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Correspondence to: Daniel S. Perrien, ACHRI, Slot 512-20B, 1120 Marshall Street, Little Rock, AR 72202. E-mail: perriendaniels@uams.edu
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Summary |
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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.
(J Histochem Cytochem 50:567574, 2002)
Key Words: mechanical stress, osteoblast, intramembranous ossification, distraction osteogenesis, limb lengthening
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Introduction |
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Distraction osteogenesis (DO), a variant of fracture healing, is a bone-lengthening procedure that utilizes an external fixator to gradually stretch a surgically induced osteotomy. At least three modes of ossification can be observed during DO: intramembranous, transchondral, and endochondral (
Beyond 7 days of distraction in the rat, the distraction gap is bridged by five biologically active, spatially oriented zones (Fig 1) (
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Bone formation during DO is extremely rapid, approximately seven times that of embryonic development. It is believed that the mechanical tension stress provided by the external fixator is responsible for the increased rate of osteogenesis. However, the exact mechanism by which the mechano-signaling stimulates osteoblastogenesis during DO is unknown.
Osteopontin (OPN) is a non-collagenous multifunctional glycoprotein routinely present in mineralized tissues (
An immunohistochemical (IHC) study of OPN expression in developing rat mandible reported various degrees of OPN expression by pre-osteoblasts, osteoblasts, and osteocytes (
In this study, osteopontin protein expression during DO in the rat was examined after 14 days of distraction, focusing on areas of intramembranous and transchondral ossification. To examine any potential relationship between OPN expression and proliferation of osteoblastic cells, OPN-stained sections were also compared to serial sections stained for PCNA. The working hypotheses for these studies were (a) that OPN would be detected at various levels in the extracellular matrix throughout the distraction gap, (b) that OPN would be detected at different intensities in fibroblasts of the central fibrous interzone, pre-osteoblasts, and osteoblasts of the primary matrix front, and osteoblasts and osteocytes within the forming microcolumns, and (c) that a relationship would be observed between proliferation of pre-osteoblasts and OPN expression in the primary matrix front where intramembranous ossification occurs.
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Materials and Methods |
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Surgical Procedures
The following procedures were approved by the institutional animal care and use committee and performed as previously described (
Specimen Preparation
Specimens were processed as described (
Immunohistochemistry for OPN
Sections were deparafinized and rehydrated through xylene and serial dilutions of EtOH to distilled H2O. They were then incubated in Antigen Retrieval Citra (Biogenex; San Ramon, CA) at 95C for 15 min. Sections were washed in PBS, pH 7.4, with 0.02% Triton X-100 (PBS) twice for 3 min each. All incubations were performed in a humidity chamber at room temperature and followed by two, 3-min washes in PBS. For labeling of OPN, the specimens were incubated with levamisol (Vector Laboratories; Burlingame, CA) for 30 min to block endogenous alkaline phosphatase activity. Primary antibody OP-199 (
Immunohistochemical Labeling of PCNA
To compare the relationship of OPN expression to proliferation, serial sections to those stained for OPN were immunohistochemically labeled for PCNA expression as previously described (
Semiquantitation of OPN- and PCNA-positive Cells
Semiquantitation of PCNA+ and OPN+ cells was performed as previously described, with slight modifications (
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Results |
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Sections incubated with OP-199 or PC-10 displayed several novel staining patterns, and no significant signal was seen in the control sections. Proliferating cells (PCNA+) were distributed in typical fashion throughout the central callus as described (
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Qualitatively, OPN expression appeared to vary according to the tissue and cell type examined. OPN was detected in the matrix and in some cells in each of the three zones. The intensity of the stain in both positive cells and matrix was weakest in the fibrous interzone compared to the other tissue types examined (Fig 2A). OPN-positive fibroblast-like cells were scattered throughout the fibrous interzone in an apparently random fashion, and matrix staining was relatively even throughout.
