Clinical value of serum bone resorption markers for predicting clinical outcomes after use of bone modifying agents in metastatic bone tumors: A prospective cohort study.

Bone modifying agents (BMAs) have become a standard treatment to prevent skeletal-related events (SREs) in bone metastases (BMs). The aim of our study is to determine the clinical value of serum bone resorption markers for predicting clinical outcomes after using BMAs in patients with BM. Patients were enrolled between May 2013 and October 2017 at the Nagoya University Hospital, Japan. We prospectively observed changes in pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP) and tartrate-resistant acid phosphatase 5b (TRACP-5b) during treatment with BMAs. The relationship between serum markers before and after treatment and clinical outcomes such as progression of bone disease (BD), SREs and overall survival (OS) were evaluated. Pearson chi-square test and Kaplan-Meier product limit methods were used for analysis. Sixty-seven patients were analyzed. The primary tumor sites were 21 lung, 16 breast and 30 others. Forty and 27 patients were treated with Denosumab and Zoledronic acid, respectively. Progression of BDs, SREs and death were observed in 10, 16 and 31 cases, respectively. The median follow-up period after using BMAs was 12.3 (range 0.3-66.3) months. ICTP at 3-4 weeks was significantly correlated with increasing BD progression, SREs and death after treatment in both the whole and lung cancer cohorts. Base line ICTP and TRACP-5b were also associated with increasing BD progression in the whole cohort. Our study showed that early posttreatment ICTP is useful for predicting BD progression, SREs and OS after use of BMAs in patients with BM and even in patients with lung cancer BM.

MRI characteristics predict the efficacy of meloxicam treatment in patients with desmoid-type fibromatosis.

INTRODUCTION: This study aimed to determine the clinical significance of MRI characteristics as a possible predictor of responsiveness to meloxicam treatment in patients with desmoid-type fibromatosis (DF). Additionally, it analysed the correlation between CTNNB1 mutation status and signal intensity of MRI. METHODS: Forty-six patients consecutively treated with meloxicam composed this study. The low-intensity area (LIA) on T2-weighted MRI was determined. We divided patients into two groups based on the efficacy of meloxicam: a clinical benefit group (CB group, including CR: complete response; PR: partial response; and SD: stable disease) and non-clinical benefit group (NB group, including PD: progressive disease). Correlations of the efficacy with LIA and CTNNB1 mutation status with LIA were investigated. RESULTS: In total, 11, 17 and 18 patients showed PR, SD and PD, respectively. The mean LIA ratio before treatment was significantly higher (P < 0.001) in the CB group than in the NB group. For predicting the efficacy, sensitivity was 68%, and specificity was 89% when setting the cut-off value as 20% for LIA. Mean changes in the LIA ratio before and after treatment were significantly higher (P = 0.01) in the CB group than in the NB group. Mean LIA ratio before treatment was significantly lower (P < 0.001) in the S45F mutation group than in the other mutation group. In multivariate analysis, the LIA ratio before treatment was a significant predictor of responsiveness (P = 0.02). CONCLUSIONS: MRI characteristics were a useful predictor of the efficacy of meloxicam in DF patients. It may be possible to predict the clinical outcome more accurately when combined with other factors, such as CTNNB1 mutantion status.

Roles of Nmnat1 in the survival of retinal progenitors through the regulation of pro-apoptotic gene expression via histone acetylation.

Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous dystrophy of the retina and mutations in the nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) gene is one of causal factors of LCA. NMNAT1 is a nuclear enzyme essential for nicotinamide adenine dinucleotide (NAD) biosynthesis pathways, but the mechanisms underlying the LCA pathology and whether NMNAT1 has a role in normal retinal development remain unclear. Thus, we examined the roles of Nmnat1 in retinal development via short hairpin (sh)-RNA-mediated downregulation. Retinal explants expressing sh-Nmnat1 showed large numbers of apoptotic retinal progenitor cells in the inner half of the neuroblastic layer. Decreased intracellular NAD content was observed and the addition of NAD to the culture medium attenuated sh-Nmnat1-induced apoptosis. Of the nuclear Sirtuin (Sirt) family, the expression of sh-Sirt1 and sh-Sirt6 resulted in a phenotype similar to that of sh-Nmnat1. Sirt proteins are histone deacetylases and the expression of sh-Nmnat1 increased the levels of acetylated histones H3 and H4 in the retina. Expression of sh-Nmnat1 resulted in significantly increased expression of Noxa and Fas, two pro-apoptotic genes. Acetylation of the genomic 5'-untranslated regions of Noxa and Fas loci was upregulated by sh-Nmnat1 expression. The co-expression of sh-Fas with sh-Nmnat1 reduced the number of apoptotic cells induced by sh-Nmnat1 expression alone. Taken together, our data suggested that the increased expression of Noxa and Fas explains, at least in part, the phenotype associated with sh-Nmnat1 in the retina. Taken together, these findings demonstrate the importance of the NAD biosynthesis pathway in normal development of the retina.

