[Frontiers in Bioscience S3, 117-125, January 1, 2011]

Injury responses and repair mechanisms of the injured growth plate

Rosa Chung1, 2, 3, Bruce K Foster2, Cory J Xian1, 2, 3

1Discipline of Physiology, University of Adelaide, Adelaide, Australia, 2Department of Orthopaedic Surgery, Women's and Children's Hospital, North Adelaide, Australia, 3Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Mechanisms of bone growth and modelling
4. Growth plate injuries, injury responses and repair mechanisms
4.1. Growth plate injuries, their classification, and effects on bone growth
4.2. Injury responses after a growth plate fracture
4.2.1. Inflammatory phase
4.2.2. Fibrogenic phase
4.2.3. Osteogenic and maturation phases
4.3. Mechanisms of bony repair of injured growth plate cartilage
4.4. Effects of injuries and the adjacent non-injured growth plate tissue
5. Perspective & conclusions
6. Acknowledgements
7. References

1. ABSTRACT

The growth plate is responsible for longitudinal growth of children's long bones. However, being a cartilaginous tissue, the growth plate has a limited ability for regeneration and thus injured growth plate is often repaired by bony tissue resulting in bone growth defects of the involved limb. Understanding the pathophysiology of growth plate bony repair and developing preventative treatments remain a challenge. This review discusses previous and recent studies investigating growth plate injury responses and repair mechanisms in a rat tibial growth plate injury model. Following an injury, inflammatory, fibrogenic, osteogenic and bone-bridge maturation repair phases have been observed on days 1-3, 3-7, 7-14 and 10 onwards, respectively. Important roles of several growth factors and cytokines (such as PDGF-BB, FGF-2, TNF-alpha and IL-1beta) have been highlighted, regulating different phases of growth plate injury repair. Studies have also shown that while intramembranous ossification is the major mechanism responsible for the bony repair, endochondral ossification, to a lesser extent, also plays a role.  Further understanding of the growth plate injury responses and bony repair mechanisms is still required.

2. INTRODUCTION

The growth plate is solely responsible for the longitudinal growth of long bones via a tightly controlled and regulated process called endochondral ossification, whereby a calcified cartilaginous template is first made and then replaced by bone (1-3). However, being the weakest region of the long bone, it is susceptible to injuries, which, depending on the location and severity, can lead to "undesirable" bony repair and a myriad of orthopaedic problems. Currently, the pathophysiology for the bony repair and bone growth defects remains unclear, and there lacks a biological treatment for the injured growth plates to prevent the bony repair and the bone growth defects. This review discusses and summarises the major molecular and cellular events involved with the growth plate injury responses and bony repair which have been reported in recent studies using rodent models.

3. MECHANISMS OF BONE GROWTH AND MODELLING

The developing long bones in children, such as the femur (thigh bone) or humerus (arm bone), can be divided into five distinguishable regions, including articular cartilage, epiphysis, physis (referred as the growth plate), metaphysis and diaphysis (4, 5). The growth plate, a cartilage-like structure situated directly below the epiphysis and present only in developing long bones, is a layer of hyaline cartilage that allows the long bone to grow longitudinally but not in width. It functions to produce mineralised cartilaginous scaffold from which trabecular bone is formed through the endochondral ossification mechanism involving chondrogenesis and osteogenesis (3, 6, 7) (Figure 1).

There are three distinguishable zones within the growth plate: the resting (reserve) zone, the proliferative zone and the hypertrophic zone (4, 6, 8). Previously, the resting zone has been thought to play no direct roles in longitudinal growth of bones as the cells within the zone (pre-chondrocytes) proliferate very slowly or do not proliferate at all (5). Histologically, the resting zone is characterised by the sparse distribution of either singular or coupled round cells that are abundant in lipid and cytoplasmic vacuoles within the matrix, indicative of its proposed role as a storehouse for nutrients (5, 6, 9). Even though the resting zone possesses the ability to produce a cartilaginous matrix, it remains relatively inactive in both cell and matrix turnover (6), with very low rates of proteoglycan and collagen-2 production (10). On the other hand, research has suggested that the cells within the resting zone act as a pool of stem cell-like cells, producing proliferative chondrocytes for the proliferative zone (11, 12). In addition, Abad et al (2002) reported that, by producing an unknown growth plate orienting morphogen, the resting zone may be responsible for influencing the columnar directional arrangement of growth plate chondrocytes within the proliferative zone (12).

There are two main functions at the proliferative zone, matrix production and cellular division, which are vital contributions to the longitudinal growth of long bones (6). Histologically, the proliferative zone is characterised by longitudinal columns of slightly flattened chondrocytes. These columns are separated from each other via the surrounding cartilage matrix, which is enriched in collagen-II (5). The extent of total longitudinal growth can be determined by the thickness of the proliferative zone, with a greater number of cells present representing a greater potential of longitudinal growth (5). At the end of the proliferative zone, the chondrocytes no longer proliferate and instead begin to undergo hypertrophy as they enter into the hypertrophic zone. Histologically, the cells within the hypertrophic zone are 5 to 10 times greater in size than those in the proliferative zone. Producing collagen-X and alkaline phosphatase, the hypertrophic zone is involved with matrix mineralization. In addition, production of vascular endothelial growth factor (VEGF-a) as well as a low oxygen tension present attract vessel invasion from metaphysis, which will turn the calcified cartilage into trabecular bone in metaphysis. Since the hypertrophic chondrocytes are larger in size and this relatively thicker zone of calcified cartilage serves as a template for bone deposition, the hypertrophic zone is the principal engine of longitudinal bone growth, and thus the variation in the rate at which the hypertrophic zone increases in thickness has been regarded as the major reason behind the differences in growth rate in different parts of the body (5).

