Immunohistochemical Study of Osteopontin Expression During Distraction Osteogenesis in the Rat

Abstract

S U M M A R Y Distraction osteogenesis (DO)is a limb-lengthening procedure that combines mechanical tension stress with fracture healing to provide a unique opportunity for detailed histological examination of bone formation. Osteopontin (OPN) is a multifunctional matricellular protein believed to play a key role in wound healing and cellular response to mechanical stress. We studied the expression of OPN during DO using standard immunohistochemical (IHC) staining techniques. In addition, we compared the expression of OPN to proliferation (PCNA-positive cells) in the DO gap. After 14 days of distraction in the rat, these stains revealed variations in OPN expression and its relationship to proliferation according to the cell type, tissue type, and mode of ossification examined. Fibroblast-like cells within the central fibrous area exhibited intermittent low levels of OPN, but no relationship was observed between OPN and proliferation. In areas of transchondral ossification, OPN expression was very high in the morphologically intermediate oval cells. During intramembranous ossification, osteoblasts appeared to exhibit a bimodal expression of OPN. Specifically, proliferating pre-osteoblasts expressed osteopontin, but OPN was not detected in the post-proliferative pre-osteoblasts/osteoblasts that border the new bone columns. Finally, intracellular OPN was detected in virtually all of the mature osteoblasts/osteocytes within the new bone columns, while detection of OPN in the matrix of the developing bone columns may increase with the maturity of the new bone. These results imply that the expression of OPN during DO may be more similar to that seen during embryogenesis than would be expected from other studies. Furthermore, the biphasic expression of OPN during intramembranous ossification may exemplify the protein's multi-functional role. Early expression may facilitate pre-osteoblastic proliferation and migration, while the latter downregulation may be necessary for hydroxyapatite crystal formation.

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“…The percentage of new mineralized bone within the distraction gap was calculated by dividing mineralized bone area by total gap area. The distracted tibiae then were decalcified in 5% formic acid, dehydrated, and embedded in paraffin, as previously described (17,18). Longitudinal sections (5-7 microns) were stained with hematoxylin and eosin.…”

Insight into the Molecular Pathophysiology of Delayed Bone Healing in a Sheep Model

“…A general caution is that the expression of all factors was studied without the measurement of corresponding gene products, although gene products have already been demonstrated in fracture callus. 5,[20][21][22][23] Further, this study lacks cell-specific measurement of gene expression. Callus tissue is a heterogenous mixture of various cells and tissues, and quantitative reverse transcriptase-PCR on total callus tissue does not address the spatial patterns of gene expression among these various cell types.…”

Autologous rabbit adipose tissue-derived mesenchymal stromal cells for the treatment of bone injuries with distraction osteogenesis

“…Osteoblasts synthesizing osteocalcin and osteopontin were labeled and shown by immunohistochemical staining in the prepared sections (36,41). The number of cells staining positive for osteocalcin, which is considered specific for ossification, was found to be 24, 31 and 37 in groups I, II and III, respectively ( Figure 7A).…”

“…To observe emission of the GFP-labeled cells, samples from group II and group III were analyzed using a fluorescent microscope. Cells positive for osteocalcin and osteopontin were counted per square millimeter in six different areas in the samples prepared by immunohistochemical staining (36).…”