Roles of neurotrophins in skeletal tissue formation and healing.

Neurotrophins and their receptors are key molecules that are known to be critical in regulating nervous system development and maintenance and have been recognized to be also involved in regulating tissue formation and healing in skeletal tissues. Studies have shown that neurotrophins and their receptors are widely expressed in skeletal tissues, implicated in chondrogenesis, osteoblastogenesis, and osteoclastogenesis, and are also involved in regulating tissue formation and healing events in skeletal tissue. Increased mRNA expression for neurotrophins NGF, BDNF, NT-3, and NT-4, and their Trk receptors has been observed in injured bone tissues, and NT-3 and its receptor, TrkC, have been identified to have the highest induction at the injury site in a drill-hole injury repair model in both bone and the growth plate. In addition, NT-3 has also recently been shown to be both an osteogenic and angiogenic factor, and this neurotrophin can also enhance expression of the key osteogenic factor, BMP-2, as well as the major angiogenic factor, VEGF, to promote bone formation, vascularization, and healing of the injury site. Further studies, however, are needed to investigate if different neurotrophins have differential roles in skeletal repair, and if NT-3 can be a potential target of intervention for promoting bone fracture healing.

Increase in late diagnosed developmental dysplasia of the hip in South Australia: risk factors, proposed solutions.

OBJECTIVES: To review evidence for the increased incidence of late diagnosed developmental dysplasia of the hip (DDH) in South Australia; to identify perinatal risk factors associated with late DDH in babies born between 2003 and 2009 in SA. DESIGN: Linkage study of data collected prospectively by the South Australian Birth Defects Register (SABDR) and the Pregnancy Outcome Statistics Unit (SA Department of Health), supplemented by medical records review. PARTICIPANTS: All children born 2003-2009 in whom DDH was diagnosed between 3 months and 5 years of age and notified to the SABDR (data inclusion range, 2003-2014). Children with teratological hip dislocations and other major congenital abnormalities were excluded. MAIN OUTCOME MEASURES: Uni- and multivariable analyses were performed to identify perinatal risk factors for late diagnosed DDH. RESULTS: The incidence of late diagnosed DDH in babies born 2003-2009 was 0.77 per 1000 live births, contrasting with the figure of 0.22 per 1000 live births during 1988-2003. Significant perinatal risk factors were birth in a rural hospital (v metropolitan public hospital: odds ratio [OR], 2.47; CI, 1.37-4.46; P = 0.003), and being the second child (v being the first-born: OR, 1.69; CI, 1.08-2.66; P = 0.023). Breech presentation was highly significant as a protective factor when compared with cephalic presentation (OR, 0.25; CI, 0.12-0.54; P < 0.001). CONCLUSIONS: The incidence of late DDH has increased in SA despite an ongoing clinical screening program. Increased awareness, education, and avoidance of inappropriate lower limb swaddling are necessary to reverse this trend.

Potential Effects of Phytoestrogen Genistein in Modulating Acute Methotrexate Chemotherapy-Induced Osteoclastogenesis and Bone Damage in Rats.

Chemotherapy-induced bone damage is a frequent side effect which causes diminished bone mineral density and fracture in childhood cancer sufferers and survivors. The intensified use of anti-metabolite methotrexate (MTX) and other cytotoxic drugs has led to the need for a mechanistic understanding of chemotherapy-induced bone loss and for the development of protective treatments. Using a young rat MTX-induced bone loss model, we investigated potential bone protective effects of phytoestrogen genistein. Oral gavages of genistein (20 mg/kg) were administered daily, for seven days before, five days during, and three days after five once-daily injections (sc) of MTX (0.75 mg/kg). MTX treatment reduced body weight gain and tibial metaphyseal trabecular bone volume (p < 0.001), increased osteoclast density on the trabecular bone surface (p < 0.05), and increased the bone marrow adipocyte number in lower metaphyseal bone (p < 0.001). Genistein supplementation preserved body weight gain (p < 0.05) and inhibited ex vivo osteoclast formation of bone marrow cells from MTX-treated rats (p < 0.001). However, MTX-induced changes in bone volume, trabecular architecture, metaphyseal mRNA expression of pro-osteoclastogenic cytokines, and marrow adiposity were not significantly affected by the co-administration of genistein. This study suggests that genistein may suppress MTX-induced osteoclastogenesis; however, further studies are required to examine its potential in protecting against MTX chemotherapy-induced bone damage.

