Full Length ArticleOsteoblast derived-neurotrophin‑3 induces cartilage removal proteases and osteoclast-mediated function at injured growth plate in rats
Graphical abstract
Introduction
Childhood bone fractures are common with about 50% of children and adolescents unfortunately experiencing a bone fracture [1]. Located at the weakest part of a long bone, the growth plate cartilage (which is responsible for bone growth) is highly prone to injuries (involved in 20% of all fractures) [2]. Yet, the injured growth plate is often “undesirably” repaired by bony tissue, causing bone growth defects (limb length discrepancy and angulations). Currently, correction of these bone defects relies on extremely invasive and sometimes ineffective surgical procedures, and there is a strong need to gain mechanistic understanding underlying the “faulty” repair and develop a preventive treatment [3]. Although previous work in animal models of growth plate repair has identified repair responses (inflammatory, fibrogenic, osteogenic and remodelling) and of some molecular pathways involved [[3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]], mechanisms for the faulty repair still require further elucidation.
In a recent study, neurotrophin‑3 (NT-3) and its high affinity receptor TrkC were found to be highly induced at the injury site and endogenous NT-3 was found to promote bony repair [18]. Increasing evidence now suggests that, apart from their known functions regulating the development/maintenance of the nervous system, neurotrophins or NTs [including nerve growth factor (NGF), brain-derived growth factor (BDNF), neurotrophin-3 (NT-3) and NT-4] and their receptors [including their common low-affinity p75 receptor and specific high-affinity Trk receptors (TrkA for NGF, TrkB for BDNF & NT-4, and TrkC for NT-3)], also have roles in the bone and cartilage as well as skeletal repair events [[18], [19], [20]]. NTs and receptors are expressed in bones [21] and bone repair cells [18,22,23]. Local application of NGF promoted bone healing [24,25]. Recently, in a rat tibial growth plate and bone injury model, we observed increased mRNA expression for neurotrophins NGF, BDNF, NT-3 and NT-4 and their Trk receptors (with NT-3 and its receptor TrkC showing the highest induction) at injury site [18]. Furthermore, NT-3 has been shown to induce the critical osteogenic factor bone morphogenetic protein (BMP-2) and key angiogenic factor vascular endothelial growth factor (VEGF), and NT-3 was found to promote bone formation and vascularization and thus bony repair at the injured growth plate. However, it remains unknown whether and how the injury site-induced NT-3 is involved in regulating the growth plate injury site remodelling.
Previous studies in rat models demonstrated that bony repair at the injured growth plate involves both cartilage and bone formation and osteoclastic remodelling of the newly formed bone trabeculae at the injury site [8,9,12]. During skeletal repair injury site remodelling, cartilage tissue initially formed needs to be removed by osteoclast/chondroclast-mediated resorption and/or degradation by cartilage proteases collagenase-3 (MMP-13) and gelatinase-9 (MMP-9) [26,27]. Then, the initial bone formed and bone converted from cartilage tissue require further remodelling (by resorption and bone formation) [26,28]. Skeletal repair events are known to be tightly regulated by locally produced molecules, such as inflammatory cytokines, osteogenic factors (particularly BMPs [29]), angiogenic factors (particularly VEGF [30]), and osteoclastogenesis regulators [including the major osteoclastogenesis stimulator RANKL (receptor activator of nuclear factor kappa-B ligand) and inhibitor osteoprotegerin (OPG)] [31]. Evidence is emerging that remodelling of the skeletal injury sites is orchestrated by crosstalk between osteoclast precursors/osteoclasts and repair cells (osteoblasts and chondrocytes, which produce cross-talking signals [26,28]). However, further work is required to identify and characterise cross-talk signals that regulate skeletal repair and injury site remodelling.
The current study investigated potential functions and action mechanisms of injury site-derived NT-3 in injury site remodelling in a rat proximal tibial growth plate repair model and in in vitro models of osteoclastogenesis and resorption.
Section snippets
Rat drill-hole growth plate injury and NT-3 intervention treatments
To investigate roles of NT-3 in injury site remodelling during growth plate injury repair, a proximal tibial drill-hole growth plate injury model was used in both hind legs of 6-week-old male normal Sprague Dawley rats as described [6], with approval from the Animal Ethics Committee of SA Pathology/CHN (South Australia). On days 3 and 7 after injury, some injured rats received intraperitoneal injection of a normal sheep serum IgG or a sheep neutralising anti-NT-3 serum IgG [32] (both at 1 mg/kg
Results
To examine potential effects on the non-injured areas/bones of the systemic treatment with rhNT-3 or the anti-NT-3, further analyses of micro-computer tomography (μ-CT) data [18] at the non-injured areas of tibia metaphysis were performed. Results showed that systemic treatment with rhNT-3 or the anti-NT-3 had no significant effects in altering the trabecular bone volume fraction and trabecular structural parameters (trabecular number, thickness and separation) at both day 10 and day 28
Discussion
Mechanisms for the faulty bony repair and bone bridge formation at injured growth plate cartilage remain largely unclear. In a recent study, NT-3 and its receptor TrkC were found to highly induced, and NT-3 was found to enhance osteogenesis and angiogenesis and promote the bony repair at the growth plate injury site [18]. In the current study, we demonstrated that injury site-derived NT-3 participates in the injury site remodelling. We demonstrated that NT-3 is important for the growth plate
Disclosure
All authors state that they have no conflicts of interest.
Acknowledgements
This work was funded in parts by project grants from the National Health and Medical Research Council (NHMRC Australia) (Nos. 1043426 and 1127396), Channel-7 Children's Research Foundation of South Australia (No. 151051), and Natural Science Foundation of China (No. 81671928). Yu-Wen Su was supported by University of South Australia President's PhD Scholarship, and Cory J. Xian was funded by NHMRC Senior Research Fellowship (No. 1042105).
Author contributions
Study design: YS, XZ and CX. Study conduct: YS. Data collection: YS, SC, LZ, RC, CF, YP, QT, and MH. Data analysis: YS and CX. Data interpretation: YS, SC, BF, LB, SG, DC, YX, LC, CP, XZ, JX, and CX. Drafting manuscript: YS and CX. Revising manuscript content: YS, SC, XZ, JX, and CX. Approving final version of manuscript: YS, SC, LZ, RC, CF, MH, YP, QT, LB, SG, DC, YX, LC, BF, CP, XZ, JX, and CX. YS and CX take responsibility for the integrity of the data analysis.
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Current address: Department of Developmental Biology, Harvard University School of Dental Medicine, Boston, MA 02115, USA.