Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0606365 on September 7, 2006

Published online before print September 7, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0606365v1
80/6/1272    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chung, R.
Right arrow Articles by Xian, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chung, R.
Right arrow Articles by Xian, C. J.
(Journal of Leukocyte Biology. 2006;80:1272-1280.)
© 2006 by Society for Leukocyte Biology

Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats

Rosa Chung*,{dagger},{ddagger}, Johanna C. Cool*, Michaela A. Scherer*, Bruce K. Foster*,§ and Cory J. Xian*,{dagger},{ddagger},§,1

* Department of Orthopaedic Surgery, Women’s and Children’s Hospital, North Adelaide, South Australia, Australia;
{dagger} Department of Pharmaceutical Biotechnology, University of South Australia, Adelaide, Australia; and Departments of
§ Paediatrics and
{ddagger} Physiology, University of Adelaide, South Australia, Australia

1 Correspondence: Department of Orthopaedic Surgery, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, SA 5006, Australia. E-mail: cory.xian{at}adelaide.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Previously, injury-induced, initial inflammatory response was shown to be an acute inflammatory event containing predominantly neutrophils. To examine potential roles of neutrophils in the bony repair, a neutrophil-neutralizing antiserum or control normal serum was administered systemically in rats with growth plate injury. The inflammatory response was found temporally associated with increased expression of neutrophil chemotactic chemokine cytokine-induced neutrophil chemoattractant-1 and cytokines TNF-{alpha} and IL-1β. Following the inflammatory response, mesenchymal infiltration, chondrogenic and osteogenic responses, and bony repair were observed at the injury site. Neutrophil reduction did not significantly affect infiltration of other inflammatory cells and expression of TNF-{alpha} and IL-1β and growth factors, platelet-derived growth factor-B and TGF-β1, at the injured growth plate on Day 1 and had no effects on mesenchymal infiltration on Day 4. By Day 10, however, there was a significant reduction in proportion of mesenchymal repair tissue but an increase (although statistically insignificant) in bony trabeculae and a decrease in cartilaginous tissue within the injury site. Consistently, in antiserum-treated rats, there was an increase in expression of osteoblastic differentiation transcription factor cbf-{alpha}1 and bone matrix protein osteocalcin and a decrease in chondrogenic transcription factor Sox-9 and cartilage matrix collagen-II in the injured growth plate. These results suggest that injury-induced, neutrophil-mediated inflammatory response appears to suppress mesenchymal cell osteoblastic differentiation but enhance chondrogenic differentiation, and thus, it may be involved in regulating downstream chondrogenic and osteogenic events for growth plate bony repair.

Key Words: fracture repair • growth factors and cytokines • bone growth


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The growth plate is responsible for the longitudinal growth of children’s long bones through a tightly controlled process, called endochondral ossification, which involves synthesis of calcified cartilage scaffold and its subsequent bone conversion [1 ]. The growth plate is divided into three zones, and the chondrocytes are in different maturation and metabolic stages. At the resting zone, the cells (chondrogenic progenitor cells or prechondrocytes) are relatively in an inactive state; after activation and differentiation, chondrocytes enter into the proliferative phase at the proliferative zone and synthesize matrix protein collagen-II (Col-II), which is essential for maintaining growth plate structure [2 ]. As the chondrocytes begin to increase in size, they enter the hypertrophic zone, where they synthesize Col-X, which allows calcification and mineralization of the extracellular matrix at the lower hypertrophic zone of the growth plate [2 , 3 ]. At their terminal differentiation stage at the growth plate-metaphyseal bone transitional zone, hypertrophic chondrocytes die by apoptosis, and the calcified lower zone of hypertrophic cartilage is invaded by blood vessels, bringing in bone-forming cell osteoblasts and resorbing cell osteoclasts, which will remodel and convert the mineralized cartilage into metaphyseal trabecular bone.

Being a cartilage tissue, the growth plate is the weakest structure in the developing long bones and thus, prone to injuries. Fractures through the growth plate may result after mechanical or physiological failure [3 ]. However, although the fracture of the damaged growth plate recovers, up to 30% of injured growth plates are usually repaired by bony tissue [4 ]. This faulty bone repair impairs normal bone longitudinal growth function and can cause orthopaedic problems in the involved long bones, including angular deformity and limb-length discrepancy [5 ]. Although growth plate injuries are common, and trauma injuries to the growth plate can significantly affect bone growth [4 ], how the bony repair occurs at the injured growth plate remains largely unclear. Our previous study has found that the bony-bridge formation at the injured growth plate involved osteoblast differentiation from marrow-derived mesenchymal cells and direct bone formation from osteoblasts [6 ]. In addition, this study has identified four different phases of injury responses in a rat growth plate injury model, namely the inflammatory, fibrogenic, osteogenic, and bone-bridge maturation-remodeling responses, occurring during Days 1–3, 3–7, 7–14, and 10–25 postinjury, respectively [6 ].

