1955. 14. Fell, H. B. & Robison, R.: The development of the calcifying mechanism in avian cartilage and osteoid tissue. Biochem J 28: No. 6,2243 15. Trowell, C . A , : A modified technique for organ culture in v i f r o . Exp Cell Res6: 246, 1954. 16. Franks, L. M.: A factor in normal human serum that inhibits epithelial growth in organ cultures. E x p Cell Res 17: 579, 1959. 17. Eagel, H.: Science 130: 432, 1959. Received June 36. 1 573 Address for reprints: Conny Edeno Department of Pathology I University of Goteborg S-413 45 Goteborg Sweden Upsala J Med Sci 79