Roles of apoptotic chondrocyte-derived CXCL12 in the enhanced chondroclast recruitment following methotrexate and/or dexamethasone treatment.

Intensive use of methotrexate (MTX) and/or dexamethasone (DEX) for treating childhood malignancies is known to cause chondrocyte apoptosis and growth plate dysfunction leading to bone growth impairments. However, mechanisms remain vague and it is unclear whether MTX and DEX combination treatment could have additive effects in the growth plate defects. In this study, significant cell apoptosis was induced in mature ATDC5 chondrocytes after treatment for 48 h with 10-5 M MTX and/or 10-6 M DEX treatment. PCR array assays with treated cells plus messenger RNA and protein expression confirmation analyses identified chemokine CXCL12 having the most prominent induction in each treatment group. Conditioned medium from treated chondrocytes stimulated migration of RAW264.7 osteoclast precursor cells and formation of osteoclasts, and these stimulating effects were inhibited by the neutralizing antibody for CXCL12. Additionally, while MTX and DEX combination treatment showed some additive effects on apoptosis induction, it did not have additive or counteractive effects on CXCL12 expression and its functions in enhancing osteoclastic recruitment and formation. In young rats treated acutely with MTX, there was increased expression of CXCL12 in the tibial growth plate, and more resorbing chondroclasts were found present at the border between the hypertrophic growth plate and metaphysis bone. Thus, the present study showed an association between induced chondrocyte apoptosis and stimulated osteoclastic migration and formation following MTX and/or DEX treatment, which could be potentially or at least partially linked molecularly by CXCL12 induction. This finding may contribute to an enhanced mechanistic understanding of bone growth impairments following MTX and/or DEX therapy.

ß-Catenin signaling is important for osteogenesis and hematopoiesis recovery following methotrexate chemotherapy in rats.

Cancer chemotherapy can significantly impair the bone formation and cause myelosuppression; however, their recovery potentials and mechanisms remain unclear. This study investigated the roles of the ß-catenin signaling pathway in bone and bone marrow recovery potentials in rats treated with antimetabolite methotrexate (MTX) (five once-daily injections, 0.75 mg/kg) with/without ß-catenin inhibitor indocyanine green (ICG)-001 (oral, 200 mg/kg/day). ICG alone reduced trabecular bone volume and bone marrow cellularity. In MTX-treated rats, ICG suppressed bone volume recovery on Day 11 after the first MTX injection. ICG exacerbated MTX-induced decreases on Day 9 osteoblast numbers on bone surfaces, their formation in vitro from bone marrow stromal cells (osteogenic differentiation/mineralization), as well as expression of osteogenesis-related markers Runx2, Osx, and OCN in bone, and it suppressed their subsequent recoveries on Day 11. On the other hand, ICG did not affect MTX-induced increased osteoclast density and the level of the osteoclastogenic signal (RANKL/OPG expression ratio) in bone, suggesting that ICG inhibition of ß-catenin does nothing to abate the increased bone resorption induced by MTX. ICG also attenuated bone marrow cellularity recovery on Day 11, which was associated with the suppressed recovery of CD34+ or c-Kit+  hematopoietic progenitor cell contents. Thus, ß-catenin signaling is important for osteogenesis and hematopoiesis recoveries following MTX chemotherapy.

Retracted: Icariin attenuates methotrexate chemotherapy-induced bone marrow microvascular damage and bone loss in rats.