As with bone fractures and soft tissue injuries, the foremost cellular response to trauma at the growth plate is the initial injury-induced inflammatory response [6 , 7 ], in which inflammatory cells (predominantly neutrophils and to a lesser extent, macrophage/monocytes and lymphocytes) were found to infiltrate into the injured growth plate. The inflammatory infiltrate was found rapid and transient, and infiltration was apparent at 8 h, peaking on Day 1 and subsiding on Day 3. Leukocyte infiltration is one of the key features in inflammation and local factors, which mediate this influx of leukocyte infiltrates, including early response cytokines such as IL-1β and TNF-{alpha}, cell-surface adhesion molecules, and chemotactic molecules such as chemokines [8 ]. Our previous immunohistochemical studies have shown that inflammatory cells at the growth plate injury site synthesized IL-1β and TNF-{alpha} [9 ], both of which are known, proinflammatory cytokines involved with the regulation of the inflammatory response to tissue injury or bone fracture [10 , 11 ]. Chemokine IL-8 is expressed by myriad cell types such as neutrophils and T cells, and as a potent neutrophil chemoattractant, it plays a vital role in neutrophil recruitment during inflammation and wound healing [12 ]. Rodents lack a homologue of IL-8 [13 ], and instead, they possess another molecule, called cytokine-induced neutrophil chemoattractant (CINC), which shares similar properties as human IL-8 [14 ]. However, whether an induction of CINC at the injured growth plate is involved in regulating the neutrophil infiltration in our rat growth plate injury model remains to be investigated.

The inflammatory response is also known to be an important source of growth factors, which together with the cytokines, may play important roles in regulating downstream events leading to tissue repair. We have recently identified up-regulation at the injured growth plate of several important growth factors known to be involved with bone fracture repair, including fibroblast growth factor-2, platelet-derived growth factor (PDGF)-B, and TGF-β1 [7 , 9 ].

As the injury-induced inflammatory response is the initial cellular event at the injured growth plate, which produces various cytokines and growth factors [6 , 7 , 9 ] that are known to play important roles in regulating bone-fracture healing, it is possible that this initial inflammatory response could be important in regulating downstream-healing events and bony repair of the injured growth plate. As the neutrophils are the predominant infiltrating, inflammatory cells [6 , 7 , 9 ], in this current study, using a rat growth plate injury model and neutrophil immunodepletion approach, we addressed whether a neutrophil-mediated, inflammatory response could be important in regulating bony-healing responses at the injured growth plate.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Growth plate injury and neutrophil-depletion trial
Thirty-six male, 8-week-old Sprague Dawley rats were subjected to experimental growth plate injuries in the proximal tibia of both hind legs. A drill-hole injury, disrupting the central part of the proximal tibial growth plate cartilage, was inflicted surgically using a dental drill under anesthesia as described [6 ]. The Animal Ethics Committee of the Children, Youth and Women’s Health Service (South Australia) approved all protocols. At 1 day prior to (Day –1), immediately after (Day 0), and at Day 4 after surgery, rats received one i.p. injection of normal rabbit serum or rabbit antirat neutrophil antiserum (Accurate Chemical and Scientific Corp., Hicksville, NY; 1:8 dilution in saline) at a vol of 2 ml/kg body weight. One group of untreated/uninjured, age-matched, normal rats (n=6) was also set up as a normal control group for gene-expression studies.

Groups of rats (n=6 per time-point and per treatment) were killed by CO2 overdose for specimen collection on Days 1, 4, and 10 postoperation, time-points that have been shown appropriate previously for observing injury-induced inflammatory, fibrogenic, and bone formation responses at the injured growth plate [6 ]. Both tibiae were dissected and cleared of soft tissue. The normal or injured growth plate cartilage (containing the injury site) from the right proximal tibia was collected as described [9 ], snap-frozen, and stored at –80°C for RNA extraction and gene expression study. The left proximal tibia containing the injury site was collected and fixed in 10% buffered formalin for 24 h and decalcified for 4 days at 4°C in formic acid-based bone decalcifier solution Immunocal (Decal Corp., Tallman, NY). Using a fine blade, the decalcified, left-proximal tibia was bisected longitudinally through the injury site and processed routinely for paraffin embedding. Sections of 5 µm were cut from paraffin tissue blocks and collected on SuperFrost Plus glass slides for immunohistochemical and histology staining.

Histological and immunohistochemical analysis
To visualize cartilage or bone formation and tissue structure within the injury site, sections were stained with alcian blue and H&E as described [6 ]. To examine treatment effects on inflammatory infiltrates, measurements of cell density of neutrophils, monocytes/macrophages, and lymphocytes were carried out on Day 1 sections within the growth plate injury sites using an image analysis program (Image-Pro Plus, Media Cybernetics, Silver Spring, MD), and the cell counts of each cell type were expressed as the average number of cells/mm2 injury site area. These different types of inflammatory cells were distinguished by their distinct, morphological structures (e.g., cell nucleus, cytoplasm, and size) on stained sections. To measure proportions of various types of repairing tissues, areas of mesenchymal tissue, cartilaginous tissue, bone trabeculae, and bone marrow were measured at injury sites in Days 4 and 10 sections, and the measurements were expressed as percentages of the total injury-site areas. To confirm the presence of cartilage or bony repair at the injured growth plate, immunostaining of associated proteins of cartilage (Col-II and Col-X) and bone (bone cell differentiation factor cbf-{alpha}1 and bone matrix protein osteocalcin) was conducted as described previously [6 ].

