Bone Growth Defects Research Articles

Inhibition of protein kinase-D promotes cartilage repair at injured growth plate in rats

IntroductionInjured growth plate cartilage is often repaired by bony tissue, causing bone growth defects in children. Currently, mechanisms for the undesirable repair remain unclear and there are no biological treatments available to prevent the associated bone growth defects. Osterix is known as a vital transcription factor for osteoblast differentiation which is critical for normal bone formation and bone repair, and osterix is known to be regulated by protein kinase-D; however it is unknown whether protein kinase-D–osterix signalling plays any roles in the bony repair of injured growth plate. MethodsUsing a rat model, this study investigated potential roles of protein kinase-D (PKD) in regulating expression of osteogenic transcription factor osterix and the growth plate bony repair. 4 days post injury at the proximal tibial growth plate, rats received four once-daily injections of vehicle or 2.35mg/kg gö6976 (a PKD inhibitor), and growth plate tissues collected at day 10 were examined histologically and molecularly. In addition, effects of PKD inhibition on osteogenic and chondrogenic differentiation were examined in vitro using rat bone marrow mesenchymal stromal cells. ResultsCompared to vehicle control, PKD inhibition caused a decrease in bone volume (p<0.05), an increase in % of mesenchymal tissue (p<0.01), and an increase in cartilaginous tissue within the injury site. Consistently, gö6976 treatment tended to decrease expression of bone-related genes (osterix, osteocalcin) and increase levels of cartilage-related genes (Sox9, collagen-2a, collagen-10a1). In support, in vitro experiments showed that gö6976 presence in the primary rat marrow stromal cell culture resulted in a decrease of alkaline phosphatase+ CFU-f colonies formed (p<0.05) and an increase in collagen-2a expression in chondrogenic pellet culture (p<0.05). ConclusionThese studies suggest that PKD is important for growth plate bony repair and its inhibition after growth plate injury may result in less bone formation and potentially more cartilage repair.

Prevention of Bone Growth Defects, Increased Bone Resorption and Marrow Adiposity with Folinic Acid in Rats Receiving Long-Term Methotrexate

The underlying pathophysiology for bone growth defects in paediatric cancer patients receiving high dose methotrexate chemotherapy remains unclear and currently there are no standardized preventative treatments for patients and survivors. Using a model in young rats, we investigated damaging effects of long-term treatment with methotrexate on growth plate and metaphyseal bone, and the potential protective effects of antidote folinic acid. This study demonstrated that chronic folinic acid supplementation can prevent methotrexate-induced chondrocyte apoptosis and preserve chondrocyte columnar arrangement and number in the growth plate. In the metaphysis, folinic acid supplementation can preserve primary spongiosa heights and secondary spongiosa trabecular volume by preventing osteoblasts from undergoing apoptosis and suppressing methotrexate-induced marrow adiposity and osteoclast formation. Systemically, plasma of folinic acid supplemented rats, in comparison to plasma from rats treated with MTX alone, contained a significantly lower level of IL-1β and suppressed osteoclast formation in vitro in normal bone marrow cells. The importance of IL-1β in supporting plasma-induced osteoclast formation was confirmed as the presence of an anti-IL-1β neutralizing antibody attenuated the ability of the plasma (from MTX-treated rats) in inducing osteoclast formation. Findings from this study suggest that folinic acid supplementation during chronic methotrexate treatment can alleviate growth plate and metaphyseal damages and therefore may be potentially useful in paediatric patients who are at risk of skeletal growth suppression due to chronic methotrexate chemotherapy.

Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area

Injury to the growth plate is common and yet the injured cartilage is often repaired with undesirable bony tissue, leading to bone growth defects in children. Using a rat tibial growth plate injury model, our previous studies have shown sequential inflammatory, fibrogenic, osteogenic and bone maturation responses involved in the bony repair. However, it remains unclear whether there is progressive accumulation of bone within the injury site and any potential degenerative changes at the adjacent non-injured area of the growth plate. This study examined effects of growth plate injury on the structure, composition and some cellular and molecular changes at the injury site and adjacent uninjured area. Micro-CT analysis revealed that while the bone volume within the injury site at day 14 was small, the bone bridge was considerably larger at the injury site by 60 days post-injury. Interestingly, formation of bone bridges in the adjacent uninjured area was detected in 60% of injured animals at day 60. Immunohistochemical analyses revealed reduced chondrocyte proliferation (PCNA labelling) but increased apoptosis (nick translation labelling) in the adjacent uninjured area. RT-PCR analysis on adjacent uninjured growth plate tissue found increased expression of osteocalcin at day 60, differential expression of apoptosis-regulatory genes and alterations in genes associated with chondrocyte proliferation/differentiation, including Sox9 and IGF-I. Therefore, this study has demonstrated progressive changes in the structure/composition of the injury site and adjacent uninjured area and identified cellular and molecular alterations or degeneration in adjacent uninjured growth plate in response to injury.

Growth plate dysfunction, reduced osteogenesis, increased osteocyte apoptosis and osteoclastogenesis in methotrexate chemotherapy-induced bone growth defects

Growth plate dysfunction, reduced osteogenesis, increased osteocyte apoptosis and osteoclastogenesis in methotrexate chemotherapy-induced bone growth defects

Methotrexate toxicity in growing long bones of young rats: a model for studying cancer chemotherapy-induced bone growth defects in children.

The advancement and intensive use of chemotherapy in treating childhood cancers has led to a growing population of young cancer survivors who face increased bone health risks. However, the underlying mechanisms for chemotherapy-induced skeletal defects remain largely unclear. Methotrexate (MTX), the most commonly used antimetabolite in paediatric cancer treatment, is known to cause bone growth defects in children undergoing chemotherapy. Animal studies not only have confirmed the clinical observations but also have increased our understanding of the mechanisms underlying chemotherapy-induced skeletal damage. These models revealed that high-dose MTX can cause growth plate dysfunction, damage osteoprogenitor cells, suppress bone formation, and increase bone resorption and marrow adipogenesis, resulting in overall bone loss. While recent rat studies have shown that antidote folinic acid can reduce MTX damage in the growth plate and bone, future studies should investigate potential adjuvant treatments to reduce chemotherapy-induced skeletal toxicities.

Musculoskeletal Manifestations of Sickle Cell Anaemia: A Pictorial Review

Sickle cell anaemia is an autosomal recessive genetic condition producing abnormal haemoglobin HbS molecules that result in stiff and sticky red blood cells leading to unpredictable episodes of microvascular occlusions. The clinical and radiological manifestations of sickle cell anaemia result from small vessel occlusion, leading to tissue ischemia/infarction and progressive end-organ damage. In this paper we discuss and illustrate the various musculoskeletal manifestations of sickle cell disease focusing primarily on marrow hyperplasia, osteomyelitis and septic arthritis, medullary and epiphyseal bone infarcts, growth defects, and soft tissue changes.

Injury responses and repair mechanisms of the injured growth plate

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.

Pathophysiology and prevention of cancer chemotherapy-induced bone growth defects

Pathophysiology and prevention of cancer chemotherapy-induced bone growth defects

Targeted Ablation of the WW Domain-Containing Oxidoreductase Tumor Suppressor Leads to Impaired Steroidogenesis

The WW domain-containing oxidoreductase (WWOX) gene encodes a 46-kDa tumor suppressor. The Wwox protein contains two N-terminal WW domains that interact with several transcriptional activators containing proline-tyrosine motifs and a central short-chain dehydrogenase/reductase domain that has been suggested to play a role in steroid metabolism. Recently, we have shown that targeted deletion of the Wwox gene in mice leads to postnatal lethality and defects in bone growth. Here, we report that Wwox-deficient mice display impaired steroidogenesis. Mutant homozygous mice are born with gonadal abnormalities, including failure of Leydig cell development in testis and reduced theca cell proliferation in ovary. Furthermore, Wwox(-/-) mice displayed impaired gene expression of key steroidogenesis enzymes. Affymetrix microarray gene analysis revealed differentially expressed related genes in steroidogenesis in knockout mice testis and ovary as compared with control mice. These results demonstrate the essential requirement for the Wwox tumor suppressor in proper steroidogenesis.