Methotrexate chemotherapy promotes osteoclast formation in the long bone of rats via increased pro-inflammatory cytokines and enhanced NF-κB activation.

Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MTX-induced pro-inflammatory cytokines and activation of NF-κB in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis indicated that, although the receptor activator of NF-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-α levels and activation of NF-κB.

Capital Femoral Epiphysis with Acute Unstable Valgus Type Slip Managed with Closed Reduction and Percutaneous Fixation: A Case Report.

Case: We present a case of acute unstable valgus slipped capital femoral epiphysis (SCFE) in an 8-year-old female who presented after a trip and fall. The patient was managed with emergent closed reduction and percutaneous screw fixation and prophylactic fixation of contralateral side after 6 weeks. At 18-month follow-up, the patient was symptom free with a good range of movement and no evidence of slip progression, chondrolysis or avascular necrosis of the femoral head. Conclusion: We demonstrate that, in this case, closed reduction and percutaneous fixation provided satisfactory outcome at 18-month follow-up. This case highlights the need for both anteroposterior and lateral radiographs.

The role of osteocyte apoptosis in cancer chemotherapy-induced bone loss.

Intensive cancer chemotherapy leads to significant bone loss, the underlying mechanism of which remains unclear. The objective of this study was to elucidate mechanisms for effect of the commonly used anti-metabolite methotrexate (MTX) on osteocytes and on general bone homeostasis. The current study in juvenile rats showed that MTX chemotherapy caused a 4.3-fold increase in the number of apoptotic osteocytes in tibial metaphysis, which was accompanied by a 1.8-fold increase in the number of tartrate-resistant acid phosphatase-positive bone resorbing osteoclasts, and a 35% loss of trabecular bone. This was associated with an increase in transcription of the osteoclastogenic cytokines IL-6 (10-fold) and IL-11 (2-fold). Moreover, the metaphyseal bone of MTX-treated animals exhibited a 37.6% increase in the total number of osteocytes, along with 4.9-fold higher expression of the DMP-1 transcript. In cultured osteocyte-like MLO-Y4 cells, MTX treatment significantly increased caspase-3-mediated apoptosis, which was accompanied by the formation of plasma membrane-born apoptotic bodies and an increase in IL-6 (24-fold) and IL-11 (29-fold) mRNA expression. Conditioned media derived from MTX-treated MLO-Y4 cells was twice as strong as untreated media in its capacity to induce osteoclast formation in primary bone marrow osteoclast precursors. Thus, our in vivo and in vitro data suggested that MTX-induced apoptosis of osteocytes caused higher recruitment of DMP-1 positive osteocytes and increased osteoclast formation, which could contribute towards the loss of bone homeostasis in vivo.

Methotrexate chemotherapy reduces osteogenesis but increases adipogenic potential in the bone marrow.

Intensive use of cancer chemotherapy is increasingly linked with long-term skeletal side effects such as osteopenia, osteoporosis and fractures. However, cellular mechanisms by which chemotherapy affects bone integrity remain unclear. Methotrexate (MTX), used commonly as an anti-metabolite, is known to cause bone defects. To study the pathophysiology of MTX-induced bone loss, we examined effects on bone and marrow fat volume, population size and differentiation potential of bone marrow stromal cells (BMSC) in adult rats following chemotherapy for a short-term (five once-daily doses at 0.75 mg/kg) or a 6-week term (5 doses at 0.65 mg/kg + 9 days rest + 1.3 mg/kg twice weekly for 4 weeks). Histological analyses revealed that both acute and chronic MTX treatments caused a significant decrease in metaphyseal trabecular bone volume and an increase in marrow adipose mass. In the acute model, proliferation of BMSCs significantly decreased on days 3-9, and consistently the stromal progenitor cell population as assessed by CFU-F formation was significantly reduced on day 9. Ex vivo differentiation assays showed that while the osteogenic potential of isolated BMSCs was significantly reduced, their adipogenic capacity was markedly increased on day 9. Consistently, RT-PCR gene expression analyses showed osteogenic transcription factors Runx2 and Osterix (Osx) to be decreased but adipogenic genes PPARγ and FABP4 up-regulated on days 6 and 9 in the stromal population. These findings indicate that MTX chemotherapy reduces the bone marrow stromal progenitor cell population and induces a switch in differentiation potential towards adipogenesis at the expense of osteogenesis, resulting in osteopenia and marrow adiposity.

