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aa-def rats revealed
shortened, irregularly distributed trabeculae beneath
the growth plate cartilage of tibiae (Fig. 1B), compared
with those of aa+ rats (Fig. 1A). These trabeculae
were fragmented and surrounded by thick
fibrous tissue. This fibrous tissue stained intensely
for ALPase, a hallmark of osteoblastic cells, and contained
many TRAPase-positive osteoclasts (Fig. 1E).
In contrast, the ascorbic acid-sufficient (aa+) bones
revealed ALPase-positive osteoblasts and TRAPasereactive
osteoclasts on the surfaces of metaphyseal
trabeculae (Fig. 1C).
Higher numbers of cells immunopositive
for PCNA were seen in the fibrous tissue
layer in aa-def bones when compared to similar areas
in aa+ metaphyses (Compare Figs. 1D and F). Highly magnified microscopy showed that osteoblasts
in aa-def bones were not attached to the bone
surfaces (Figs. 2A, B). Only few osteoblasts were
lying on the surfaces of the metaphyseal trabeculae
at the chondro-osseous junction; instead, fibroblastlike
cells surrounded the fragmented trabeculae in
this area (Fig. 2A). Trabecular surfaces stained intensely
for toluidine-blue (Fig. 2A), osteopontin, and
osteocalcin (Figs. 2C, D). Under TEM, an electrondense
linear structure could be found in a similar
location (Fig. 2B). The fibroblast-like cells included
round and enlarged rER which contained evenly
electron-dense material (Fig. 2E).
Amorphous organic
material including fine fibrillar structures were
associated with A. Thus, in the metaphysis of aa-def tibia, osteoblasts
tended to detach from the bone matrix, and many
osteoblasts and fibroblast-like cells in the fibrous tissue
layer showed enlarged rER, indicating the accumulation
of organic materials inside the rER.

[Matrix mineralization in the ascorbic acid-deficient bone]

Bone mineralization in the primary trabeculae close
to the chondro-osseous junction was examined by
calcein labeling, and aa-def bones showed a small
amount of bone deposition onto mineralized cartilage
(Compare insets of Figs. 3A, B).
Bone matrix
could be seen surrounding the cartilage cores of primary
trabeculae in aa+ rats (Fig. 3A),whereas aadef
cartilage was often seen without a surrounding
bone matrix (Fig. 3B). Under TEM, in contrast
with normal striated collagen fibrils of aa+ animals
(Fig. 3C), amorphous, feather-like, or fibrillar structures
were seen associated with osteoblasts with
round-shaped rER in aa-def specimens (Fig. 3D).
Thus, the ultrastructure of collagen fibrils was
shown to be different between aa-def and aa+
groups.
　Next, mineralization in the metaphyseal trabeculae
was assessed. In a state of ascorbic acid sufficiency,
von Kossa staining revealed numerous granular
structures adjacent to mature osteoblasts in the primary
trabeculae (Figs. 4A, B). Accordingly, TEM
observations portrayed numerous matrix vesicles
and mineralized nodules, some of which were associated
with collagen fibrils (Fig. 4C). At a higher
magnification, mineral crystals could be seen growing towards the collagen fibrils from the mineralized
nodules (Fig. 4D).
Crystal deposition unrelated to
mineralized nodules was not seen in normal collagen
fibrils. In aa-def metaphyses, von Kossa staining
verified poor mineralization and a markedly
reduced trabecular number (Figs. 5A, B). Fragmented
trabeculae neighboring the growth plate cartilage
showed granular structures stained with von Kossa’s
solution.
TEM imaging from corresponding areas
demonstrated numerous matrix vesicles and mineralized
nodules (Figs. 5C, D).
Therefore, bone mineralization
still seemed to take place even in the
circumstance of ascorbic acid deficiency. At higher
magnification,