Single metastasis of myxoid liposarcoma from the thigh to thyroid gland: a case report.

BACKGROUND: Thyroid metastasis of soft tissue sarcoma is very rare, and the diagnosis is especially difficult when only a single lesion is present. CASE PRESENTATION: A 50-year-old man was diagnosed with myxoid liposarcoma of the right thigh and treated with wide resection. Two and a half years after the surgery, a growing low-density area was incidentally observed in the right lobe of his thyroid gland on follow-up chest computed tomography. Fine needle aspiration biopsy was performed twice, and the thyroid mass was suspected of being a sarcoma metastasis. He was treated by hemithyroidectomy, and the lesion was pathologically confirmed as a metastasis of myxoid liposarcoma. CONCLUSION: We experienced single thyroid gland metastasis in patients with myxoid liposarcoma in whom a growing mass is observed in the thyroid gland after radical surgery of the primary site.

Predictors of complications in heat-treated autograft reconstruction after intercalary resection for malignant musculoskeletal tumors of the extremity.

BACKGROUND: Biological reconstruction with recycled heat-treated autografts has been an option for a segmental skeletal defect after intercalary resection for malignant musculoskeletal tumors in the extremity. This study was undertaken to evaluate the clinical outcomes in patients treated with this procedure and identify factors affecting the incidence of complications. METHODS: We retrospectively reviewed 24 patients treated with heat-treated autografts after intercalary resection at our institution between 1992 and 2015. RESULTS: The survival rate of the heat-treated autografts was 70.1% at 10 years. Of the 48 host-graft junctions in the 24 patients, nonunion occurred in 18 junctions (38%). In the univariate analysis, location in the upper extremity, intercalary grafts without vascularized fibula autografts (VFG), and junction at the diaphysis significantly increased the rate of nonunion (P = 0.003, P = 0.003, and P = 0.031, respectively). Location in the upper extremity was an independent factor associated with nonunion in the multivariate analysis (P = 0.006). Upper extremity location and intercalary grafts without VFG were also significant factors for bone absorption (P = 0.042 and P < 0.001, respectively). CONCLUSIONS: Our results can provide useful information to devise possibly novel clinical approaches to patients requiring intercalary reconstruction of the extremity.

A2E-associated cell death and inflammation in retinal pigmented epithelial cells from human induced pluripotent stem cells.

Accumulation of lipofuscin in the retinal pigmented epithelium (RPE) is observed in retinal degenerative diseases including Stargardt disease and age-related macular degeneration. Bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is a major component of lipofuscin. A2E has been implicated in RPE atrophy and retinal inflammation; however, mice with A2E accumulation display only a mild retinal phenotype. In the current study, human iPSC-RPE (hiPSC-RPE) cells were generated from healthy individuals to examine effects of A2E in human RPE cells. hiPSC-RPE cells displayed RPE-specific features, which include expression of RPE-specific genes, tight junction formation and ability to carry out phagocytosis. hiPSC-RPE cells demonstrated cell death and increased VEGF-A production in a time-dependent manner when they were cocultured with 10µM of A2E. PCR array analyses revealed upregulation of 26 and 12 pro-inflammatory cytokines upon A2E and H2O2 exposure respectively, indicating that A2E and H2O2 can cause inflammation in human retinas. Notably, identified gene profiles were different between A2E- and H2O2- treated hiPSC-RPE cells. A2E caused inflammatory changes observed in retinal degenerative diseases more closely as compared to H2O2. Collectively, these data obtained with hiPSC-RPE cells provide evidence that A2E plays an important role in pathogenesis of retinal degenerative diseases in humans.

Suppression of hyaluronan synthesis attenuates the tumorigenicity of low-grade chondrosarcoma.

Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of an HA synthesis inhibitor, 4-methylumbelliferone (MU), on ECM formation as well as antitumor effects in chondrosarcoma. We investigated the effects of MU on rat chondrosarcoma (RCS) cells with a grade I histological malignancy in vitro and in vivo grafted model. HA binding protein (HABP) stainability on and around the RCS cells was effectively reduced with treatment of MU. ECM formation was markedly suppressed by MU at a dose of 1.0 mM. Cell proliferation was significantly reduced by MU at 24 h. Cell motility and invasion were suppressed in a dose-dependent manner by MU. No significant changes in mRNA expression of Has1-3 were observed. Furthermore, MU inhibited the growth of grafted tumors in vivo. Histologically, chondrosarcoma cells of control tumors showed a cell-clustering structure. HABP stainability was markedly decreased in the MU-treated group. These results suggest that MU exhibits antitumor effects on low-grade chondrosarcoma, via inhibition of HA accumulation and ECM formation. MU, which is an approved drug in bile therapy, could be a new off-label medication for chondrosarcomas. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1573-1580, 2018.

Conditional knockdown of hyaluronidase 2 in articular cartilage stimulates osteoarthritic progression in a mice model.

The catabolism of hyaluronan in articular cartilage remains unclear. The aims of this study were to investigate the effects of hyaluronidase 2 (Hyal2) knockdown in articular cartilage on the development of osteoarthritis (OA) using genetic manipulated mice. Destabilization of the medial meniscus (DMM) model of Col2a promoter specific conditional Hyal2 knockout (Hyal -/- ) mice was established and examined. Age related and DMM induced alterations of articular cartilage of knee joint were evaluated with modified Mankin score and immunohistochemical staining of MMP-13, ADAMTS-5, KIAA11199, and biotinylated- hyaluronan binding protein staining in addition to histomorphometrical analyses. Effects of Hyal2 suppression were also analyzed using explant culture of an IL-1α induced articular cartilage degradation model. The amount and size of hyaluronan in articular cartilage were higher in Hyal2 -/- mice. Hyal2 -/- mice exhibited aggravated cartilage degradation in age-related and DMM induced mice. MMP-13 and ADAMTS-5 positive chondrocytes were significantly higher in Hyal2 -/- mice. Articular cartilage was more degraded in explant cultures obtained from Hyal2 -/- mice. Knockdown of Hyal2 in articular cartilage induced OA development and progression possibly mediated by an imbalance of HA metabolism. This suggests that Hyal2 knockdown exhibits mucopolysaccharidosis-like OA change in articular cartilage similar to Hyal1 knockdown.

Docosahexaenoic acid promotes differentiation of photoreceptor cells in three-dimensional neural retinas.

Retinal tissues generated from human pluripotent stem cells can be an excellent tool for investigating pathogenesis of retinal diseases and developing new pharmacologic therapies. Moreover, patient derived retinal tissues could allow for retinal transplantation therapy for degenerative retinal diseases. However, obtaining retinal tissues with matured photoreceptor outer segments, which are essential for photoreceptor functions, is currently challenging. Here we investigated the effects of docosahexaenoic acid (DHA) for maturation of photoreceptor outer segments at the late stage and visual chromophore analog, 9-cis-retinal for the early stage of differentiation to three-dimensional (3D)-retinal tissues from human embryonic stem cells (hESCs), respectively. In the presence of DHA, differentiated 3D-retinal tissues demonstrated improved maturation of photoreceptor outer segments and increased number of photoreceptor cells compared with tissues without DHA. Increased mRNA expression of mature photoreceptor markers was additionally documented in retinal tissues cultured with DHA. Conversely supplementation with 9-cis-retinal failed to improve differentiation of retinal tissues perhaps due to chronic aldehyde toxicity. The current study demonstrated that the addition of DHA to culture medium can help promote differentiation of photoreceptor outer segments in vitro and utilization of this methodology may lead to future therapies for patients with blinding diseases.

New Treatments for Stargardt Disease and Related Retinal Degenerative Diseases.

Stargardt disease is a progressive hereditary retinal disease which is currently incurable. Although ABCA4 has been identified as a major causative gene, patients with genetic mutations in other genes (PRPH2, ELOVL4, and PROM1) display similar clinical phenotypes as Stargardt disease. Recent advances in genetic and molecular studies have greatly enhanced our understanding of the disease and have led to identification of targetable pathophysiological progresses and new molecular clinical applications. Here we summarize the current understanding of the pathophysiology and potential therapies for Stargardt disease and other related retinal degenerative diseases.

Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium.

Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by two-photon microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk-/- mice also accumulated fluorescent products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk-/- mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.

Acute Stress Responses Are Early Molecular Events of Retinal Degeneration in Abca4-/-Rdh8-/- Mice After Light Exposure.