Deregulation of the CXCL12/CXCR4 axis in methotrexate chemotherapy-induced damage and recovery of the bone marrow microenvironment.

Cancer chemotherapy disrupts the bone marrow (BM) microenvironment affecting steady-state proliferation, differentiation and maintenance of haematopoietic (HSC) and stromal stem and progenitor cells; yet the underlying mechanisms and recovery potential of chemotherapy-induced myelosuppression and bone loss remain unclear. While the CXCL12/CXCR4 chemotactic axis has been demonstrated to be critical in maintaining interactions between cells of the two lineages and progenitor cell homing to regions of need upon injury, whether it is involved in chemotherapy-induced BM damage and repair is not clear. Here, a rat model of chemotherapy treatment with the commonly used antimetabolite methotrexate (MTX) (five once-daily injections at 0.75 mg/kg/day) was used to investigate potential roles of CXCL12/CXCR4 axis in damage and recovery of the BM cell pool. Methotrexate treatment reduced marrow cellularity, which was accompanied by altered CXCL12 protein levels (increased in blood plasma but decreased in BM) and reduced CXCR4 mRNA expression in BM HSC cells. Accompanying the lower marrow CXCL12 protein levels (despite its increased mRNA expression in stromal cells) was increased gene and protein levels of metalloproteinase MMP-9 in bone and BM. Furthermore, recombinant MMP-9 was able to degrade CXCL12 in vitro. These findings suggest that MTX chemotherapy transiently alters BM cellularity and composition and that the reduced cellularity may be associated with increased MMP-9 expression and deregulated CXCL12/CXCR4 chemotactic signalling.

Enhanced BMP signalling causes growth plate cartilage dysrepair in rats.

Growth plate cartilage injuries often result in bony repair at the injury site and premature mineralisation at the uninjured region causing bone growth defects, for which underlying mechanisms are unclear. With the prior microarray study showing upregulated bone morphogenetic protein (BMP) signalling during the injury site bony repair and with the known roles of BMP signalling in bone healing and growth plate endochondral ossification, this study used a rat tibial growth plate drill-hole injury model with or without systemic infusion of BMP antagonist noggin to investigate roles of BMP signalling in injury repair responses within the injury site and in the adjacent "uninjured" cartilage. At days 8, 14 and 35 post-injury, increased expression of BMP members and receptors and enhanced BMP signalling (increased levels of phosphorylated (p)-Smad1/5/8) were found during injury site bony repair. After noggin treatment, injury site bony repair at days 8 and 14 was reduced as shown by micro-CT and histological analyses and lower mRNA expression of osteogenesis-related genes Runx2 and osteocalcin (by RT-PCR). At the adjacent uninjured cartilage, the injury caused increases in the hypertrophic zone/proliferative zone height ratio and in mRNA expression of hypertrophy marker collagen-10, but a decrease in chondrogenesis marker Sox9 at days 14 and/or 35, which were accompanied by increased BMP signalling (increased levels of pSmad1/5/8 protein and BMP7, BMPR1a and target gene Dlx5 mRNA). Noggin treatment reduced the hypertrophic zone/proliferative zone height ratio and collagen-10 mRNA expression, but increased collagen-2 mRNA levels at the adjacent growth plate. This study has identified critical roles of BMP signalling in the injury site bony repair and in the hypertrophic degeneration of the adjacent growth plate in a growth plate drill-hole repair model. Moreover, suppressing BMP signalling can potentially attenuate the undesirable bony repair at injury site and suppress the premature hypertrophy but potentially rescue chondrogenesis at the adjacent growth plate.