Real-time quantitative RT-PCR analysis of gene expression
Real-time quantitative RT-PCR assays were carried out to examine expression of genes regulating the inflammatory response (TNF-{alpha}, IL-1β, and CINC-1), growth factors important for cartilage/osteogenic tissue formation (TGF-β1 and PDGF-B), cartilage-specific molecules (chondrogenic transcription factor Sox-9 and cartilage protein Col-IIa), osteoblast differentiation transcription factor cbf-{alpha}1, and bone matrix protein osteocalcin. Total RNA from frozen growth plate samples was isolated using TRIZOL solution (Sigma, NSW, Australia) as described [9 ]. Extracted RNA was treated with DNase (Promega, NSW, Australia), and its quality/quantity was determined by spectrophotometry. As a result of the small amount of total RNA, which can be obtained from each individual rat growth plate sample, and the large numbers of genes, which need to be analyzed in this study, purified RNA from six rats in each treatment time-point group was pooled (equal amount from each rat) to make up a total amount of 5 µg. cDNA of the pooled RNA was synthesized using random decamers (Geneworks, SA, Australia) and Superscript-II RNase H RT (Stratagene, La Jolla, CA), according to the manufacturer’s instructions.

A SYBR Green real-time PCR assay was used to analyze the expression of genes stated in Table 1 in these synthesized cDNA samples. Cyclophilin-A was used as the internal reference, as we have previously found that its expression does not change after growth plate injury, and its expression level is suitable for analyzing expression of growth factors, cytokines, and matrix molecules in the growth plate [9 ]. PCR assays for each gene and cyclophilin were run in parallel in triplicate using their specific primer pairs [9 ]. All PCR assays were carried out using a Rotorgene 72-well PCR machine (Corbett Research, NSW, Australia) using similar reaction conditions and cycling parameters as described in our previous study [9 ]. Relative gene expression was calculated using the 2–{Delta}{Delta}CT method, where threshold cycle (CT) values from triplicate runs were averaged and calibrated in relation to cyclophilin CT values. Levels of gene expression (fold changes) in injured growth plate samples were presented in relation to expression levels in normal, uninjured samples (normal).


View this table:
[in this window]
[in a new window]

  		Table 1. Rat Primer Sequences Used in This Study for Real-Time PCR Assays

 
Statistical analysis
Data (except for real-time RT-PCR results) were expressed as mean ± SEM, and statistical significance of results between groups and time-points was analyzed by two-way ANOVA tests. Statistical significance was assumed when P < 0.05. As the RT-PCR data were generated from pooled RNA samples, data are presented as means of individual pooled samples (without SEM), and thus, no statistical analysis was carried out.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects on injury-induced inflammatory response
Compared with normal serum-treated control rats, which showed significant inflammatory infiltration at the growth plate injury site, rats treated with a rabbit antirat neutrophil serum had an obvious decrease in the density of inflammatory cells present in the wound area (Fig. 1A vs. 1B ). Quantification of the numbers of inflammatory cells per unit area of growth plate injury site showed a significant 60% decrease in the number of neutrophils in antiserum-treated rats (P<0.01; Fig. 1C ), which confirms effectiveness of the antibody treatment in reducing the number of neutrophils. No significant differences were seen in the numbers of macrophage/monocytes and lymphocytes in the two treatment groups, although there tended to have slightly more macrophages/monocytes in the antiserum-treated rats (Fig. 1C) .


Figure 1
View larger version (94K):
[in this window]
[in a new window]

  		Figure 1. Inflammatory response at the injured growth plate. (A) Influx of inflammatory cells, particularly neutrophils (arrows), at the injury site on Day 1 in a normal serum-treated rat. (B) Reduced infiltration of neutrophils within the growth plate injury site on Day 1 in an antineutrophil serum-treated rat, although infiltrated lymphocytes (solid arrow) and macrophage/monocytes (open arrow) were still present. (C) Significant decrease in density of neutrophils within injury site of antiserum-treated group compared with normal serum group (**, P<0.01, n=6). (D) Up-regulated expression (12- to 14-fold) of CINC-1 gene during the inflammatory phase (Day 1) in the injured growth plate compared with noninjured control. Similarly, there was up-regulated gene expression of inflammatory cytokines TNF-{alpha} (E) and IL-1β (F) during the inflammatory response phase after growth plate injury. (A, B) Original scale bar = 25 µm.

 
As neutrophils are the predominant inflammatory cells at the growth plate injury site (Fig. 1C) , and CINC-1 is a known, key neutrophil chemoattractant in rodents, in this study, levels of CINC-1 mRNA expression at an injured growth plate were analyzed by quantitative real-time RT-PCR. Consistent with the prominent neutrophil infiltration on Day 1, the injured growth plate levels of CINC-1 expression were elevated 12- to 14-fold compared with the noninjured, normal control (Fig. 1D) . By Day 4, however, levels of CINC-1 returned to normal levels, which is consistent with clearance of neutrophils at this time-point. This temporal change in CINC-1 levels suggests the important role of CINC-1 in attracting neutrophils during the inflammatory response. It is interesting that 60% neutrophil reduction with antiserum treatment seemed to increase CINC-1 mRNA expression slightly at Days 1 and 4 (Fig. 1D) , probably as a result of a negative-feedback mechanism.