With the mineralisation and angiogenesis, chondrocytes within the lower hypertrophic zone particularly at the chondro-osseo junction are destined for apoptotic cell death, which causes calcified tissue/bone absorbing cells (osteoclasts or chondroclasts) to zone in and dissolve the calcified cartilage (9). The influx of bone building cells (osteoblasts) deposits bone matrix to replace the previously absorbed tissue to form trabecular bone (6, 8, 13, 14). Therefore, with vascularisation and coordinated action of osteoclasts/chondroclasts and osteoblasts, the calcified hypertrophic cartilage is modelled and remodelled into the metaphyseal trabecular bone, in which mineralised growth plate cartilage is first being replaced by primary woven bone (primary spongiosa) and then further modelled and remodelled into more mature laminar trabecular bone (secondary spongiosa) (8). In mature bone, the metaphysis is where the epiphysis and diaphysis meet.

4. GROWTH PLATE INJURIES, INJURY RESPONSES AND REPAIR MECHANISMS

4.1. Growth plate injuries, their classifications, and effects on bone growth

Due to accidents in sports and play, skeletal fractures are common in children. Since the growth plate is the least rigid region of the long bone, its injuries are common, and it has been estimated that around 20% bone fractures involve growth plate (15). The Salter-Harris classification system has been used to distinguish the different types of growth plate injuries and relationship between the characteristics of the fractures and their prognoses (6, 16-18). Current literature indicates the most common types of fractures occurring in the distal tibias of younger children is type I (around 40%), which in most cases has a reasonably good prognosis as the cells responsible for interstitial growth of the growth plate as well as the epiphyseal blood supply remain undisturbed (18-20). Similarly, the prognoses for future growth in type II fractures are also quite good. Other types of fractures, types III, IV, and V, however, may/will all result in bony formation at the injured site (21). It has been estimated that in up to 30% of all children with growth plate related injuries, undesirable formation of bony tissue and bone bridge at the injury site hinders normal growth of the developing long bone in the affected limb (22, 23), which results in significant orthopaedic problems such as limb length discrepancy and bone angulation deformity (22, 24).

4.2. Injury responses after a growth plate fracture

The cellular and molecular mechanisms for the bony repair of the injured growth plate remain largely unknown. An earlier study identified four different phases of injury responses in a rat growth plate injury model (25) - the inflammatory, fibrogenic, osteogenic and bone bridge maturation remodelling responses occurring during days 1-3, 3-7, 7-14, 10-25, respectively. Similarly, this pattern of growth plate injury repair was demonstrated in a murine growth plate injury model (26). In addition, similar injury responses were also observed in various growth plate injury models including mice, rabbits, pigs and sheep (26-29). Following from these studies, there have been some additional in vivo mechanistic studies using a rat tibial growth plate injury model (30-33).

4.2.1. Inflammatory phase

Common to bone fractures and soft tissue injuries, the first response after a growth plate injury is the inflammatory phase (25, 34, 35). During this initial phase there is an influx of inflammatory cells - predominately neutrophils together with macrophages/monocytes and lymphocytes entering into the growth plate injury site. This rapid influx of inflammatory cells has been shown to commence approximately 8 hours after the injury in a rat growth plate injury model, peaking at day 1 and gradually subsiding by day 3. Consistent with the abundant numbers of neutrophils seen within the infiltrate, the gene expression of rat neutrophil chemokine CINC-1 (similar to human interleukin-8) was shown to be significantly increased during the peak of the inflammatory phase (day 1) (31). By the end of the inflammatory phase (day 4) the levels of CINC-1 had decreased back to almost basal levels. Along with the influx of inflammatory cells entering the injury site, the infiltrate also secretes a myriad of growth factors and cytokines that are thought to regulate further downstream responses during growth plate injury repair. Pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1beta), which are known regulators of inflammation after tissue injury and bone fractures, were seen in their mRNA expression levels during the inflammatory phase - peaking between 8 hours to day 1 post injury (34) (Figure 2). Follow-up studies also showed a significant increase of these cytokines at day 1 post injury in a rat growth plate injury model (30, 31). Growth factors insulin-like growth factor (IGF-I) and transforming growth factor (TGF-beta) were also found to be upregulated during this early phase of injury repair (34).

Previous studies have examined the potential role of the inflammatory phase in mediating the cascade of downstream events leading to the bony bridge formation after growth plate injury. As one of the key regulators of the inflammatory response, p38 mitogen activated protein kinase (MAPK) has been shown to be increased in activation at the injured growth plate (33) (Figure 2). Furthermore, Zhou et al (2006) found that TNF-alpha was needed for the activation of p38 at the injured growth plate as p38 activation was blocked in rats treated with a TNF-alpha antagonist (33). TNF-alpha inhibition also resulted in a reduced mesenchymal infiltration, proliferation as well as a reduced expression of FGF-2, indicating the potential role of TNF-alpha in mesenchymal infiltration and proliferation within the growth plate injury site (33). Similarly, Gerstenfeld et al (2001) found that in bone fractures, blocking TNF-alpha signalling resulted in a significant delay in bone callus formation (36). The role of TNF-alpha has also been studied in other types of tissue repair. Consistent with the finding of TNF-alpha role in mesenchymal cell infiltration into growth plate injury site (33), Fu et al (2009) reported that TNF-alpha had a strong chemotaxis role for mesenchymal stem cell migration during wound repair (37), and thus abrogation of TNF-alpha resulted in an obvious delay in MSC migration and wound healing. Overall, these studies highlight the importance of TNF-alpha during tissue repair.