Damaging effects of chronic low-dose methotrexate usage on primary bone formation in young rats and potential protective effects of folinic acid supplementary treatment

Methotrexate (MTX) is a most commonly used anti-metabolite in cancer treatment and as an anti-rheumatic drug. While MTX chemotherapy at a high dose is known to cause bone growth defects in growing bones, effects of its chronic use at a low dose on growing skeleton remain less clear. Here, we examined effects on bone growth of long-term MTX chemotherapy at a low dose in young rats, and potential protective effects of supplementary treatment with antidote folinic acid (given ip at 1 mg/kg 6 h after MTX). After two cycles of 5 once-daily MTX injections (at 0.75 mg/kg, 5 days on/9 days off/5 days on), histological analysis showed that MTX at this dose caused significant reduction in heights of growth plate and primary spongiosa bone on day 22 compared to controls (P < 0.05). In contrast, a similar dosing regimen but at a lower dose (0.4 mg/kg) caused only slight or no reduction in heights of both regions. However, after the induction phase at this 0.4 mg/kg dosing, continued use of MTX at a low dose (once weekly at 0.2 mg/kg) caused a reduction in primary spongiosa height and bone volume on weeks 9 and 14, which was associated with an increased osteoclast formation and their bone surface density as well as a decreased osteoblast bone surface density in the primary spongiosa. Folinic acid supplementation was shown able to prevent the MTX effects in the primary spongiosa. These results suggest that acute use of MTX can damage growth plate and primary bone at a high dose, but not at a low dose. However, long-term use of MTX at a low dose can reduce primary bone formation probably due to decreased osteoblastic function but increased osteoclastic formation and function, and supplementary treatment with folinic acid may be potentially useful in protecting bone growth during long-term low-dose MTX chemotherapy.

026 Altered Skin Wound Healing in Homeobox Gene Msx-2 Knockout Mice

Homeobox genes are transcription factor that form nested patterns in specific regions throughout the body. Homeobox genes are important for wound healing study because some have been shown to be related to scarless fetal wound healing. Muscle segment homeobox-2 (Msx-2) is a homeobox gene commonly expressed at sites of epithelial-mesenchymal interactions during tissue morphogenesis. Overexpression of Msx-2 leads to craniosynostosis, epidermal dysplasia, and abnormal hair growth. A knockout of this gene causes pleiotropic defects in bone growth, cyclic alopecia, and a thinner dermis. While Msx-2 has been found to play many critical roles in development, no functional study has been conducted on Msx-2 and wound healing. We hypothesize that Msx-2 regulates skin morphogenesis during wound repair which was tested by studying excisional skin wound closure. Isolated keratinocytes and dermal fibroblasts of Msx-2 WT and KO mice were also studied to determine the effects of Msx-2. Results showed that Msx-2 mRNA was induced in epidermis and dermis during wound repair. Additionally, Msx-2 KO mice exhibited enhanced re-epithelialization and faster wound closure than the WT control. These repair phenotypes may be attributed to keratinocyte motility and fibroblast interaction with collagen matrix as, in vitro, Msx-2 KO keratinocytes exhibited increased motility, and fibroblasts show stronger collagen matrix contraction and expressed higher levels of α2β1 than WT control. Msx-2 KO fibroblasts were refractory to BMP4 in collagen matrix contraction, demonstrating a disruption in the Msx-2 pathway. In conclusion, Msx-2 may regulate skin morphogenesis during repair at both epithelial and mesenchymal levels. This work was supported by NIH grant R01GM055081(TLT).