Characterisation and developmental potential of ovine bone marrow derived mesenchymal stem cells.

Since discovery, significant interest has been generated in the potential application of mesenchymal stem cells or multipotential stromal cells (MSC) for tissue regeneration and repair, due to their proliferative and multipotential capabilities. Although the sheep is often used as a large animal model for translating potential therapies for musculoskeletal injury and repair, the characteristics of MSC from ovine bone marrow have been inadequately described. Histological and gene expression studies have previously shown that ovine MSC share similar properties with human and rodents MSC, including their capacity for clonogenic growth and multiple stromal lineage differentiation. In the present study, ovine bone marrow derived MSCs positively express cell surface markers associated with MSC such as CD29, CD44 and CD166, and lacked expression of CD14, CD31 and CD45. Under serum-deprived conditions, proliferation of MSC occurred in response to EGF, PDGF, FGF-2, IGF-1 and most significantly TGF-alpha. While subcutaneous transplantation of ovine MSC in association with a ceramic HA/TCP carrier into immunocomprimised mice resulted in ectopic osteogenesis, adipogenesis and haematopoietic-support activity, transplantation of these cells within a gelatin sponge displayed partial chondrogenesis. The comprehensive characterisation of ovine MSC described herein provides important information for future translational studies involving ovine MSC.

Roles of Wnt signalling in bone growth, remodelling, skeletal disorders and fracture repair.

Wnt signalling has an essential role in regulating bone formation and remodelling during embryonic development and throughout postnatal and adult life. Specifically, Wnt signalling regulates bone formation by controlling embryonic cartilage development and postnatal chondrogenesis, osteoblastogenesis, osteoclastogenesis, endochondral bone formation, and bone remodelling. Abnormalities in the function of Wnt genes give rise to or contribute to the development of several pathological bone conditions, including abnormal bone mass, osteosarcomas and bone loss in multiple myeloma. Furthermore, Wnt signalling is activated during bone fracture repair and plays a crucial role in regulating bone regeneration.

Folinic acid attenuates methotrexate chemotherapy-induced damages on bone growth mechanisms and pools of bone marrow stromal cells.

Chemotherapy often induces bone growth defects in pediatric cancer patients; yet the underlying cellular mechanisms remain unclear and currently no preventative treatments are available. Using an acute chemotherapy model in young rats with the commonly used antimetabolite methotrexate (MTX), this study investigated damaging effects of five once-daily MTX injections and potential protective effects of supplementary treatment with antidote folinic acid (FA) on cellular activities in the tibial growth plate, metaphysis, and bone marrow. MTX suppressed proliferation and induced apoptosis of chondrocytes, and reduced collagen-II expression and growth plate thickness. It reduced production of primary spongiosa bone, volume of secondary spongiosa bone, and proliferation of metaphyseal osteoblasts, preosteoblasts and bone marrow stromal cells, with the cellular activities being most severely damaged on day 9 and returning to or towards near normal levels by day 14. On the other hand, proliferation of marrow pericytes was increased early after MTX treatment and during repair. FA supplementation significantly suppressed chondrocyte apoptosis, preserved chondrocyte proliferation and expression of collagen-II, and attenuated damaging effects on production of calcified cartilage and primary bone. The supplementation also significantly reduced MTX effects on proliferation of metaphyseal osteoblastic cells and of bone marrow stromal cells, and enhanced pericyte proliferation. These observations suggest that FA supplementation effectively attenuates MTX damage on cellular activities in producing calcified cartilage and primary trabecular bone and on pools of osteoblastic cells and marrow stromal cells, and that it enhances proliferation of mesenchymal progenitor cells during bone/bone marrow recovery.

Osteoblast derived-neurotrophin­3 induces cartilage removal proteases and osteoclast-mediated function at injured growth plate in rats.

Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin­3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW264.7 osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW264.7 cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts).