Since the major inflammatory cells involved with the inflammatory phase are found to be neutrophils (25), a follow-up study examined the role of the neutrophil-mediated inflammatory response in growth plate injury repair by utilising an anti-serum to deplete the majority of neutrophils (31). As a result of the depletion, an increase in expression of osteogenesis genes such as osteocalcin and Runt- related transcription factor 2 (Runx2 or also commonly referred to as core binding factor alpha-1 or cbf alpha-1) was seen. In addition, neutrophil depletion also decreased the expression of chondrogenesis-related genes such as Sox-9 and collagen -2 (31). This study indicates that neutrophils play a role of initiating the growth plate injury response and consequently may enhance chondrogenic differentiation. During both soft tissue and bone healing repair, neutrophil recruitment has also been found to be vital, as they play an active role in the clearance of undesirable bacteria and microdebris within the injured zone (38, 39).

Furthermore, one previous study observed significant upregulated gene expression of injury-induced key inflammatory mediators cyclo-oxygenase-2 enzyme (COX-2) and inducible nitric oxide synthase (iNOS) during the inflammatory phase at the injured growth plate (Figure 2) and found that inhibition of COX-2 or iNOS by specific inhibitors caused an increased proportion of undifferentiated mesenchymal tissue but a decrease in chondrogenic differentiation within the injury site (30). This study confirms that the injury-induced inflammatory response in general at the growth plate injury site is necessary for enhancing the chondrogenic differentiation of mesenchymal cells. Overall, these studies suggest that the injury-induced inflammatory response has an important role early in regulating growth plate injury repair as it initiates and regulates a cascade of downstream events which lead to the bony repair at the growth plate injury site. Similarly during bone fracture healing, these two inflammatory mediators (COX-2 and iNOS) have been found to be important for triggering the cascade of events leading to tissue repair. More specifically, numerous bone fracture studies have demonstrated that inhibiting COX-2 resulted in a delay in bone formation and fracture healing (40-43), highlighting the importance of injury-induced inflammatory response and COX-2 enzyme during tissue repair.

Other studies have also shown that during the inflammatory phase there were increases in the levels of several members of the bone morphogenic protein (BMP) family. The BMPs have been known for having roles in chondrogenic and osteoblastogenic differentiation as well as encouraging mesenchymal cell proliferation and migration (44, 45). Ngo et al (2006) observed the presence and upregulation of BMP-3 and BMP-4 within the growth plate injury sites of young rats (46) (Figure 2). BMP-4 also appeared to be produced by inflammatory cells- indicating their role in mediating the initial inflammatory event in regulating mesenchymal cell migration and differentiation (46). BMP-4's proposed role in regulating mesenchymal cell migration and differentiation during skeletal repair was also echoed in another earlier study which examined BMP-4 potential role and level of expression in regenerating tissue of a rabbit leg-lengthening model (47).

4.2.2. Fibrogenic phase

Following the initial inflammatory phase in the rat growth plate injury model is the fibrogenic phase - occurring during days 3-7 post injury (25). The fibrogenic response involves the influx of fibrous vimentin-immunopositive mesenchymal cells into the injury site (25). This response was also observed in mice, whereby approximately 7 days post injury, there was presence of undifferentiated, spindle- shaped cells near the superior and inferior areas of the growth plate injury site (26). Although it is yet to be confirmed, previous findings of osteogenesis as well as chondrogenesis from these infiltrated cells (25, 30, 31, 48) suggest that these filtrating cells may contain pre-determined chondroprogenitor and osteoprogenitor cells as well as multipotent mesenchymal stem cells. The infiltration of such stromal progenitor cells (originating from periosteum, the circulation as well as from the bone marrow) following the inflammatory response has been confirmed in bone fractures, which is critical for the formation of the bridging "soft callus" as the next stage of the fracture repair process (27, 35, 49).

During the influx of fibrogenic cells in both injured growth plate and bone, mRNA levels of growth factors FGF-2 and PDGF-BB have been found to be significantly upregulated, indicating the possible involvement of both growth factors in regulating this mesenchymal reaction phase in both bone or growth plate injury repair (Figure 2) (34, 48, 50). FGF-2 has functions in various biological responses such as cell proliferation, differentiation and migration (51). During bone fracture healing, various cells such as monocytes, macrophages, mesenchymal cells, osteoblasts and chondrocytes produce FGF-2 (52). Along with its well known roles in mesenchymal cell migration and proliferation (53, 54), FGF-2 has been found to inhibit chondrocyte differentiation (55), alkaline phosphastase activity (56, 57) as well as stimulating bone resorption in vitro (58, 59), suggesting its role in suppressing skeletal cell differentiation during bone fracture repair. Interestingly, a more recent in vitro study has shown that FGF-2 was able to increase the osteogenic and chondrogenic differentiation potentials of mesenchymal cells via suppression of key signalling from TGF-beta (54, 60). However, although it has been suggested that FGF-2 may play a possible role in mesenchymal and osteoprogenitor cell proliferation, migration and differentiation, further studies are required to investigate the functions of the upregulated FGF-2 at the injured growth plate during the fibrogenic phase (33, 34).