Methotrexate (MTX), a widely used antimetabolite in paediatric cancer to treatment, has been widely reported to cause bone loss and bone marrow (BM) microvascular (particularly sinusoids) damage. Investigations must now investigate how MTX-induced bone loss and microvasculature damage can be attenuated/prevented. In the present study, we examined the potency of icariin, an herbal flavonoid, in reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with five daily MTX injections (0.75 mg/kg) with and without icariin oral supplementation until Day 9 after the first MTX injection. Histological analyses showed a significant reduction in the bone volume/tissue volume (BV/TV) fraction (%) and trabecular number in the metaphysis trabecular bone of MTX-treated rats, but no significant changes in trabecular thickness and trabecular spacing. However, the BV/TV (%) and trabecular number were found to be significantly higher in MTX + icariin-treated rats than those of MTX alone-treated rats. Gene expression analyses showed that icariin treatment maintained expression of osteogenesis-related genes but suppressed the induction of adipogenesis-related genes in bones of MTX-treated rats. In addition, icariin treatment attenuated MTX-induced dilation of BM sinusoids and upregulated expression of endothelial cell marker CD31 in the metaphysis bone of icariin + MTX-treated rats. Furthermore, in vitro studies suggest that icariin treatment can potentially enhance the survival of cultured rat sinusoidal endothelial cells against cytotoxic effect of MTX and promote their migration and tube formation abilities, which is associated with enhanced production of nitric oxide.

Release of CXCL12 From Apoptotic Skeletal Cells Contributes to Bone Growth Defects Following Dexamethasone Therapy in Rats.

Dexamethasone (Dex) is known to cause significant bone growth impairment in childhood. Although previous studies have suggested roles of osteocyte apoptosis in the enhanced osteoclastic recruitment and local bone loss, whether it is so in the growing bone following Dex treatment requires to be established. The current study addressed the potential roles of chemokine CXCL12 in chondroclast/osteoclast recruitment and bone defects following Dex treatment. Significant apoptosis was observed in cultured mature ATDC5 chondrocytes and IDG-SW3 osteocytes after 48 hours of 10-6 M Dex treatment, and CXCL12 was identified to exhibit the most prominent induction in Dex-treated cells. Conditioned medium from the treated chondrocytes/osteocytes enhanced migration of RAW264.7 osteoclast precursor cells, which was significantly inhibited by the presence of the anti-CXCL12 neutralizing antibody. To investigate the roles of the induced CXCL12 in bone defects caused by Dex treatment, young rats were orally gavaged daily with saline or Dex at 1 mg/kg/day for 2 weeks, and received an intraperitoneal injection of anti-CXCL12 antibody or control IgG (1 mg/kg, three times per week). Aside from oxidative stress induction systemically, Dex treatment caused reductions in growth plate thickness, primary spongiosa height, and metaphysis trabecular bone volume, which are associated with induced chondrocyte/osteocyte apoptosis and enhanced chondroclast/osteoclast recruitment and osteoclastogenic differentiation potential. CXCL12 was induced in apoptotic growth plate chondrocytes and metaphyseal bone osteocytes. Anti-CXCL12 antibody supplementation considerably attenuated Dex-induced chondroclast/osteoclast recruitment and loss of growth plate cartilage and trabecular bone. CXCL12 neutralization did not affect bone marrow osteogenic potential, adiposity, and microvasculature. Thus, CXCL12 was identified as a potential molecular linker between Dex-induced skeletal cell apoptosis and chondroclastic/osteoclastic recruitment, as well as growth plate cartilage/bone loss, revealing a therapeutic potential of CXCL12 functional blockade in preventing bone growth defects during/after Dex treatment. © 2018 American Society for Bone and Mineral Research.

Flavonoid genistein protects bone marrow sinusoidal blood vessels from damage by methotrexate therapy in rats.

Cancer chemotherapy can cause significant damage to the bone marrow (BM) microvascular (sinusoidal) system. Investigations must now address whether and how BM sinusoidal endothelial cells (SECs) can be protected during chemotherapy. Herein we examined the potential protective effects of genistein, a soy-derived flavonoid, against BM sinusoidal damage caused by treatment with methotrexate (MTX). The groups of young adult rats were gavaged daily with genistein (20 mg/kg) or placebo. After 1 week, rats also received daily injections of MTX (0.75 mg/kg) or saline for 5 days and were killed after a further 4 days. Histological analyses showed that BM sinusoids were markedly dilated ( p < 0.001) in the MTX-alone group but were unaffected or less dilated in the genistein+MTX group. In control rats, genistein significantly enhanced expression of vascular endothelial growth factor (VEGF; p < 0.01), particularly in osteoblasts, and angiogenesis marker CD31 ( p < 0.001) in bone. In MTX-treated rats, genistein suppressed MTX-induced apoptosis of BM SECs ( p < 0.001 vs MTX alone group) and tended to increase expression of CD31 and VEGF ( p < 0.05). Our in vitro studies showed that genistein in certain concentrations protected cultured SECs from MTX cytotoxic effects. Genistein enhanced tube formation of cultured SECs, which is associated with its ability to induce expression of endothelial nitric oxide synthase and production of nitric oxide. These data suggest that genistein can protect BM sinusoids during MTX therapy, which is associated, at least partially, with its indirect effect of promoting VEGF expression in osteoblasts and its direct effect of enhancing nitric oxide production in SECs.

