The Effects of Ascorbic Acid on the Growth of the Fetal Rat Bone in Ca++ - free Culture Medium

Suh, Kuen Tak; Yoo, Chong Il

Abstract

In order to study the effect of ascorbic acid on the growth of the fetal rat long bones in calcium free culture medium, fetal femurs from rat fetus on 19th day of gestation were cultured for 1, 3, 5, 7 and 9 days in medium described below. Culture media used were MEM, Ca++ - free MEM, Ca++ - free MEM supplemented with 100ug/ml ascorbic acid and Ca++ - free MEM supplemented with 400ug/ml ascorbic acid. The fetal femurs were obtained by separating from the rat fetuses and were washed with sterilized Ca++ , K+ - free buffer solution. The bones were transferred to Millipore filters cemented to stainless steel grids that were placed in culture dishes. The media were pipetted into the culture dishes until it just wetted the Millipore filters so that the bones were maintained at the medium-air interface. For the light microscopy, the cultured bones were washed with phosphate buffer and fixed in 10% neutral buffered formalin and embedded in paraplast. They were sectioned at a thinckness of 6âˆ’8um without decalcification, and were stained with hematoxylin-eosin, van Gieson, and von Kossa method. For the electron microscopy, the bone tissues were prefixed in mixture of 2% paraforma1dehyde and 2.5% glutaraldehyde and postfixed in 1% osmium tetraoxide solution. They were dehydrated in graded alcohol, transferred to aceton, and then dried with liquid CO2 by critical point dryer(Ladd, Burlington, Vermont). These dried tissues were coated with gold and observed with JSM-35C scanning electron microscope. The result were as follows : 1. The fetal rat femurs cultured in Ca++ - free MEM showed that the differentiation of chondrocytes was delayed, the ossification of the cartilage was not obvious and the formation of periosteum was poor, as compared to those of the control group. 2. The chondrocytes of the bones cultured in Ca++ - free MEM with 100ug/ml ascorbic acid revealed the pictures of progressive differentiation similar to those seen in the control group. The length of bone marrow and the thickness of the periosteum increased. 3. The bone tissues cultured in Ca++ - free MEM with 400ug/ml ascorbic acid showed active formation of new osteoid, increased bone matrix and abundant collagen fibers distributed extensively. 4. The bone trabeculaeof diaphysis showed the similar pictures to those of the epiphysis and metaphysis. In Ca++ - free MEM with ascorbic acid, the collagen fibrils increased and numerous spherules were formed. 5. The spherules were noted to be 2 types. One was the calcospherite associated with collagen fibrils, and the other was spherical cytoplasmic outgrowth covered by collaged fibrils.