PDGFs have been documented to have many different roles including cell migration, cell proliferation and angiogenesis in wound healing (61-64). In particular, it is also a potent chemotactant for fibroblasts and smooth muscle cells (65). During bone fracture repair, PDGFs have been found to be essential for triggering the initial events leading to the migration and proliferation of fibroblasts and osteoblasts (52). Similarly, Zhou et al (2004) found that gene expression levels of PDGF-BB were significantly upregulated following the inflammatory phase in a rat growth plate injury model (34). A recent study by Chung et al (2009) found that inhibition of PDGF-R signalling during the fibrogenic phase reduced proliferation and the level of infiltration of mesenchymal cells by day 4 after injury and the amounts of bony or cartilage tissues at the injury site by day 14, suggesting a critical role of PDGF in the fibrogenic phase of growth plate repair (32).

4.2.3. Osteogenic and maturation phases

Following the fibrogenic phase, the subsequent osteogenic response involves some bone cell differentiation among some of the infiltrated mesenchymal cells, as indicated by positive immunohistochemical staining of Runx-2 and alkaline phosphatase (markers of osteoblast differentiation and maturation, respectively) (25, 30, 31, 33). Furthermore, the presence of active bone deposition containing bone matrix protein osteocalcin on the new trabecular bone surface within the growth plate injury site is indicative of the bony tissue formation (25, 30). During the remodelling and maturation of the bony bridge, bone trabeculae are found to be separated by abundant marrow cells, and were surrounded by flattened spindle-like inactive osteoblasts in resting phase - producing little or no osteocalcin which is characteristic of inactive bone formation (25). In addition, resorptive cells osteoclasts are sometimes seen on some areas of newly formed trabeculae at the injury site (48), suggesting that osteoclastic bone resorption is involved in the bone bridge matuation phase at the injured growth plate. While the molecular mechanisms regulating this maturation phase remains unclear, upregulation of TNF-alpha, IGF-I and BMP-7 at the injured growth plate (Figure 2) suggest their involvement in the bony bridge remodelling (34, 46). Consistently, TNF-alpha upregulation has been observed during the remodelling phase in bone fracture repair (66), and TNF-alpha has been shown to be important in regulating bone remodelling by promoting differentiation of bone resorptive cells, osteoclasts (67). Similarly, BMP-7 upregulation is known to be important for bone formation and remodelling at the bone fracture sites (68). Further studies are required to characterise the molecular and cellular mechanisms regulating the bony bridge maturation/remodelling at the growth plate injured site.

4.3. Mechanisms of bony repair of injured growth plate cartilage

Studies in both murine and rat growth plate injury models by Lee et al (2000) and Xian et al (2004) respectively showed that the bony bridge formation occurring after injury was a result from direct bone formation mainly via intramembranous ossification (25, 26). In support, Lee et al (2000) saw no changes in the levels of endochondral ossification-related molecules including collagen-2, Indian hedgehog (Ihh) and vascular endothelial growth factor (VEGF) at the time points examined (26), and Xian et al revealed Runx2+ osteoblastic differentiation and bony trabecular formation from infiltrated mesenchymal cells (25). Similarly, Zhou et al (2004) reported no up-regulation of chondrogenic transcription factor Sox-9 and cartilage matrix protein collagen-2 at the injured growth plate in this rat model (34). However, more recent studies in the rat growth plate injury model (some with different post-injury time points examined) have found that apart from direct bone formation being as the major bony repair mechanism present, endochondral ossification, despite to a lesser extent, also occurred as a potential mechanism underlying the bony repair. Arasapam et al (2006) found increased expression of some cartilage related genes including collagen-2, collagen-10 and Sox-9 together with increased levels of some bone related genes (30). This indicates the presence of the formation of both cartilage and bone within the growth plate injury site and hence involvement of both endochondral and intramembranous ossification mechanisms during the bony repair. Similarly, Chung et al also found endochondral ossification involvement during bone bridge formation showing presence of cartilage-related molecules, Sox-9 and collagen- 2 and -10 at the growth plate injury site and positive immunostaining of both collagen-2 and -10 in cartilage-like tissue derived from infiltrated mesenchymal cells (31, 32). Further mechanistic studies are required to understand the bone formation pathways underlying the bony repair of injured growth plate.

4.4. Effects of injuries on the adjacent non-injured growth plate tissue

While most growth plate injury studies have focused and looked at the events occurring purely within the injury site, very few have investigated the potential effects of injuries on adjacent growth plate chondrocytes. An earlier study looking at the effects of growth plate trauma observed the intrusion of growth plate cartilage tissue into the metaphyseal region, and found that these islands of trapped cartilage disrupt the continuing bone growth of the surrounding tissue (69). Consequently, there was abnormal widening and irregularities of the remaining growth plate and hence potentially resulting in the deformities and discrepancies seen in many patients as a result from their growth plate related injuries (69). More recently, Coleman et al (2010) utilised micro-CT imaging to characterise changes occurring within the injured growth plates of rats as well as the effect on the whole tibial bone itself (70). Interestingly, Coleman et al (2010) observed that bone volume present within the injury site did not directly correlate with overall reduced bone growth by 35 days after the injury. Furthermore, using micro-CT imaging, by the time a bone bridge has formed, significant damage could already be detected in the remaining non-injured growth plate, including cellular disorganisation as well as a significant decrease in overall growth plate thickness and volume. Interestingly, Coleman et al (2010) also observed that tethers, which usually form with age as the growth plate begins to close (71), were present earlier in the adjacent growth plate after injury (70). These studies highlight the potential involvement of the adjacent remaining growth plate during growth plate injury repair and its contribution to limb length discrepancies and bone angulation deformities that form after growth plate injuries (69, 70). Further mechanistic studies are required to gain a better understanding how the bone bridge formation within the injury site and changes in the adjacent non-injured growth plate tissue contribute to the final undesirable bony repair and bone growth defects after a growth plate injury.