In the primary matrix front, OPN expression varied according to the type of ossification examined. In areas of transchondral ossification, the intensity of the matrix staining was weak and relatively even throughout. Positively stained cells in the transchondral primary matrix front appeared to express moderate to high levels of OPN (Fig 2D). In fact, the overall intensity of OPN in the positive oval cells was higher than in any other cell type (Table 1). OPN appeared to be evenly distributed throughout the cytoplasm of transchondral cells, and unstained nuclei were common (Fig 2D).
In the intramembranous primary matrix front, a distinct biphasic pattern of OPN expression was observed (Fig 2F). A thin dense line of OPN+ cells was seen where the primary matrix front borders the fibrous interzone. In addition, a second line of OPN- cells, corresponding to nonproliferative differentiating preosteoblasts, was present between the OPN+ line and the edge of the developing microcolumns where OPN appeared to be upregulated again. Comparison of OPN-stained sections with those stained for PCNA revealed close relationships between the OPN+ line and the proliferation front as well as the OPN- line and the post-proliferative cells of the primary matrix front immediately adjacent to the microcolumns (Fig 2H). Matrix staining in the intramembranous primary matrix front also appeared to follow this pattern.
The concentration of OPN+ cells in the microcolumns was notably higher than in columns formed by transmembranous ossification (Table 1). However, this probably reflects differences in total cell density in these columns rather than differences in the incidence of OPN expression. No other notable differences in OPN expression were seen in the transchondral and intramembranous microcolumns. In this region of the callus, the matrix and cells stained with an intensity that varied from moderate to high, whereas sinusoids between the microcolumns showed little or no OPN+ staining (Fig 2I). At the leading edge of the microcolumns, where they border the primary matrix front, the matrix was moderately positive for OPN. However, the intensity of the matrix staining in the microcolumns grew progressively greater with increasing proximity to the host cortex (Fig 2K). This may indicate that the concentration of OPN in the new bone matrix increases with the maturity of the new osteoid.
Semiquantatively, the concentration of both OPN+ and PCNA+ cells varied according the tissue and cell type examined (Table 1). The concentration of PCNA+ and OPN+ cells was highest in the intramembranous primary matrix front and lowest in the fibrous interzone. The concentration of OPN+ cells appeared comparable to the number of PCNA+ cells in both the fibrous interzone and the intramembranous primary matrix front. Interestingly, OPN+ cells were significantly more concentrated than PCNA+ cells in the transchondral primary matrix front.
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Discussion |
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This study demonstrates the expression of OPN during DO in adult (4-month-old) rats by IHC. It provides evidence that cells in the fibrous interzone, intramembranous and transchondral primary matrix front, and the developing microcolumns express OPN. The intracellular and extracellular intensity of the OPN stain and concentration of OPN+ cells is highly variable between the three zones. Comparison of OPN-labeled sections to those labeled for PCNA revealed a close relationship between proliferation and OPN expression only in the primary matrix front where intramembranous ossification occurred.
During DO, the tissue composition of the distraction callus, or gap, undergoes three major changes. As in typical fracture healing, a hematoma is formed at the time of osteotomy. During the first 710 days of distraction, the hematoma is gradually replaced by fibrous tissue and initial bone formation begins at the proximal and distal host cortices and marrow spaces. As the stretching continues, the new bone columns continue to form along the tension vector, growing towards the center of the gap. When the desired amount of lengthening is achieved, the distraction is stopped and the external fixator remains in place to stabilize the callus until the new bone is bridged/fused, consolidated, and the medullary canal is formed in the lengthened segment.
The work presented here utilized specimens harvested immediately after 14 days of active distraction. These specimens provide the opportunity for detailed histological examination of the osteogenesis in a temporospatially organized manner isolated from processes of bone resorption. The concentration of OPN+ cells, intensity of intracellular staining, and intensity of matrix staining in the FIZ were all notably lower than in the other tissue types examined. In contrast, an ISH study during DO in the rat did not detect OPN in the cells of the fibrous interzone (
Intracellular OPN appeared to be localized in discrete foci either within the cytoplasm or on the membrane of the fibroblast-like cells during DO (Fig 2A). An in vitro study of fetal calvarial fibroblasts reported two patterns of intracellular OPN expression (
The comparison of OPN to PCNA in the fibrous interzone did not reveal any relationship between proliferation and OPN expression. This was expected because of the apparent random and unorganized pattern of proliferation in the fibrous interzone. A double labeling technique is needed to draw any firm conclusions about the relationship of OPN to proliferation in this zone.