Our previous studies have implicated potential involvement of proinflammatory IL-1β and TNF-{alpha} in the inflammatory response at the injured growth plate injury. Consistently, in the current study, levels of TNF-{alpha} (Fig. 1E) and IL-1β (Fig. 1F) were increased approximately twofold compared with uninjured controls during the inflammatory process on Day 1 and returned normal by Day 4. It is interesting that there was approximately 1.5-fold more TNF-{alpha} in the injured growth plate of rats treated with antineutrophil serum compared with normal, serum-treated controls (Fig. 1E) . As macrophage/monocytes are known to be the major TNF-{alpha}-producing inflammatory cells, the slight increase in the number of macrophage/monocytes at the injured growth plate on Day 1 in antiserum-treated rats is consistent with this higher level of TNF-{alpha} expression. However, no differences were found in IL-1β expression levels between the two groups.

Effects on tissue repair at injured growth plate
Following the injury-induced inflammatory response, by Days 4 and 10, the presence of fibrogenic, chondrogenic, osteogenic, and remodeling responses was revealed by the influx of mesenchymal cells (fibrous cells), formation of cartilaginous tissue (alcian blue positively stained round or oval-shaped cells), and bone trabeculae (pinkly stained smooth solid surface) and the presence of bone marrow (with marrow cell masses) at the injury site (Fig. 2A 2B 2C 2D ). Although mesenchymal cells were still present in Day 10 injury sites, mesenchymal tissue area proportions were reduced significantly on Day 10 compared with Day 4 samples in normal serum-treated rats (P<0.05) and antiserum-treated rats (P<0.01; Fig. 3A ). It is interesting that on Day 10, there was a smaller amount of mesenchymal tissue at the injury site in antiserum-treated rats compared with normal serum-treated rats (P<0.05; Fig. 3A ). On both time-points and in rats from both treatment groups, cartilaginous tissue was present, and the cartilaginous tissue proportions were lower (although statistically insignificant) in the antiserum-treated group compared with the normal serum-treated group (P>0.05; Fig. 3B ). Obvious formation of bone trabeculae and bone marrow did not appear until Day 10. It is interesting that although statistically not significant (P>0.05), in the Day 10 samples, there tended to be greater amounts of bony trabeculae (Fig. 3C) and bone marrow (Fig. 3D) seen in the antiserum-treated group than in the normal serum-treated group.


Figure 2
View larger version (180K):
[in this window]
[in a new window]

  		Figure 2. Effects of antineutrophil serum treatment on injury repair tissues formed at the injured growth plate at different time-points. Injury sites from Day 4 normal serum-treated rats (A) and Day 4 antiserum-treated rats (C) showed larger amounts of mesenchymal infiltrates (M) and smaller amounts of cartilaginous tissue (Ca). Injury sites from the Day 10 normal-serum group (B) and Day 10 antiserum group (D) showed considerable amounts of bone trabeculae (BT) and mesenchymal tissue but smaller amounts of cartilaginous tissue and bone marrow (BM). The remaining, adjacent growth plate cartilage is indicated by open arrows. (A) Original scale bar = 250 µm (which applies to B–D).

 

Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Figure 3. Proportions of different repair tissues formed at the injured growth plate at Days 4 and 10. Quantitative histology image analysis measurements of area percent (over total growth plate injury site areas, n=6) of each type of repair tissue: mesenchymal tissue (A), cartilaginous tissue (B), bone trabeculae (C), and bone marrow (D). Proportions of mesenchymal tissue decreased significantly on Day 10 compared with Day 4 in normal serum-treated rats (*, P<0.05) or antiserum-treated rats (**, P<0.01), and antiserum-treated rats had significantly smaller amounts of mesenchymal tissue compared with normal serum-treated rats on Day 10.

 
Expression analysis of genes involved in fibrogenic, cartilaginous, and osteogenic repair at the injured growth plate
To examine the effects of growth plate injury and neutrophil reduction on genes involved with fibrogenic, chondrogenic, and osteogenic responses, mRNA expressions at the injured growth plate were examined by quantitative real-time RT-PCR. Consistent with previous studies [9 ], TGF-β1 showed a 3.5-fold up-regulation of expression during the inflammatory phase (Day 1), and its levels decreased toward normal levels by Day 10 in both treatment groups (Fig. 4A ). PDGF-B also showed a 2- to 2.5-fold increase in expression on Day 1, and its levels returned to basal level by Day 4 in both treatment groups (Fig. 4B) . Neutrophil reduction at the injured growth plate did not seem to affect expression of these two fibrogenic growth factors.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		Figure 4. mRNA expression of some genes involved in fibrogenic, cartilaginous, and osteogenic repair at the injured growth plate. Quantitative real-time RT-PCR expression data for TGF-β1 (A), PDGF-B (B), Sox-9 (C), Col-IIa (D), cbf-{alpha}1 (E), and osteocalcin (F) are expressed as fold change in relation to noninjured, normal control after being normalized to the internal standard cyclophilin-A.

 
Sox-9 is a chondrogenic differentiation transcription factor, and Col-II is a cartilage-specific matrix molecule. In normal serum-treated rats, there were small increases in expression levels of Sox-9 (Fig. 4C) and Col-II (Fig. 4D) on Days 4 and 10, confirming the presence of chondrogenic response during healing of the injured growth plate, as also observed by the histology analysis described above (Fig. 2) . Consistent with the slight decrease in the cartilaginous tissue-repair proportion (Fig. 3) , the antineutrophil serum treatment appeared to suppress up-regulation of these two chondrogenic genes, particularly on Day 10, suggesting that neutrophils may enhance chondrogenic response in the growth plate injury repair.