5. PERSPECTIVE & CONCLUSION

Growth plate cartilage injuries in children are common and are a significant clinical problem since they can often lead to undesirable bony repair and bone growth defects. Currently, pathophysiological mechanisms for the undesirable bony repair at the injured growth plate still remain elusive and understanding the detailed events for growth plate injury responses and bony repair is imperative towards the development of an alternative and effective biological therapy for inducing growth plate cartilage regeneration. However, studies in the last 10 years using rodent growth plate injury models have observed sequential injury repair phases (inflammatory, fibrogenic, osteogenic and maturation) and differential gene expression (including various growth factors and cytokines and inflammatory mediators) occurring after growth plate injury. Interestingly, it has been shown that the initial inflammatory response appears to play an important role in regulating downstream healing events and bony repair at the injured growth plate. In addition, after the inflammatory response, infiltration of mesenchymal progenitor cells occurs, which contributes to both intramembranous and endochondral bone formation mechanisms for the bony repair at the injured growth plate. Therefore, growth plate injury responses and bony repair mechanisms appear to be similar to the events that happen during bone fracture healing.

6. ACKNOWLEDGEMENTS

The work from the author's lab discussed here was funded in parts by grants from Bone Growth Foundation (to CJX and BKF), Channel-7 Children's Research Foundation of South Australia (to CJX and BKF), and from the Australian National Health and Medical Research Council (NHMRC) (to CJX and BKF). RC was funded by University of Adelaide Faculty of Science PhD Scholarship, and Bone Growth Foundation and Sansom Institute (UniSA) Top-up Scholarships.

7. REFERENCES

1. R. T. Ballock and R. J. O'Keefe: The biology of the growth plate. J Bone Joint Surg Am, 85-A(4), 715-26 (2003)
PMid:12672851

2. S. Provot and E. Schipani: Molecular mechanisms of endochondral bone development. Biochem Biophys Res Commun, 328(3), 658-65 (2005)
doi:10.1016/j.bbrc.2004.11.068
PMid:15694399

3. C. J. Xian: Roles of epidermal growth factor family in the regulation of postnatal somatic growth. Endocr Rev, 28(3), 284-96 (2007)
doi:10.1210/er.2006-0049
PMid:17322455

4. C. J. Xian, Foster, B.K.: The biological aspects of children's fractures. In: Fractures in Children. Ed J. Beaty, Kasser, J. Lippincott Williams and Wilkins, Philadephia (2006)

5. F. Forriol and F. Shapiro: Bone development: interaction of molecular components and biophysical forces. Clin Orthop Relat Res(432), 14-33 (2005)

6. J. P. Iannotti: Growth plate physiology and pathology. Orthop Clin North Am, 21(1), 1-17 (1990)
PMid:2404230

7. X. Yang, Karsenty, G.: Transcription factors in bone: developmental and pathological aspects. Trends Mol Med, 8, 340- 345 (2002)
doi:10.1016/S1471-4914(02)02340-7

8. G. J. Tortora, Grabowski, S.R.: Principles of anatomy and physiology. John Wiley & Sons Inc, New York (2000)

9. F. Burdan, J. Szumilo, A. Korobowicz, R. Farooquee, S. Patel, A. Patel, A. Dave, M. Szumilo, M. Solecki, R. Klepacz and J. Dudka: Morphology and physiology of the epiphyseal growth plate. Folia Histochem Cytobiol, 47(1), 5-16 (2009)
doi:10.2478/v10042-009-0007-1
PMid:19419931

10. J. Melrose, S. M. Smith, M. M. Smith and C. B. Little: The use of Histochoice for histological examination of articular and growth plate cartilages, intervertebral disc and meniscus. Biotech Histochem, 83(1), 47-53 (2008)
doi:10.1080/10520290801990414

11. E. B. Hunziker: Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Tech, 28(6), 505-19 (1994)
doi:10.1002/jemt.1070280606
PMid:7949396

12. V. Abad, J. L. Meyers, M. Weise, R. I. Gafni, K. M. Barnes, O. Nilsson, J. D. Bacher and J. Baron: The role of the resting zone in growth plate chondrogenesis. Endocrinology, 143(5), 1851-7 (2002)
doi:10.1210/en.143.5.1851
PMid:11956168

13. B. C. van der Eerden, M. Karperien and J. M. Wit: Systemic and local regulation of the growth plate. Endocr Rev, 24(6), 782-801 (2003)
doi:10.1210/er.2002-0033
PMid:14671005

14. D. J. Hadjidakis and Androulakis, II: Bone remodeling. Ann N Y Acad Sci, 1092, 385-96 (2006)
doi:10.1196/annals.1365.035
PMid:17308163

15. T. Mizuta, W. M. Benson, B. K. Foster, D. C. Paterson and L. L. Morris: Statistical analysis of the incidence of physeal injuries. J Pediatr Orthop, 7(5), 518-23 (1987)
PMid:3497947

16. J. H. Brown and S. A. DeLuca: Growth plate injuries: Salter-Harris classification. Am Fam Physician, 46(4), 1180-4 (1992)
PMid:1414883