PURPOSE: Mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8) mimic features of human Stargardt disease and age-related macular degeneration. RNA-sequencing of whole eyes was done to study early gene expression changes in Abca4-/-Rdh8-/- mice. METHODS: Abca4-/-Rdh8-/- mice at 4 weeks of age were exposed to intense light. Total RNA was extracted from whole eyes and used to generate RNA libraries that were paired-end sequenced on the Illumina HiSeq 2500 device. Differentially expressed genes were annotated using Gene set enrichment analysis (GSEA). Selected genes in enriched pathways exhibiting differential expression were validated using quantitative qRT-PCR and ELISA. RESULTS: Transcriptome analysis of the whole eye identified 200 genes that were differentially expressed 24 hours after light exposure compared to no light in Abca4-/-Rdh8-/- mice. Expression of several visual cycle and photoreceptor genes were decreased, indicative of photoreceptor/RPE cell death. Gene categories of early stress response genes, inflammatory cytokines, immune factors, and JAK STAT components were upregulated. Lipocalin 2 (Lcn2) was the most upregulated early stress response gene identified. Protein LCN2 was produced by RPE cells and the neural retina after intense light exposure as well as in cultured RPE cells from mice and humans incubated with lipopolysaccharide or photoreceptor outer segments. CONCLUSIONS: Identification of important mediators involved in the crosstalk between the acute stress response and immune activation in RPE cells and the neural retina, such as LCN2, provide novel molecular targets for reducing cellular stress during retinal degeneration.

Giant cell tumor of bone arising in long bones possibly originates from the metaphyseal region.

Giant cell tumor of bone (GCTB) is a primary benign bone tumor with a locally aggressive character. Definitive descriptions of the site of origin for this type of tumor are not available. The aim of the present study was to evaluate the site of origin of GCTB of long bones with regards to epiphyseal lines by means of radiographic examination. For that purpose, plain X-ray scans of 71 GCTBs arising in long bones were retrospectively reviewed. The tumor locations were the distal femur in 31 cases, proximal femur in 11 cases, proximal tibia in 13 cases, distal radius in 6 cases, proximal humerus in 5 cases and proximal fibula in 5 cases. The vertical center (VC) of the tumor was determined with X-ray anteroposterior view, and the correlation between the VC and the epiphyseal line, and between the distance from the epiphyseal line to the VC and tumor area or volume were analyzed using a regression model equation based on scatter plot diagrams. The VC of the tumor was located in the metaphyseal region in 57 cases, in the epiphyseal line in 11 cases and in the epiphyseal region in 3 cases. In cases of GCTB located in the distal femur or proximal tibia, significant correlations between the distance from the VC to the epiphyseal line and tumor area or volume were identified. The site of origin of GCTB was estimated to be located in the metaphyseal region. GCTB often occurs in mature patients, which renders it challenging to estimate the true site of origin of this lesion, since the metaphyseal line has disappeared in mature patients. The results of the present study suggest that GCTB possibly originates in the metaphyseal region.

Increased expression and activation of cathepsin K in human osteoarthritic cartilage and synovial tissues.

Few studies have analyzed Cathepsin K (CatK) expression in human osteoarthritic tissues. We investigated CatK expression and activation in human articular cartilage using clinical specimens. Human osteoarthritic cartilage was obtained during surgery of total hip arthroplasty (n = 10), and control cartilage was from that of femoral head replacement for femoral neck fracture (n = 10). CatB, CatK, CatL, CatS, and Cystatin C (CysC) expressions were evaluated immunohistochemically and by real-time PCR. Intracellular CatK protein was quantified by ELISA. Intracellular CatK activity was also investigated. Osteoarthritis (OA) chondrocytes were strongly stained with CatK, particularly in the superficial layer and more damaged areas. CatB, CatL, CatS, and CysC were weakly stained. CatK mRNA expression was significantly higher in OA group compared to that in control group (p = 0.043), whereas those of CatB, CatL, CatS, and CysC did not differ significantly. Mean CatK concentration (4.83 pmol/g protein) in OA chondrocytes was higher than that (3.91 pmol/g protein) in control chondrocytes (p = 0.001). CatK was enzymatically more activated in OA chondrocytes as compared with control chondrocytes. This study, for the first time, revealed increased CatK expression and activation in human OA cartilage, suggesting possible crucial roles for it in the pathogenesis of osteoarthritic change in articular cartilage.

Characteristics of cultured desmoid cells with different CTNNB1 mutation status.