Effects of etoposide and cyclophosphamide acute chemotherapy on growth plate and metaphyseal bone in rats.

Pediatric cancer chemotherapy is known to cause bone growth arrest and osteoporotic changes, and yet the underlying mechanisms remain largely unknown. This project investigated effects of acute chemotherapy with topoisomerase inhibitor etoposide (Eto, 80 mg/kg), alkylating agent cyclophosphamide (Cyc, 240 mg/kg) or their combination (Cyc 120 mg/kg + Eto 50 mg/kg) on structural and cellular changes in the growth plate cartilage and metaphyseal bone, two important regions responsible for bone growth and bone mass accumulation. On day 3 after a single injection with either of the three treatments, although the total growth plate thickness was not significantly altered, the cellularity and height of the proliferative zone were significantly reduced. It was shown that while Eto suppressed chondrocyte proliferation, Cyc induced apoptosis in the growth plate proliferative zone. In the metaphysis, although osteoblastic cell surface was decreased in all three treated groups, the trabecular bone bone volume (BV/TV%) was not significantly altered on day 3. On the other hand, the acute chemotherapy reduced heights of both primary and secondary spongiosa trabecular bone. Therefore, Eto and/ or Cyc chemotherapy altered survival or proliferation of growth plate chondrocytes and metaphyseal osteoblastic cells and reduced heights of metaphyseal spongiosa trabecular bone, which may contribute to chemotherapy side effects of these two drugs on bone lengthening and bone mass accumulation.

Cellular mechanisms for methotrexate chemotherapy-induced bone growth defects.

Methotrexate (MTX) is a commonly used anti-metabolite in childhood oncology and is known to cause bone growth arrest and osteoporosis; yet the underlying mechanisms for MTX-induced bone growth defects remain largely unclear. This study characterized damaging effects in young rats of acute chemotherapy with 5 once-daily doses of MTX (0.75 mg/kg) on the cellular activities in the growth plate in producing calcified cartilage and trabecular bone and on activities of osteoblastic cells in the metaphysis. MTX treatment significantly induced chondrocyte apoptosis. MTX also suppressed chondrocyte proliferation and reduced collagen-II mRNA expression and total thickness of the growth plate, with the damage being most obvious on day 9 after the first injection, and with the growth plate histological structure returning normal on day 14. In the adjacent metaphyseal bone, mirroring the decrease in the width of the growth plate, production of primary spongiosa bone was markedly reduced and bone volume of the secondary spongiosa was decreased. Furthermore, MTX treatment significantly induced osteocyte apoptosis in the primary spongiosa and reduced proliferation of osteoblasts and preosteoblasts particularly in the secondary spongiosa. These observations suggest that methotrexate chemotherapy may cause bone growth defects by arresting cellular activities in the growth plate in producing calcified cartilage and primary trabecular bone and by decreasing pools of metaphyseal osteoblastic cells. However, this short-term MTX treatment only caused transit suppressions on growth plate cartilage and trabecular bone, as most cellular and histological parameters had recovered by day 14 or 21.

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

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-1beta. 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-1beta and growth factors, platelet-derived growth factor-B and TGF-beta1, 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-alpha1 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.

TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats.