In the normal growth plate cartilage, osteoblast differentiation transcription factor cbf-{alpha}1 is normally expressed in the hypertrophic zone, which is associated with its additional function in stimulating chondrocyte maturation [15 ]. In the present study, we showed that after growth plate injury and prior to bone-bridge formation, the expression levels of cbf-{alpha}1 in the injured growth plate had been suppressed on Day 4 in normal, serum-treated animals compared with noninjured controls (Fig. 4E) . However, in antineutrophil antiserum-treated rats, the levels of cbf-{alpha}1 were greater than those in normal serum-treated animals on Day 4 (Fig. 4E) , which is consistent with the greater osteogenic repair outcome on Day 10 at the injured growth plate of antiserum-treated animals (Fig. 3C) . On Day 10 during bony repair, mRNA levels of cbf-{alpha}1 were increased compared with those on Day 4 in both treatment groups, which is consistent with the higher levels of bone formation on Day 10 compared with Day 4 (Fig. 3C) .

Osteocalcin, also expressed by normal growth plate involved in the calcification of hypertrophic cartilage matrix [16 ], is produced by mature osteoblasts during an osteogenic event and during bony-bridge formation at the injured growth plate [6 ]. Consistent with a higher level of cbf-{alpha}1 on Day 4 in the antiserum-treated group compared with the normal serum-treated group, there was a slightly greater level of osteocalcin expression on Day 4 in the antiserum-treated group, although on Day 10, the expression levels appeared not to be different between the two treatment groups (Fig. 4F) .

Expression of cartilage- and bone-related molecules at the growth plate injury site
To confirm the presence of cartilaginous repair at the growth plate injury site, immunostaining of Col-II and Col-X was performed. Col-II is found normally in the proliferative zone of the growth plate, as it was also observed in the current study (Fig. 5A ). Consistent with the alcian blue staining, which revealed the presence of cartilaginous tissue within the growth plate injury site on Day 4 (Fig. 2A) , there was positive Col-II immunostaining found among some chondrocyte-like cells within the injury site of Day 4 rats (Fig. 5B) . Furthermore, immunostaining of Col-X, which is normally found in the growth plate hypertrophic zone, as it was also observed in the current study (Fig. 5C) , was also present among some chondrocyte-like cells within the growth plate injury site on Day 4 (Fig. 5D) . It is interesting that on Day 10, when the bone bridge was formed, there was no obvious immunostaining of Col-II and Col-X. As Col-II is a marker for chondrocyte differentiation, and Col-X is a collagen involved in the chondrocyte maturation during endochondral bone formation, positive staining for Col-II and -X within the growth plate injury site suggests that the endochondral ossification mechanism is involved in the bone-bridge formation at the growth plate injury site.


Figure 5
View larger version (138K):
[in this window]
[in a new window]

  		Figure 5. Immunostaining of cartilage- or bone-specific proteins at the injured growth plate in rats treated with normal serum. (A) Col-II-positive staining in the proliferation zone of growth plate cartilage (arrow) in normal growth plate. (B) Positive Col-II staining in the growth plate injury site (solid arrow), with adjacent growth plate cartilage indicated by an open arrow. (C) The hypertrophic zone of the normal growth plate cartilage showed positive Col-X immunostaining (arrow). (D) Positive Col-X immunostaining in growth plate injury site (solid arrow). (E, F) Some mesenchymal, preosteoblast, or osteoblast-like cells surrounding or lining the bone trabeculae express cbf-{alpha}1 immunoreactivity at the growth plate injury site on Day 4 (E) and Day 10 (F). (G) Normal metaphyseal bone showing positive osteocalcin immunostaining in some osteoblasts lining the bone trabeculae (arrows). (H, I) Some osteoblast-like cells surrounding or lining the bone trabeculae express osteocalcin at the growth plate injury site on Day 4 (H) and Day 10 (I). (J) Suggesting specificity for the immunostaining observed, there was no staining seen on sections when a normal IgG was used instead of the primary antibodies in the immunostaining procedures. (A, B, G–J) Original scale bar = 50 µm; (C–F) original bar = 25 µm.

 
Consistent with the previous observation about the involvement of the intramembranous ossification mechanism for the bony repair of the injured growth plate [6 ] and the presence of cbf-{alpha}1 mRNA expression at the injured growth plate (Fig. 4E) , cbf-{alpha}1-immunopositive mesenchymal, preosteoblastic, or osteoblastic-like cells were present at the injury site on Day 4 (Fig. 5E) and Day 10 (Fig. 5F) , suggesting bone cell differentiation prior to and during bony repair of the injured growth plate. To examine the expression of bone matrix proteins during bony repair at the growth plate injury site, immunohistochemical analysis of osteocalcin was performed. Osteocalcin is expressed in osteoblasts lining the normal metaphyseal trabecular bone, as it was also observed in the current study (Fig. 5G) . After growth plate injury, many osteoblasts or osteoblast-like cells surrounding or lining bony islands or trabeculae expressed osteocalcin at the injury site on Day 4 (Fig. 5H) and Day 10 (Fig. 5I) . No obvious differences were observed in Col-II, Col-X, cbf-{alpha}1, and osteocalcin immunostaining in rats treated with normal serum or antineutrophil serum (data not shown). Suggesting specificity for the immunostaining observed, there was no staining seen on sections when a normal IgG was used instead of the primary antibodies in the immunostaining procedures (Fig. 5J) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fractured growth plate is often repaired by bony tissue rather than by cartilage regeneration, which causes orthopaedic problems such as limb-length discrepancy and/or angulation deformities in children. Our previous studies using a rat model have found that the bony repair is preceded by several sequential, injury-induced responses, namely, inflammatory, fibrogenic, and osteogenic responses at the injured growth plate [6 ]. It is increasingly thought that the initial inflammatory process, together with various associated inflammatory cytokines and growth factors, may play an important role in regulating downstream responses, leading to tissue repair [17 ]. Our previous studies have shown that the initial inflammatory response at the injured growth plate is rapid and transient and is characterized by infiltration of inflammatory cells, particularly neutrophils peaking on Day 1 and subsiding on Day 3 after injury [6 ]. To investigate the potential role of neutrophil-mediated inflammatory response in the bony repair responses at the injured growth plate, the current study has examined neutrophil infiltration and expression of its chemotactic factor, CINC-1, and effects of neutrophil depletion on the injury responses in a rat growth plate injury model [6 ].