17. R. B. Salter, Harris, W.R.: Injuries involving the epiphyseal plate. Journal of Bone and Joint Surgery, 45-A (1963)

18. J. T. Leary, M. Handling, M. Talerico, L. Yong and J. A. Bowe: Physeal fractures of the distal tibia: predictive factors of premature physeal closure and growth arrest. J Pediatr Orthop, 29(4), 356-61 (2009)
PMid:19461377

19. S. J. Mubarak, J. R. Kim, E. W. Edmonds, M. E. Pring and T. P. Bastrom: Classification of proximal tibial fractures in children. J Child Orthop, 3(3), 191-7 (2009)
doi:10.1007/s11832-009-0167-8
PMid:19308478    PMCid:2686808

20. R. M. Kay and G. A. Matthys: Pediatric ankle fractures: evaluation and treatment. J Am Acad Orthop Surg, 9(4), 268-78 (2001)
PMid:11476537

21. C. J. Basener, C. T. Mehlman and T. G. DiPasquale: Growth disturbance after distal femoral growth plate fractures in children: a meta-analysis. J Orthop Trauma, 23(9), 663-7 (2009)
doi:10.1097/BOT.0b013e3181a4f25b
PMid:19897989

22. J. A. Ogden: Growth slowdown and arrest lines. J Pediatr Orthop, 4(4), 409-15 (1984)
PMid:6470109

23. A. Barmada, T. Gaynor and S. J. Mubarak: Premature physeal closure following distal tibia physeal fractures: a new radiographic predictor. J Pediatr Orthop, 23(6), 733-9 (2003)
doi:10.1097/00004694-200311000-00010
PMid:14581776

24. J. M. Wattenbarger, H. E. Gruber and L. S. Phieffer: Physeal fractures, part I: histologic features of bone, cartilage, and bar formation in a small animal model. J Pediatr Orthop, 22(6), 703-9 (2002)
doi:10.1097/00004694-200211000-00002
PMid:12409892

25. C. J. Xian, F. H. Zhou, R. C. McCarty and B. K. Foster: Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site. J Orthop Res, 22(2), 417-26 (2004)
doi:10.1016/j.orthres.2003.08.003
PMid:15013105

26. M. A. Lee, T. P. Nissen and N. Y. Otsuka: Utilization of a murine model to investigate the molecular process of transphyseal bone formation. J Pediatr Orthop, 20(6), 802-6 (2000)
doi:10.1097/00004694-200011000-00021
PMid:11097259

27. D. Jaramillo, F. Shapiro, F. A. Hoffer, C. S. Winalski, M. F. Koskinen, R. Frasso and A. Johnson: Posttraumatic growth-plate abnormalities: MR imaging of bony-bridge formation in rabbits. Radiology, 175(3), 767-73 (1990)
PMid:2343128

28. T. Wirth, S. Byers, R. W. Byard, J. J. Hopwood and B. K. Foster: The implantation of cartilaginous and periosteal tissue into growth plate defects. Int Orthop, 18(4), 220-8 (1994)
doi:10.1007/BF00188326
PMid:8002111

29. E. A. Makela, S. Vainionpaa, K. Vihtonen, M. Mero and P. Rokkanen: The effect of trauma to the lower femoral epiphyseal plate. An experimental study in rabbits. J Bone Joint Surg Br, 70(2), 187-91 (1988)
PMid:3346285

30. G. Arasapam, M. Scherer, J. C. Cool, B. K. Foster and C. J. Xian: Roles of COX-2 and iNOS in the bony repair of the injured growth plate cartilage. J Cell Biochem, 99(2), 450-61 (2006)
doi:10.1002/jcb.20905
PMid:16619262

31. R. Chung, Cool, JC, Scherer, MA, Foster, BK, Xian, CJ.: Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats. J Leukoc Biol., 80(6), 1272-80 (2006)
doi:10.1189/jlb.0606365
PMid:16959896

32. R. Chung, B. K. Foster, A. C. Zannettino and C. J. Xian: Potential roles of growth factor PDGF-BB in the bony repair of injured growth plate. Bone, 44(5), 878-85 (2009)
doi:10.1016/j.bone.2009.01.377
PMid:19442606

33. F. H. Zhou, B. K. Foster, X. F. Zhou, A. J. Cowin and C. J. Xian: TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats. J Bone Miner Res, 21(7), 1075-88 (2006)
doi:10.1359/jbmr.060410
PMid:16813528

34. F. H. Zhou, Foster, B.K., Sander, G., Xian, C.J.: Expression of proinflammatory cytokines and growth factors at the injured growth plate cartilage in young rats. Bone, 35, 1307- 1315 (2004)
doi:10.1016/j.bone.2004.09.014
PMid:15589211

35. A. Schindeler, M. M. McDonald, P. Bokko and D. G. Little: Bone remodeling during fracture repair: The cellular picture. Semin Cell Dev Biol, 19(5), 459-66 (2008)
doi:10.1016/j.semcdb.2008.07.004

36. L. C. Gerstenfeld, T. J. Cho, T. Kon, T. Aizawa, J. Cruceta, B. D. Graves and T. A. Einhorn: Impaired intramembranous bone formation during bone repair in the absence of tumor necrosis factor-alpha signaling. Cells Tissues Organs, 169(3), 285-94 (2001)
doi:10.1159/000047893
PMid:11455125