Transient Inhibition of the Hedgehog Pathway in Young Mice Causes Permanent Defects in Bone Structure

The Hedgehog (Hh) pathway plays critical roles in normal development and in tumorigenesis. We generated Gli-luciferase transgenic mice to evaluate the Smo inhibitor, HhAntag, by whole animal functional imaging. HhAntag rapidly reduced systemic luciferase activity in 10- to 14-day-old mice following oral dosing. Although pathway activity was restored 2 days after drug removal, brief inhibition caused permanent defects in bone growth. HhAntag inhibited proliferation and promoted differentiation of chondrocytes, leading to dramatic expansion of the hypertrophic zone. After drug removal, osteoblasts invaded the cartilage plate, mineralization occurred, and there was premature fusion of the growth plate resulting in permanent disruption of bone epiphyses.

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.

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.

Mechanisms for methotrexate chemotherapy-induced bone growth defects and protective effects of folinic acid supplementary treatment

Mechanisms for methotrexate chemotherapy-induced bone growth defects and protective effects of folinic acid supplementary treatment

Roles of COX‐2 and iNOS in the bony repair of the injured growth plate cartilage

Growth plate injuries often lead to bone growth defects, which primarily occur due to bony repair at injury sites. Bony repair is preceded by an injury-induced inflammatory response, which could play a role in regulating the repair process. Here, roles of two inflammatory mediators, cyclo-oxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), in the injury responses were analysed by examining their gene expression and effects of blocking their activities, respectively, with celecoxib and aminoguanidine during 2 days prior to and until 7 days after injury in a rat tibial growth plate injury model. Quantitative RT-PCR assays revealed upregulated expression of COX-2 on days 1 and 4 and iNOS on day 1. Histological analysis of injury sites revealed significant reductions in inflammatory infiltrate (particularly neutrophils) on day 1 in treated groups compared to saline control. While bony tissue proportions at injury sites were unaffected by either treatment, mesenchymal tissue proportions were larger but cartilaginous tissue proportions were smaller on day 8 (though statistically insignificant), and bone remodelling appeared delayed with a smaller bone marrow proportion on day 14 in both treatment groups. These findings suggest that COX-2 and iNOS mediate injury-induced inflammatory response, and may play a role in enhancing mesenchymal cell differentiation to cartilaginous cells and in promoting bone remodelling during bony repair of growth plate injury sites. Furthermore, increased expression of cartilage-related (collagen-2, collagen-10, SOX-9) and bone-related molecules (osteocalcin, cbfalpha-1) suggest involvement of both endochondral and direct bone formation mechanisms during bony repair.

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

Growth inhibition of human embryonic and fetal rat bones in organ culture by rubella virus.

Paired organ cultures of metacarpal, metatarsal, and long bones of previable human embryos of 7 to 12 weeks' gestation and tibias of 17-day rat fetuses with inoculated with live or ultraviolet-inactivated rubella virus or control fluids and the growth of the bones was measured by increase in wet weight. In several cultures the ability of the human bones to incorporate 35S, a measure of rate of mucopolysaccharide synthesis, was tested. Growth of human and rat bones was retarded in cultures inoculated with live virus but not in cultures inoculated with inactivated virus or control fluids. Mean 35S uptake was increased by approximately 25% in virus-inoculated cultures of bones of 9- to 12-week human embryos. No histological abnormalities were seen. These findings suggest that (1) defective bone growth in congenital rubella is a direct effect of viral infection of bone, (2) a disorder of mucopolysaccharide syntheses may contribute to the osseous lesions that occur in this disease, and (3) organ cultures of human embryonic and fetal rat bones may serve as convenient models for studying the pathogenesis of this virus-induced congenital osteopathy.