Consistent with our previous studies about growth plate injury [6 , 7 ] and other studies about acute inflammation in general [18 ], neutrophils are one of the most numerous cell types at the site of acute inflammation. Many previous studies have demonstrated that local induction of chemokine IL-8 in humans or its equivalent in rats, CINC-1, has been associated with the direct influx of neutrophils to the site of inflammation [19 ]. In the current study, a large induction of CINC-1 expression was revealed in injured growth plate on Day 1, followed by a rapid decline by Day 4, which coincides with the influx and clearance of inflammatory cells, particularly neutrophils at the growth plate injury site. In addition, confirming our previous findings [9 ], the current study also found up-regulated expression of two important proinflammatory cytokines TNF-{alpha} and IL-1β on Day 1 at the injured growth plate. Produced by activated macrophages, lymphocytes, and neutrophils [20 ], IL-Iβ and TNF-{alpha} also play important roles in stimulating leukocytes to immigrate into the site of inflammation [21 ]. Findings from this study suggest a corelationship between neutrophil infiltration and induction of chemokine CINC-1 and cytokines TNF-{alpha} and IL-1β at the injured growth plate.

During the inflammatory response on Day 1, expression of TGF-β1 and PDGF-B was found up-regulated at the injured growth plate. Consistently, levels of TGF-β1 and PDGF-B are known to be increased early after bone fracture [22 ], and it has been shown that cytokines TNF-{alpha} and IL-1β can stimulate the release of these two fibrogenic growth factors during injury repair [23 ]. TGF-β1, a potent chemoattractant for monocytes and growth and differentiation factor for mesenchymal cells, is involved in regulating inflammatory response and fracture healing [24 , 25 ]. PDGF-B is a fibrogenic growth factor, involved in promoting proliferation of primitive mesenchymal cells and their differentiation into chondrocytes or osteoblasts [9 , 25 ]. Up-regulation of TGF-β1 and PDGF-B during Day 1 and returning to near basal levels by Day 4 suggest their potential roles in regulating the inflammatory response and/or being mediators of the initial inflammatory event in regulating subsequent healing responses.

In our current study, prominent mesenchymal cell infiltration to the injury site was found on Day 4, and by Day 10, bone trabeculae were dominant, with some well-formed bone marrow, confirming the rapid time course of bony-bridge formation and remodeling during growth plate fracture repair [6 ]. Consistent with our previous studies [6 , 9 ], in the current study, intramembranous ossification during the bony repair of the injured growth plate was revealed by the histological staining of bony trabeculae, molecular expression of bone cell differentiation transcription factor cbf-{alpha}1 and bone matrix protein osteocalcin, and localization of cbf-{alpha}1 among infiltrated mesenchymal cells and in osteoblasts lining the bony trabeculae. In addition, on Days 4 and 10, a small proportion of cartilaginous repair tissue was also observed, which is consistent with up-regulated expression of chondrogenic transcription factor Sox-9 and cartilage matrix molecule Col-IIa and the positive immunolocalization of Col-II and Col-X at the injury site, suggesting that apart from the intramembranous ossification, the endochondral ossification mechanism also contributed to the growth plate cartilage bony repair.

As neutrophils are the most numerous inflammatory cell types at the growth plate injury site and as this initial inflammatory response was found to be associated with up-regulated expression of various cytokines (TNF-{alpha} and IL-1β) and growth factors (TGF-β and PDGF-B) at the injured growth plate, the current study has explored the potential roles of neutrophils in the bony repair of injured growth plate. Using an antirat neutrophil antiserum, a significant 60% reduction in neutrophil count was achieved at the growth plate injury site. It is interesting that 60% neutrophil reduction did not significantly influence the infiltration of the other two inflammatory cell types (macrophage/monocytes and lymphocytes) nor did it affect levels of IL-1β and TNF-{alpha} gene expression during the inflammatory phase at the injured growth plate. Although neutrophils express IL-1β and TNF-{alpha}, macrophages appear to be the main source of IL-1β and TNF-{alpha} production [20 , 23 , 26 ]. After 60% neutrophil reduction, the lack of significant changes in infiltration of macrophage/monocytes and lymphocytes perhaps could at least partially explain the lack of obvious changes in TNF-{alpha} and IL-1β expression at the injured growth plate.