37. X. Fu, B. Han, S. Cai, Y. Lei, T. Sun and Z. Sheng: Migration of bone marrow-derived mesenchymal stem cells induced by tumor necrosis factor-alpha and its possible role in wound healing. Wound Repair Regen, 17(2), 185-91 (2009)
doi:10.1111/j.1524-475X.2009.00454.x
PMid:19320886

38. R. T. Franceschi: Biological approaches to bone regeneration by gene therapy. J Dent Res, 84(12), 1093-103 (2005)
doi:10.1177/154405910508401204
PMid:16304438

39. M. H. Kim, W. Liu, D. L. Borjesson, F. R. Curry, L. S. Miller, A. L. Cheung, F. T. Liu, R. R. Isseroff and S. I. Simon: Dynamics of neutrophil infiltration during cutaneous wound healing and infection using fluorescence imaging. J Invest Dermatol, 128(7), 1812-20 (2008)
doi:10.1038/sj.jid.5701223
PMid:18185533    PMCid:2617712

40. X. Zhang, E. M. Schwarz, D. A. Young, J. E. Puzas, R. N. Rosier and R. J. O'Keefe: Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest, 109(11), 1405-15 (2002)
PMid:12045254    PMCid:151001

41. K. M. Brown, M. M. Saunders, T. Kirsch, H. J. Donahue and J. S. Reid: Effect of COX-2-specific inhibition on fracture-healing in the rat femur. J Bone Joint Surg Am, 86-A(1), 116-23 (2004)
PMid:14711953

42. L. C. Gerstenfeld and T. A. Einhorn: COX inhibitors and their effects on bone healing. Expert Opin Drug Saf, 3(2), 131-6 (2004)
doi:10.1517/14740338.3.2.131
PMid:15006719

43. C. J. Xian and X. F. Zhou: Treating skeletal pain: limitations of conventional anti-inflammatory drugs, and anti-neurotrophic factor as a possible alternative. Nat Clin Pract Rheumatol, 5(2), 92-8 (2009)
doi:10.1038/ncprheum0982
PMid:19182815

44. S. Zoricic, I. Maric, D. Bobinac and S. Vukicevic: Expression of bone morphogenetic proteins and cartilage-derived morphogenetic proteins during osteophyte formation in humans. J Anat, 202(Pt 3), 269-77 (2003)
PMid:12713267    PMCid:1571079

45. D. Chen, M. Zhao and G. R. Mundy: Bone morphogenetic proteins. Growth Factors, 22(4), 233-41 (2004)
doi:10.1080/08977190412331279890
PMid:15621726

46. T. Q. Ngo, M. A. Scherer, F. H. Zhou, B. K. Foster and C. J. Xian: Expression of bone morphogenic proteins and receptors at the injured growth plate cartilage in young rats. J Histochem Cytochem, 54(8), 945-54 (2006)
doi:10.1369/jhc.6A6939.2006
PMid:16651391

47. G. Li, S. Berven, H. Simpson and J. T. Triffitt: Expression of BMP-4 mRNA during distraction osteogenesis in rabbits. Acta Orthop Scand, 69(4), 420-5 (1998)
doi:10.3109/17453679808999060

48. C. Chung and J. A. Burdick: Influence of three-dimensional hyaluronic Acid microenvironments on mesenchymal stem cell chondrogenesis. Tissue Eng Part A, 15(2), 243-54 (2009)
doi:10.1089/ten.tea.2008.0067
PMid:19193129    PMCid:2678568

49. G. L. Garces, I. Mugica-Garay, N. Lopez-Gonzalez Coviella and E. Guerado: Growth-plate modifications after drilling. J Pediatr Orthop, 14(2), 225-8 (1994)
PMid:8188839

50. K. Tatsuyama, Maezawa, Y., Baba, H., Imamura, Y., Fukuda, M.: Expression of various growth factors for cell proliferation and cytodifferentiation during fracture repair. European Journal of Histochem., 44, 269- 278 (2000)
PMid:11095098

51. J. Farre, S. Roura, C. Prat-Vidal, C. Soler-Botija, A. Llach, C. E. Molina, L. Hove-Madsen, J. J. Cairo, F. Godia, R. Bragos, J. Cinca and A. Bayes-Genis: FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells. Growth Factors, 25(2), 71-6 (2007)
doi:10.1080/08977190701345200
PMid:17852409

52. H. Fujii, R. Kitazawa, S. Maeda, K. Mizuno and S. Kitazawa: Expression of platelet-derived growth factor proteins and their receptor alpha and beta mRNAs during fracture healing in the normal mouse. Histochem Cell Biol, 112(2), 131-8 (1999)
doi:10.1007/s004180050399
PMid:10460466

53. L. A. Solchaga, K. Penick, V. M. Goldberg, A. I. Caplan and J. F. Welter: Fibroblast growth factor-2 enhances proliferation and delays loss of chondrogenic potential in human adult bone-marrow-derived mesenchymal stem cells. Tissue Eng Part A, 16(3), 1009-19

54. T. Ito, R. Sawada, Y. Fujiwara and T. Tsuchiya: FGF-2 increases osteogenic and chondrogenic differentiation potentials of human mesenchymal stem cells by inactivation of TGF-beta signaling. Cytotechnology, 56(1), 1-7 (2008)
doi:10.1007/s10616-007-9092-1
PMid:19002835    PMCid:2151969

55. J. Wroblewski and C. Edwall-Arvidsson: Inhibitory effects of basic fibroblast growth factor on chondrocyte differentiation. J Bone Miner Res, 10(5), 735-42 (1995)
doi:10.1002/jbmr.5650100510
PMid:7639109