Two types of ossification were examined in the primary matrix front: transchondral and intramembranous. There were distinct differences in both OPN and PCNA expression patterns between the two modes of ossification. The concentration of both OPN+ and PCNA+ cells was dramatically higher in areas of intramembranous bone formation, whereas the intensity of intracellular staining for OPN was dramatically higher in the morphologically intermediate transchondral cells than in the pre-osteoblasts/osteoblasts of the intramembranous matrix front. There appeared to be little difference in the intensity of OPN labeling in the extracellular matrix. Therefore, OPN may have a different role(s) during initial matrix formation according to the mode of ossification.
The biphasic pattern of OPN expression seen in areas of intramembranous bone formation is similar to that seen in other models (
Biphasic OPN expression during intramembranous osteogenesis is probably representative of its multifunctionality during osteogenesis. The binding of extracellular OPN by vß3 or other integrins on the cell surface may activate several signaling cascades leading to increased proliferation, cell motility, and survival signals (
In the transchondral primary matrix front, the intense intracellular OPN stain combined with the low extracellular OPN signal suggests that OPN may play a different role compared to intramembranous ossification. The pattern of OPN expression in these cells was identical to the mRNA pattern reported (
The similarity of OPN expression in the microcolumns formed by either intramembranous or transchondral ossification indicates that the mode of initial matrix formation has little or no effect on the expression of OPN by osteoblasts/osteocytes after they become embedded in new osteoid. OPN was detected in osteoblasts and the extracellular matrix of the microcolumns approximately 200 µm from the border with the primary matrix front. This suggests that the protein is produced and secreted by embedded osteoblasts relatively soon after initial matrix formation. At the leading edge of the microcolumns the intensity of the extracellular stain is relatively weak but continues to grow with proximity to the host cortex and/or marrow space (reflective of the maturity or relative age of the new bone during DO), indicating that secretion of OPN continues throughout the maturation of the new bone. This high level of extracellular OPN is probably a chemotaxic and adhesive agent for osteoclasts during the subsequent formation of the medullary canal within the lengthened segment. However, it may also be important for continued maturation of the new bone as other matrix components bind to OPN. Finally, the apparent high level of OPN in the developing bone matrix indicates that although OPN may be able to impair hydroxyapatite crystsallization, it may not interfere with crystallization in the later stages of bone maturation. Such a change in protein function could be achieved by enzymatic cleavage or other post-translational modifications that would limit OPN's ability to inhibit crystal formation.
In agreement with our hypotheses, the results reported here indicate that OPN is present at some level in fibroblasts, pre-osteoblasts, osteoblasts, transchondral cells, and osteocytes in the distraction callus of the rat tibia after 14 days of lengthening. However, this expression appears to vary greatly among cell types and stages of osteogenesis. Comparison of OPN to PCNA on serial sections revealed a distinct relationship between OPN expression and proliferation where intramembranous ossification occurred. In this area OPN expression was biphasic with one peak corresponding to proliferation and a second corresponding to bone matrix maturation. Labeling of OPN was also seen in the extracellular matrix of each zone examined. Taken in the context of other published data, these results suggest that OPN may play distinctly different roles in each of the three callus zones and according to the mode of bone formation. This implies that OPN plays an integral role in the mechanical induction and maintenance of rapid bone formation during DO. Future studies addressing the questions raised here may further our understanding of these mechanisms.
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Acknowledgments |
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Supported by NIH grants AR44987 and AA12223, by the Brooks Medical Research Fund, by the Laboratory for Limb Regeneration ResearchArkansas Children's Hospital Research Institute, and by the Departments of Orthopedics and Pediatrics, University of Arkansas for Medical Sciences.
We especially thank Dr Ceclia Giachelli for her generous provision of the OP-199 antibody used in this experiment.
Received for publication February 8, 2001; accepted November 14, 2001.
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