UNLABELLED: TNF-alpha is known to inhibit osteoblast differentiation in vitro and yet it is essential for bone fracture repair. Roles of TNF-alpha in the bony repair of injured growth plate were examined in young rats treated with a TNF-alpha antagonist. The results show that TNF-alpha mediates p38 activation, which influences the recruitment, proliferation, and osteoblast differentiation of mesenchymal cells and negatively regulates bone formation at the injured growth plate. INTRODUCTION: TNF-alpha inhibits expression of osteoblast differentiation factor cbfa1 and osteoblast differentiation in vitro and yet TNF-alpha signaling is essential for bone fracture healing. Roles of TNF-alpha in the bony repair of injured growth plate cartilage are unknown. MATERIALS AND METHODS: Roles of TNF-alpha in the activation of p38 mitogen activated protein (MAP) kinase and the subsequent bony repair of the injured growth plate were examined in young rats receiving the TNF-alpha inhibitor ENBREL or saline control. Activation of p38 was determined by Western blot analysis and immunohistochemistry. Inflammatory cell counts on day 1, measurements of repair tissue proportions, and counting of proliferative mesenchymal cells on day 8 at growth plate injury site were carried out (n = 6). Expression of inflammatory cytokines TNF-alpha and IL-1beta, fibrogenic growth factor (FGF)-2, cbfa1, and bone protein osteocalcin at the injured growth plate was assessed by quantitative RT-PCR. Effects of TNF-alpha signaling on proliferation, migration, and apoptosis of rat bone marrow mesenchymal cells (rBMMCs) and the regulatory roles of p38 in these processes were examined using recombinant rat TNF-alpha, ENBREL, and the p38 inhibitor SB239063 in cultured primary rBMMCs. RESULTS: p38 activation was induced in the injured growth plate during the initial inflammatory response, and activated p38 was immunolocalized in inflammatory cells at the injury site and in the adjacent growth plate. In addition, activation of p38 was blocked in rats treated with TNF-alpha antagonist, suggesting a role of TNF-alpha in p38 activation. Whereas TNF-alpha inhibition did not alter inflammatory infiltrate and expression of TNF-alpha and IL-1beta at the injured growth plate on day 1, it reduced mesenchymal infiltrate and cell proliferation and FGF-2 expression on day 8. Consistently, TNF-alpha increased proliferation and migration of rBMMCs in vitro, whereas p38 inhibition reduced rBMMC proliferation and migration. At the injured growth plate on day 8, TNF-alpha inhibition increased expression of cbfa1 and osteocalcin and increased trabecular bone formation at the injury site. There was a significant inverse correlation between TNF-alpha and cbfa1 expression levels, suggesting a negative relationship between TNF-alpha and cbfa1 in this in vivo model. CONCLUSIONS: These observations suggest that TNF-alpha activates p38 MAP kinase during the inflammatory response at the injured growth plate, and TNF-alpha-p38 signaling seems to be required for marrow mesenchymal cell proliferation and migration at the growth plate injury site and in cell culture. Furthermore, TNF signaling has an inhibitory effect on bone formation at the injured growth plate by suppressing bone cell differentiation and bone matrix synthesis at the injury site.

Expression of bone morphogenic proteins and receptors at the injured growth plate cartilage in young rats.

The injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Bone morphogenic proteins (BMPs) are important for bone fracture repair, and as a step to characterize potential involvement of BMPs in bony repair of injured growth plate, expression of BMPs and receptors (BMP-R) was examined by quantitative RT-PCR and immunohistochemistry in rat injured tibial growth plate. During the inflammatory response on day 1, slightly increased expression of BMP-3, BMP-4, BMP-R1a, and BMP-R2 was observed, with immunostaining seen among inflammatory cells at the injury site. During mesenchymal infiltration and osteogenic responses on days 3-14, moderately increased expression of BMP-2, -3, -4, -7, and BMP-R1a was found, with immunostaining observed among infiltrated mesenchymal cells and differentiated osteoblasts lining bony trabeculae. During maturation phase on days 14-25, only BMP-7 was seen upregulated slightly and was localized in osteoblasts and marrow cells at the injury site. The temporospatial expression of BMPs and receptors at the injured growth plate suggests potential involvement of BMP-3 and -4 in regulating the inflammatory response or as its mediators in modulating downstream events, and BMP-2, -3, -4, and -7 in the fibrogenic and osteogenic responses, and BMP-7 in bone remodeling at the injured growth plate.

Neurotrophin-3 Induces BMP-2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats.

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill-hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT-3, and NT-4 and their Trk receptors. NT-3 and its receptor TrkC showed the highest induction. NT-3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT-3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT-3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP-2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT-3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP-2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT-3 osteogenic effect in vitro because it can be almost completely abrogated by co-addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT-3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co-treatment with anti-VEGF. This study suggests that NT-3 may be an osteogenic and angiogenic factor upstream of BMP-2 and VEGF in bony repair, and further studies are required to investigate whether NT-3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.