56. S. B. Rodan, G. Wesolowski, K. A. Thomas, K. Yoon and G. A. Rodan: Effects of acidic and basic fibroblast growth factors on osteoblastic cells. Connect Tissue Res, 20(1-4), 283-8 (1989)
doi:10.3109/03008208909023898
PMid:2612159

57. E. Tanaka, Y. Ishino, A. Sasaki, T. Hasegawa, M. Watanabe, D. A. Dalla-Bona, E. Yamano, T. M. van Eijden and K. Tanne: Fibroblast growth factor-2 augments recombinant human bone morphogenetic protein-2-induced osteoinductive activity. Ann Biomed Eng, 34(5), 717-25 (2006)
doi:10.1007/s10439-006-9092-x
PMid:16568346

58. H. Kawaguchi, C. C. Pilbeam, J. R. Harrison and L. G. Raisz: The role of prostaglandins in the regulation of bone metabolism. Clin Orthop Relat Res(313), 36-46 (1995)

59. H. Kawaguchi, M. Katagiri and D. Chikazu: Osteoclastic bone resorption through receptor tyrosine kinase and extracellular signal-regulated kinase signaling in mature osteoclasts. Mod Rheumatol, 14(1), 1-5 (2004)
doi:10.1007/s10165-003-0257-2
PMid:17028797

60. T. Ito, R. Sawada, Y. Fujiwara, Y. Seyama and T. Tsuchiya: FGF-2 suppresses cellular senescence of human mesenchymal stem cells by down-regulation of TGF-beta2. Biochem Biophys Res Commun, 359(1), 108-14 (2007)
doi:10.1016/j.bbrc.2007.05.067
PMid:17532297

61. R. Cospedal, H. Abedi and I. Zachary: Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases. Cardiovasc Res, 41(3), 708-21 (1999)
doi:10.1016/S0008-6363(98)00232-6

62. J. Fiedler, Roderer, G., Gunther, K.P., Brenner, R.E.: BMP-2, BMP-4 and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells. J Cell Biochem, 87(3), 305- 12 (2002)
doi:10.1002/jcb.10309
PMid:12397612

63. E. J. Watts and M. T. Rose: Platelet-derived growth factor acts via both the Rho-kinase and p38 signaling enzymes to stimulate contraction in an in vitro model of equine wound healing. Domest Anim Endocrinol (2009)

64. C. Hellberg, A. Ostman and C. H. Heldin: PDGF and Vessel Maturation. Recent Results Cancer Res, 180, 103-14 (2010)
doi:10.1007/978-3-540-78281-0_7
PMid:20033380

65. C. H. Heldin: Platelet-derived growth factor--an introduction. Cytokine Growth Factor Rev, 15(4), 195-6 (2004)
doi:10.1016/j.cytogfr.2004.03.001

66. T. Kon, T. J. Cho, T. Aizawa, M. Yamazaki, N. Nooh, D. Graves, L. C. Gerstenfeld and T. A. Einhorn: Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing. J Bone Miner Res, 16(6), 1004-14 (2001)
doi:10.1359/jbmr.2001.16.6.1004
PMid:11393777

67. M. C. Horowitz, Y. Xi, K. Wilson and M. A. Kacena: Control of osteoclastogenesis and bone resorption by members of the TNF family of receptors and ligands. Cytokine Growth Factor Rev, 12(1), 9-18 (2001)
doi:10.1016/S1359-6101(00)00030-7

68. E. Canalis, A. N. Economides and E. Gazzerro: Bone morphogenetic proteins, their antagonists, and the skeleton. Endocr Rev, 24(2), 218-35 (2003)
doi:10.1210/er.2002-0023
PMid:12700180

69. J. G. Craig, K. E. Cramer, D. D. Cody, D. O. Hearshen, R. Y. Ceulemans, M. T. van Holsbeeck and W. R. Eyler: Premature partial closure and other deformities of the growth plate: MR imaging and three-dimensional modeling. Radiology, 210(3), 835-43 (1999)
PMid:10207489

70. R. M. Coleman, J. E. Phillips, A. Lin, Z. Schwartz, B. D. Boyan and R. E. Guldberg: Characterization of a small animal growth plate injury model using microcomputed tomography. Bone

71. E. A. Martin, E. L. Ritman and R. T. Turner: Time course of epiphyseal growth plate fusion in rat tibiae. Bone, 32(3), 261-7 (2003)
doi:10.1016/S8756-3282(02)00983-3

Abbreviations: TNF-alpha: Tumour necrosis factor- alpha; Runx2: Runx-related transcription factor 2; cbf alpha-1: core binding factor- alpha1; IL-1 beta: Interluekin-1beta; PDGF-BB: platelet derived growth factor-BB; FGF-2: basic fibrogenic growth factor; COX-2: Cyclo oxygenase-2; iNOS: nitric oxide synthases; CINC-1: cytokine induced neutrophil chemoattractant; BMP: Bone morphogenic protein; TGF-beta: Transforming growth factor-beta; VEGF: Vascular endothelial growth factor

Key Words: Growth plate, Growth plate injury, Bone growth defects, Injury responses, Repair Mechanisms, Review

Send correspondence to: Rosa Chung, Sansom Institute for Health Research, University of South Australia, City East Campus, GPO Box 2471, Adelaide, SA 5001, Australia, Tel: 618-8302-2716, Fax: 618-8302-1087; E-mail:rosa.chung@unisa.edu.au