Consistent with the lack of changes in expression of chemotactic cytokines TNF-{alpha} and IL-1β and growth factors TGF-β1 and PDGF-B [27 ] during the inflammatory response, on Day 4, straight after the inflammatory response, the extent of mesenchymal cell infiltration at the growth plate injury site was found not affected by neutrophil-neutralization treatment. These data suggest a lack of a direct, quantitative relationship between neutrophil-mediated inflammatory response and the subsequent mesenchymal infiltration response at the injured growth plate. However, by Day 10, a significantly lower proportion of mesenchymal tissue remained at the growth plate injury site in animals with 60% neutrophil depletion. It is interesting that although statistically not significant, proportions of cartilaginous repair were reduced, but bone trabeculae were more evident in these rats. Consistently, there was a reduction in levels of expression of Sox-9 and Col-IIa, suggesting that neutrophil depletion may have reduced the rate of cartilage tissue repair through reduced chondrogenesis. Conversely, expression levels of cbf-{alpha}1 and osteocalcin were greater on Day 4 in these animals, suggesting that a higher level of bone cell differentiation was apparent among the infiltrated mesenchymal cells. From these observations, it is possible that 60% neutrophil depletion may have enhanced the rate of bone formation from mesenchymal cells through osteoblast differentiation and intramembranous ossification [6 ] but conversely, has reduced the rate of cartilage tissue repair. Why the neutrophil-mediated inflammatory response appears to be inhibitory to the bone formation but beneficial to cartilage healing in this growth plate injury model remains to be investigated. Consistent with our observations, an early study has demonstrated that an increased number of neutrophils in the bone marrow in IL-6-transgenic mice led to a decrease in density of osteoblasts, in their differentiation from bone marrow stromal cells, and in formation and turnover of primary spongiosa trabecular bone [28 ].

In summary, after growth plate injury, neutrophil-predominant, inflammatory response is the first healing response, which is associated with increased expression of neutrophil chemotactic chemokine CINC-1, proinflammatory cytokines TNF-{alpha} and IL-1β, and fibrogenic growth factors TGF-β1 and PDGF-B. Following the inflammatory response, mesenchymal cell infiltration, osteogenic and chondrogenic responses, and endochondral and intramembranous bone formation mechanisms were found involved with the bony repair of the injured growth plate cartilage. Although 60% neutrophil depletion in rats with growth plate injury did not affect mesenchymal cell infiltration on Day 4, it significantly reduced proportions of mesenchymal repair tissue on Day 10, and it tended to increase bony but reduce cartilage repair proportions. Consistently, these changes in bone versus cartilage repair proportions appeared to concord with changes in levels of expression of osteogenic or chondrogenic differentiation transcription factors and matrix proteins. These results suggest that an injury-induced, neutrophil-mediated inflammatory response may be involved in regulating downstream chondrogenic and osteogenic events, leading to bony tissue formation at the growth plate injury site; it appears to play a role in suppressing mesenchymal cell osteoblastic differentiation and increasing chondrogenic differentiation in the repair of injured growth plate cartilage.


   ACKNOWLEDGEMENTS
 
The project was funded by Bone Growth Foundation (BGF) and in part by grants from Channel-7 Children’s Research Foundation of South Australia (to C. J. X. and B. K. F.) and from the Australian National Health and Medical Research Council (NHMRC; to C. J. X.).