Desmoid tumors are benign mesenchymal neoplasms with a locally aggressive nature. The mutational status of ß-catenin gene (CTNNB1) is presumed to affect the tumorous activity of the cells. In this study, we isolated three kinds of desmoid cell with different CTNNB1 status, and compared their characteristics. Cells were isolated from three patients with abdominal wall desmoid during surgery, all of which were resistant to meloxicam treatment. The mutational status of the CTNNB1 exon 3 was determined for both parental tumor tissues and isolated cultured cells. ß-catenin expression was determined with immunohistochemistry. Responsiveness to meloxicam was investigated with MTS assay together with COX-2 immunostaining. mRNA expressions of downstream molecules of Wnt/ß-catenin pathway were determined with real-time RT-PCR. Three kinds of cell isolated from desmoid tumors harboring different CTNNB1 mutation status (wild type, T41A, and S45F), all exhibited a spindle shape. These isolated cells could be cultured until the 20th passage with unchanged proliferative activity. Nuclear accumulation of ß-catenin was observed in all cultured cells, particularly in those with S45F. Proliferating activity was significantly suppressed by meloxicam (25 µmol/L, P < 0.007) in all three cell cultures, of which parental desmoid was resistant to meloxicam clinically. The mRNA expressions of Axin2, c-Myc, and Cyclin D1 differently increased in the three cultured cell types as compared with those in human skin fibroblast cells (HDF). Inhibitors of Wnt/ß-catenin pathway downregulated Axin2, c-Myc, and Cyclin D1 significantly. Isolated and cultured desmoid tumor cells harboring any one of the CTNNB1 mutation status had unique characteristics, and could be useful to investigate desmoid tumors with different mutation status of CTNNB1.

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium.

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7(flox/flox);VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7(flox/flox);VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4(-/-) and Abca4(+/+) backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.

Epilepsy phenotypes in siblings with Norrie disease.

Norrie disease is an X-linked recessive disorder that is characterized by congenital blindness. Although epileptic seizures are observed in some patients with Norrie disease, little is known about this phenomenon. Here, we report the manifestation of epilepsy in siblings with Norrie disease to increase our knowledge of epilepsy in this condition. Three brothers with congenital blindness were diagnosed with Norrie disease after genetic analyses indicated the deletion of exon 2 of the NDP gene. The eldest brother had suffered from epileptic seizures since the age of 11years, and his seizures were resistant to antiepileptic drugs. Although the second brother had no epileptic seizures, the youngest sibling had experiences epileptic seizures since the age of 8years. His seizures were controlled using lamotrigine and levetiracetam. An electroencephalography (EEG) revealed epileptiform discharges in the occipital areas in all three brothers. A study of these patients will increase our knowledge of epilepsy in patients with Norrie disease.

Inhibition of hyaluronan synthesis alters sulfated glycosaminoglycans deposition during chondrogenic differentiation in ATDC5 cells.

In chondrogenic differentiation, expression and collaboration of specific molecules, such as aggrecan and type II collagen, in extracellular matrix (ECM) are crucial. However, few studies have clarified the roles of hyaluronan (HA) in proteoglycan aggregation during chondrogenic differentiation. We assessed the roles of HA in sulfated glycosaminoglycans deposition during chondrogenic differentiation by means of 4-methylumbelliferone (4-MU), an HA synthase inhibitor, using ATDC5 cells. ATDC5 cells were treated with 0.5 mM 4-MU for 7 or 21 days after induction of chondrogenic differentiation with insulin. Depositions of sulfated glycosaminoglycans were evaluated with Alcian blue staining. mRNA expression of ECM molecules was determined using real-time RT-PCR. The deposition of aggrecan and versican was investigated with immunohistochemical staining using specific antibodies. Effects of 4-MU on HA concentrations were analyzed by HA binding assay. 4-MU suppressed the positivity of Alcian blue staining, although this delay was reversible. Interestingly, stronger positivity of Alcian blue staining was observed at day 21 in cultures with 4-MU discontinuation than in the control. 4-MU significantly increased the mRNA expression of aggrecan, versican, and type II collagen, which was consistent with increased deposition of aggrecan and versican. The HA concentration in ECM and cell-associated region was significantly suppressed with 4-MU treatment. We conclude that the inhibition of HA synthesis slows sulfated glycosaminoglycans deposition during chondrogenic differentiation despite the increased deposition of other ECM molecules. Transient starvation of HA with 4-MU accelerates chondrogenic ECM formation, suggesting its potential to stimulate chondrogenic differentiation with adequate use.