Long Chain Omega-3 Polyunsaturated Fatty Acid Supplementation Protects Against Adriamycin and Cyclophosphamide Chemotherapy-Induced Bone Marrow Damage in Female Rats.

Although bone marrow and bone toxicities have been reported in breast cancer survivors, preventative strategies are yet to be developed. Clinical studies suggest consumption of long chain omega-3 polyunsaturated fatty acids (LCn3PUFA) can attenuate age-related bone loss, and recent animal studies also revealed benefits of LCn3PUFA in alleviating bone marrow and bone toxicities associated with methotrexate chemotherapy. Using a female rat model for one of the most commonly used anthracycline-containing breast cancer chemotherapy regimens (adriamycin + cyclophosphamide) (AC) chemotherapy, this study investigated potential effects of daily LCn3PUFA consumption in preserving bone marrow and bone microenvironment during chemotherapy. AC treatment for four cycles significantly reduced bone marrow cellularity and increased marrow adipocyte contents. It increased trabecular bone separation but no obvious changes in bone volume or bone cell densities. LCn3PUFA supplementation (375 mg/100 g/day) attenuated AC-induced bone marrow cell depletion and marrow adiposity. It also partially attenuated AC-induced increases in trabecular bone separation and the cell sizes and nuclear numbers of osteoclasts formed ex vivo from bone marrow cells isolated from AC-treated rats. This study suggests that LCn3PUFA supplementation may have beneficial effects in preventing bone marrow damage and partially protecting the bone during AC cancer chemotherapy.

Crystal shape controlled H2 storage rate in nanoporous carbon composite with ultra-fine Pt nanoparticle.

This study demonstrates that the hydrogen storage rate (HSR) of nanoporous carbon supported platinum nanocatalysts (NC) is determined by their heterojunction and geometric configurations. The present NC is synthesized in an average particle size of ~1.5 nm by incipient wetness impregnation of Pt4+ at carbon support followed by annealing in H2 ambient at 102-105 °C. Among the steps in hydrogen storage, decomposition of H2 molecule into 2 H atoms on Pt NC surface is the deciding factor in HSR that is controlled by the thickness of Pt NC. For the best condition, HSR of Pt NC in 1~2 atomic layers thick (4.7 µg/g min) is 2.6 times faster than that (1.3 µg/g min) of Pt NC with higher than 3 atomic layers thick.

Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering.

Mimicking the zonal organization of native articular cartilage, which is essential for proper tissue functions, has remained a challenge. In this study, a thermoresponsive copolymer of chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAAm) was synthesized as a carrier of mesenchymal stem cells (MSCs) to provide a support for their proliferation and differentiation. Microengineered three-dimensional (3D) cell-laden CS-g-PNIPAAm hydrogels with different microstripe widths were fabricated to control cellular alignment and elongation in order to mimic the superficial zone of natural cartilage. Biochemical assays showed six- and sevenfold increment in secretion of glycosaminoglycans (GAGs) and total collagen from MSCs encapsulated within the synthesized hydrogel after 28 days incubation in chondrogenic medium. Chondrogenic differentiation was also verified qualitatively by histological and immunohistochemical assessments. It was found that 75 ± 6% of cells encapsulated within 50 µm wide microstripes were aligned with an aspect ratio of 2.07 ± 0.16 at day 5, which was more organized than those observed in unpatterned constructs (12 ± 7% alignment and a shape index of 1.20 ± 0.07). The microengineered constructs mimicked the cell shape and organization in the superficial zone of cartilage whiles the unpatterned one resembled the middle zone. Our results suggest that microfabrication of 3D cell-laden thermosensitive hydrogels is a promising platform for creating biomimetic structures leading to more successful multi-zonal cartilage tissue engineering. Biotechnol. Bioeng. 2017;114: 217-231. © 2016 Wiley Periodicals, Inc.

Methotrexate-induced bone marrow adiposity is mitigated by folinic acid supplementation through the regulation of Wnt/ß-catenin signalling.

Antimetabolite Methotrexate (MTX) is commonly used in childhood oncology. As a dihydrofolate reductase inhibitor it exerts its action through the reduction of cellular folate, thus its intensive use is associated with damage to soft tissues, bone marrow, and bone. In the clinic, MTX is administered with folinic acid (FA) supplementation to alleviate some of this soft tissue damage. However, whether and how FA alleviates damage to the bone and bone marrow requires further investigation. As the Wnt/ß-catenin signalling pathway is critical for commitment and differentiation of mesenchymal stem cells down the osteogenic or adipogenic lineage, its deregulation has been found associated with increased marrow adiposity following MTX treatment. In order to elucidate whether FA supplementation prevents MTX-induced bone marrow adiposity by regulating Wnt/ß-catenin signalling, young rats were given saline or 0.75 mg/kg MTX once daily for 5 days, receiving saline or 0.75 mg/kg FA 6 h after MTX. FA rescue alleviated the MTX-induced bone marrow adiposity, as well as inducing up-regulation of Wnt10b mRNA and ß-catenin protein expression in the bone. Furthermore, FA blocked up-regulation of the secreted Wnt antagonist sFRP-1 mRNA expression. Moreover, secreted sFRP-1 protein in the bone marrow and its expression by osteoblasts and adipocytes was found increased following MTX treatment. This potentially indicates that sFRP-1 is a major regulator of defective Wnt/ß-catenin signalling following MTX treatment. This study provides evidence that folate depletion caused by MTX chemotherapy results in increased bone marrow adiposity, and that FA rescue alleviates these defects by up-regulating Wnt/ß-catenin signalling in the bone.

Fish oil in comparison to folinic acid for protection against adverse effects of methotrexate chemotherapy on bone.

Methotrexate (MTX) chemotherapy is known to cause bone loss which lacks specific preventative treatments, although clinically folinic acid is often used to reduce MTX toxicity in soft tissues. This study investigated damaging effects of MTX injections (0.75 mg/kg/day for 5 days) in rats and potential protective benefits of fish oil (0.25, 0.5, or 0.75 ml/100 g/day) in comparison to folinic acid (0.75 mg/kg) in the tibial metaphysis. MTX treatment significantly reduced height of primary spongiosa and volume of trabecular bone while reducing density of osteoblasts. Consistently, MTX reduced osteogenic differentiation but increased adipogenesis of bone marrow stromal cells, accompanied by lower mRNA expression of osteogenic transcription factors Runx2 and Osx, but an up-regulation of adipogenesis-related genes FABP4 and PPAR-γ. MTX also increased osteoclast density, bone marrow osteoclast formation, and mRNA expression of proinflammatory cytokines IL-1, IL-6, TNF-α, and RANKL/OPG ratio in bone. Fish oil (0.5 or 0.75 ml/100 g) or folinic acid supplementation preserved bone volume, osteoblast density, and osteogenic differentiation, and suppressed MTX-induced cytokine expression, osteoclastogenesis, and adipogenesis. Thus, fish oil at 0.5 ml/100 g or above is as effective as folinic acid in counteracting MTX-induced bone damage, conserving bone formation, suppressing resorption and marrow adiposity, suggesting its therapeutic potential in preventing bone loss during MTX chemotherapy.

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-1ß and suppressed osteoclast formation in vitro in normal bone marrow cells. The importance of IL-1ß in supporting plasma-induced osteoclast formation was confirmed as the presence of an anti-IL-1ß 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.

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.