Received June 1, 2006; revised July 12, 2006; accepted July 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yang, X., Karsenty, G. (2002) Transcription factors in bone: developmental and pathological aspects Trends Mol. Med. 8,340-345[CrossRef][Medline]
  2. Van der Eerden, B. C. J., Karperien, M., Wit, J. M. (2003) Systemic and local regulation of the growth plate Endocr. Rev. 24,782-801[Abstract/Free Full Text]
  3. Iannotti, J. P., Goldstein, S., Huhn, J., Lipiello, L., Kaplan, F. S. (1994) Growth plate and bone development Simon, S. R. eds. Orthopaedic Basic Science ,185-218 American Academy of Orthopaedic Surgeons Rosemont, IL.
  4. Xian, C. J., Foster, B. K. (2006) The biologic aspects of children’s fractures Beaty, J. Kasser, J. eds. Fractures in Children ,21-50 Lippincott Williams and Wilkins Philadelphia, PA.
  5. Craig, J. G., Cramer, K. E., Cody, D. D., Hearshen, D. O., Ceulemans, R. Y., van Holsbeeck, M. T., Eyler, W. R. (1999) Premature partial closure and other deformities of the growth plate: MR imaging and three- dimensional modeling Radiology 210,835-843[Abstract/Free Full Text]
  6. Xian, C. J., Zhou, F. H., McCarty, R. C., Foster, B. K. (2004) Intramembranous ossification mechanism for bony bridge formation at the growth plate cartilage injury site J. Orthop. Res. 22,417-426[CrossRef][Medline]
  7. Arasapam, G., Scherer, M., Cool, J. C., Foster, B. K., Xian, C. J. (2006) Roles of COX-2 and iNOS in the bony repair of the injured growth plate cartilage J. Cell. Biochem. 99,450-461[CrossRef][Medline]
  8. Keane, M. P., Strieter, R. M. (2000) Chemokine signaling in inflammation Crit. Care Med. 28(Suppl. 4),N13-N26[CrossRef][Medline]
  9. Zhou, F. H., Foster, B. K., Sander, G., Xian, C. J. (2004) Expression of proinflammatory cytokines and growth factors at the injured growth plate cartilage in young rats Bone 35,1307-1315[Medline]
  10. Kon, T., Cho, T-J., Aizawa, T., Yamazaki, M., Nooh, N., Graves, D., Gerstenfeld, L. C., Einhorn, T. A. (2001) Expression of osteoprotegerin, receptor activator of NF-{kappa}B ligand (osteoprotegrin ligand) and related proinflammatory cytokines during fracture healing J. Bone Miner. Res. 16,1004-1013[CrossRef][Medline]
  11. Barone, F. C., Feuerstein, G. Z. (1999) Inflammatory mediators and stroke: new opportunities for novel therapeutics J. Cereb. Blood Flow Metab. 19,819-834[Medline]
  12. Lin, F., Nguyen, C. M-C., Wang, S-J., Saadi, W., Gross, S. P., Jeon, N. J. (2004) Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration Biochem. Biophys. Res. Commun. 319,576-581[CrossRef][Medline]
  13. Cugini, D., Azzollini, N., Gagliardini, E., Cassis, P., Bertini, R., Colotta, F., Noris, M., Remuzzi, G., Benigni, A. (2005) Inhibition of the chemokine receptor CXCR2 prevents kidney graft function deterioration due to ischemia/reperfusion Kidney Int. 67,1753-1761[CrossRef][Medline]
  14. Ramos, C. D. L., Heluy-Neto, N. E., Ribeiro, R. A., Ferreira, S. H., Cunha, F. Q. (2003) Neutrophil migration induced by IL-8 activated mast cells is mediated by CINC-1 Cytokine 21,214-223[CrossRef][Medline]
  15. Tchetina, E., Mwale, F., Poole, A. R. (2003) Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation J. Bone Miner. Res. 18,844-851[CrossRef][Medline]
  16. Gerstenfeld, L. C., Shapiro, F. D. (1996) Expression of bone-specific genes by hypertrophic chondrocytes: implication of the complex functions of the hypertrophic chondrocyte during endochondral bone development J. Cell. Biochem. 62,1-9[CrossRef][Medline]
  17. Sykaras, N., Opperman, L. A. (2003) Bone morphogenetic proteins (BMPs): how do they function and what can they offer the clinician? J. Oral Sci. 45,57-73[Medline]
  18. Murphy, F. J., Hayes, I., Cotter, T. G. (2003) Targeting inflammatory diseases via apoptotic mechanisms Curr. Opin. Pharmacol. 3,412-419[CrossRef][Medline]
  19. Mukaida, N., Harada, A., Matsushima, K. (1998) Interluekin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1) chemokines essentially involved in inflammatory and immune reactions Cytokine Growth Factor Rev. 9,9-23[CrossRef][Medline]
  20. Lo, Y-J., Liu, C-M., Wong, M-Y., Hou, L-T., Chang, W-K. (1999) Interleukin 1β secreting cells in inflamed gingival tissue of adult periodontitis patients Cytokine 11,626-633[CrossRef][Medline]
  21. Moser, B., Wolf, M., Walz, A., Loetscher, P. (2004) Chemokines: Multiple levels of leukocyte migration control Trends Immunol. 25,75-84[CrossRef][Medline]
  22. Cho, T-J., Gerstenfeld, L. C., Einhorn, T. A. (2002) Differential temporal expression of members of the transforming growth factor β superfamily during murine fracture healing J. Bone Miner. Res. 17,513-520[CrossRef][Medline]
  23. Tonks, A. J., Cooper, R. A., Jones, K. P., Blair, S., Parton, J., Tonks, A. (2003) Honey stimulates inflammatory cytokine production from monocytes Cytokine 21,242-247[CrossRef][Medline]
  24. Joyce, M. E., Jingushi, S., Bolander, M. E. (1990) Transforming growth factor-β in the regulation of fracture repair Orthop. Clin. North Am. 21,199-209[Medline]
  25. Tatsuyama, K., Maezawa, Y., Baba, H., Imamura, Y., Fukuda, M. (2000) Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair of bone Eur. J. Histochem. 44,269-278[Medline]
  26. McRitchie, D. I., Isowa, N., Edelson, J. D., Xavier, A. M., Cai, L., Man, H-E., Wang, Y-T., Keshavjee, S. H., Slutsky, A. S., Liu, M. (2000) Production of tumor necrosis factor-{alpha} by primary cultured rat alveolar epithelial cells Cytokine 12,644-654[CrossRef][Medline]
  27. Kovacs, E. J., DiPietro, L. A. (1994) Fibrogenic cytokines and connective tissue production FASEB J. 8,854-861[Abstract]
  28. Kitamura, H., Kawata, H., Takahashi, F., Higuchi, Y., Furuichi, T., Ohkawa, H. (1995) Bone marrow neutrophilia and suppressed bone turnover in human interleukin-6 transgenic mice. A cellular relationship among hematopoietic cells, osteoblasts, and osteoclasts mediated by stromal cells in bone marrow Am. J. Pathol. 147,1682-1692[Abstract]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0606365v1
80/6/1272    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chung, R.
Right arrow Articles by Xian, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chung, R.
Right arrow Articles by Xian, C. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS