Exosomes from chondrocytes overexpressing miR-214-3p facilitate M2 macrophage polarization and angiogenesis to relieve Legg Calvé-Perthes disease.

OBJECTIVE: Legg-Calvé-Perthes disease (LCPD) is a partial or total necrosis of femoral head bone caused by blood supply disorder and its etiology is not clear. Studies have revealed that microRNA-214-3p (miR-214-3p) plays a vital role in LCPD, however, its exact mechanism is still unclear. In this study, we investigated the potential role of chondrocytes-derived exosomes carrying miR-214-3p (exos-miR-214-3p) in the pathogenesis of LCPD. METHODS: RT-qPCR was performed to evaluate miR-214-3p expression level in femoral head cartilage, serum and chondrocytes of patients with LCPD, as well as dexamethasone (DEX)-exposed TC28 cells. Effects of exos-miR-214-3p on the proliferation and apoptosis were verified via MTT assay, TUNEL staining and caspase3 activity assay. The M2 macrophage markers were assessed by flow cytometry, RT-qPCR and Western blot. Moreover, angiogenic effects of human umbilical vein endothelial cells (HUVECs) were tested using CCK-8 and tube formation assays. Bioinformatics prediction, luciferase assay and ChIP were applied to verify the association between ATF7, RUNX1 and miR-214-3p. RESULTS: miR-214-3p was found to be decreased in patients with LCPD and DEX-treated TC28 cells, of which overexpression promoted cell proliferation and suppressed apoptosis. Mechanistically, exos-miR-214-3p facilitated M2 polarization by ATF7/TLR4 axis and HUVECs angiogenesis via RUNX1/VEGFA axis. CONCLUSION: miR-214-3p alleviates LCPD by promoting M2 polarization of macrophages and angiogenesis.

Rspo1 inhibited apoptosis of glucocorticoid-induced osteoblasts via Wnt/ß-catenin pathway in Legg-Calve-Perthes disease.

BACKGROUND: The pathogenesis of Legg-Calve-Perthes disease (LCPD), a juvenile form of avascular necrosis of the femoral head (ANFH), is not fully understood. OBJECTIVES: The purpose of this work was to study the regulatory effect of R-spondin 1 (Rspo1) on osteoblastic apoptosis and evaluate the pre-clinical efficacy of recombinant human protein Rspo1 (rhRspo1) in treatment of LCPD. MATERIAL AND METHODS: This is an experimental study. In vivo rabbit ANFH model was established. Human osteoblast cell line hFOB1.19 (hFOB) was used to overexpress and silence Rspo1 in vitro. Additionally, hFOB cells were induced with glucocorticoid (GC) and methylprednisolone (MP), and treated with rhRspo1. The expressions of Rspo1, ß-catenin, Dkk-1, Bcl-2, and caspase-3, and the apoptosis rate of hFOB cells were examined. RESULTS: The expressions of Rspo1 and ß-catenin were lower in ANFH rabbits. The expression of Rspo1 was decreased in GC-induced hFOB cells. Compared to the control group, after 1 µM MP induction for 72 h, the expressions of ß-catenin and Bcl-2 were higher, while Dkk-1, caspase-3 and cleaved caspase-3 expressions were lower in Rspo1 overexpression and rhRspo1-treated groups. The apoptosis rate of GC-induced hFOB cells was decreased in Rspo1 overexpression and rhRspo1-treated groups compared to the control group. CONCLUSIONS: R-spondin 1 inhibited GC-induced osteoblast apoptosis via Wnt/ß-catenin pathway, which might be associated with the development of ANFH. Moreover, rhRspo1 had a potential pre-clinical therapeutic effect on LCPD.

MicroRNA sequence analysis of plasma exosomes in early Legg-Calvé-Perthes disease.

The pathogenesis of Legg-Calvé-Perthes disease (LCPD) has not been fully elucidated, and studies on epigenetic changes that may contribute to the pathogenesis of LCPD are rare. MicroRNAs (miRNAs) are epigenetic modifications that play a critical role in gene regulation. This study aimed to determine the expression profiles of circulating exosomal miRNAs and examine the role of exosomal miRNAs in LCPD. Exosomes were extracted from the plasma of three patients with LCPD and three matched healthy volunteers. Total exosomal miRNAs were isolated, and next-generation sequencing and bioinformatic approaches were performed. The top 10 most differentially upregulated miRNAs were identified, and qRT-PCR validation was performed using additional 10 matches. In Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, plasma exosomes were used in verifying osteoclastogenesis and the endothelial dysfunction phenotypes involved. The elevated miRNAs in LCPD plasma exosomes were tested for osteoclastogenesis and endothelial dysfunction in vitro. Sequencing results revealed the expression profiles of plasma exosomal miRNAs with differential expression from the DESeq-identified miRNA profiles in LCPD versus controls in a pairwise comparison. Gene Ontology and KEGG pathway analyses indicated that the predicted target genes of different miRNAs were mainly enriched in the endothelial and osteoclast cells related to signaling pathways. Functional phenotype experiments showed that the plasma exosomes in the LCPD group promoted osteoclastogenesis and endothelial cell dysfunction. qRT-PCR experiments showed that nine miRNAs in circulating exosomes in LCPD patients were higher than those in the healthy controls. miR-3133, miR-4644, miR-4693-3p, and miR-4693-5p promoted endothelial dysfunction, and miR-3133, miR-4693-3p, miR-4693-5p, miR-141-3p and miR-30a promoted osteoclastogenesis in vitro. This study demonstrated that plasma exosomes from LCPD promote endothelial cell dysfunction and osteoclastogenesis likely through their miRNAs, which might contribute to the development of LCPD.

A case control study to determine the association between Perthes' disease and the recalled use of tobacco during pregnancy, and biological markers of current tobacco smoke exposure.

AIMS: It is well established that there is a strong association between Perthes' disease and worsening socioeconomic deprivation. It has been suggested that the primary determinant driving this association is exposure to tobacco smoke. This study aimed to examine this hypothesis. PATIENTS AND METHODS: A hospital case-control study (n = 149/146) examined the association between tobacco smoke exposure and Perthes' disease, adjusting for area-level socioeconomic deprivation. Tobacco smoke exposure was assessed by parental questionnaire of smoking habits during pregnancy, and by quantitative assay of current exposure using the urinary cotinine-creatinine ratio, which is a widely used and validated measure of tobacco smoke exposure. RESULTS: The odds of Perthes' disease significantly increased with reported in utero exposure after adjustment for socioeconomic deprivation (maternal smoking odds ratio (OR) 2.06, 95% confidence interval (CI) 1.17 to 3.63; paternal smoking OR 2.09, 95% CI 1.26 to 3.46). The cotinine-creatinine ratio was significantly greater in cases, OR 1.63 (95% CI 1.09 to 2.43), suggesting a greater 'dose' of current tobacco exposure. CONCLUSION: An association exists between tobacco smoke exposure and Perthes' disease but we remain unable to disentangle the association with socioeconomic deprivation. Cite this article: Bone Joint J 2017;99-B:1102-8.

Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease­Serbian experience.

UNLABELLED: Perthes disease is one of the most common forms of pediatric femoral head osteonecrosis with an unknown etiology. Coagulation factors were the first genetic factors suspected to have a role in the pathogenesis of this disease, but studies showed inconsistent results. It is described that inflammation is present during early stages of Perthes disease, but its genetic aspect has not been studied extensively. Little is known regarding the status of apoptotic factors during the repair process that leads to the occurrence of hip deformity in patients. Therefore, the aim of this study was to analyze major mediators involved in coagulation, inflammation, and apoptotic processes as possible causative factors of Perthes disease. The study cohort consisted of 37 patients. Gene variants of TNF-α, FV, FII, and MTHFR genes were determined by PCR-RFLP, while IL-3 and PAI-1 were genotyped by direct sequencing. The expression level of Bax, Bcl-2, Bcl2L12, Fas and FasL was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique. Our results showed a significantly increased level of expression of pro-apoptotic factor Bax along with significantly higher Bax/Bcl-2 ratio in the patient group. CONCLUSION: The results presented indicate that apoptosis could be one of the factors contributing to the lack of balanced bone remodeling process in Perthes patients.

Legg-Calvé-Perthes disease produces chronic hip synovitis and elevation of interleukin-6 in the synovial fluid.

Legg-Calvé-Perthes disease (LCPD) is a childhood hip disorder of ischemic osteonecrosis of the femoral head. Hip joint synovitis is a common feature of LCPD, but the nature and pathophysiology of the synovitis remain unknown. The purpose of this study was to determine the chronicity of the synovitis and the inflammatory cytokines present in the synovial fluid at an active stage of LCPD. Serial MRI was performed on 28 patients. T2-weighted and gadolinium-enhanced MR images were used to assess synovial effusion and synovial enhancement (hyperemia) over time. A multiple-cytokine assay was used to determine the levels of 27 inflammatory cytokines and related factors present in the synovial fluid from 13 patients. MRI analysis showed fold increases of 5.0 ± 3.3 and 3.1 ± 2.1 in the synovial fluid volume in the affected hip compared to the unaffected hip at the initial and the last follow-up MRI, respectively. The mean duration between the initial and the last MRI was 17.7 ± 8.3 months. The volume of enhanced synovium on the contrast MRI was increased 16.5 ± 8.5 fold and 6.3 ± 5.6 fold in the affected hip compared to the unaffected hip at the initial MRI and the last follow-up MRI, respectively. In the synovial fluid of the affected hips, IL-6 protein levels were significantly increased (LCPD: 509 ± 519 pg/mL, non-LCPD: 19 ± 22 pg/mL; p = 0.0005) on the multi-cytokine assay. Interestingly, IL-1ß and TNF-α levels were not elevated. In the active stage of LCPD, chronic hip synovitis and significant elevation of IL-6 are produced in the synovial fluid. Further studies are warranted to investigate the role of IL-6 on the pathophysiology of synovitis in LCPD and how it affects bone healing.

Increased soluble selectins as a reflection of activated platelets and endothelium in Legg-Calve-Perthes disease.

BACKGROUND AND AIM: Legg-Calve-Perthes disease (LCPD) is still an enigma. Hemostatic abnormalities have been indicated in the pathogenesis. We had previously demonstrated enhanced tissue factor pathway inhibitor response, increased global fibrinolytic capacity, and an increase in thrombomodulin in patients with LCPD compared with healthy individuals. These studies emphasized the role of vascular endothelium in pathogenesis of the LCPD. P-selectin is expressed on activated platelets and endothelial cells, and E-selectin is expressed on activated endothelial cells. The aim of this study was to assess circulating E-selectin and P-selectin levels in LCPD patients, which might reflect an endothelium activation and/or injury. MATERIALS AND METHODS: The study included 85 pediatric patients. Group I consisted of 55 patients with LCPD and group II (control) consisted of 30 healthy children. Peripheral venous blood concentrations of E-selectin and P-selectin levels were measured with a commercially available assay. RESULTS: Mean age was 8.41±2.73 years in group I and 8.83±2.92 years in group II. Both E-selectin and P-selectin levels were higher in LCPD patients in comparison with the age-matched controls. E-selectin was 54.92±18.84 pg/mL in group I, 45.54±15.31 pg/mL in group II and P-selectin was 46.40±20.35 pg/mL in group I, 36.92±9.84 pg/mL in group II (P=0.022 and P=0.019, respectively). CONCLUSIONS: On the basis of our results, two important endothelium and platelet markers, E-selectin and P-selectin, are upregulated in LCPD. Our results suggested that activated platelets and possibly endothelial activation, as reflected by enhanced P-selectin/E-selectin kinetics, might contribute to the microvascular thrombosis and/or inflammation of LCPD.

Acute BMP2 upregulation following induction of ischemic osteonecrosis in immature femoral head.

Juvenile ischemic osteonecrosis of the femoral head (IOFH) is one of the most serious hip conditions causing the femoral head deformity. Little is known about BMP signaling following ischemic osteonecrosis. In this study, we found acute BMP2 upregulation in the femoral head cartilage 24h after ischemic induction using our immature pig IOFH model. Similarly, in our ischemic osteonecrosis mouse model, BMP2 expression and BMP signaling were enhanced in the articular cartilage surrounding the necrotic bone. BMP2 was increased in cartilage explants and primary chondrocytes under hypoxia (1% O(2)) compared with normoxia (21% O(2)). Addition of the hypoxia inducible factor 1 (HIF1) activator DFO significantly increased BMP2 while HIF1 silencing (siHIF1) only partially reduced BMP2, suggesting other mechanisms of BMP2 upregulation being present. Hypoxia is known to induce the production of free oxygen radicals, which are converted to hydrogen peroxide (H(2)O(2)) by superoxide dismutase 2 (SOD2). As an alternative mechanism, we investigated the effect of H(2)O(2)/SOD2 production on BMP2 upregulation. Chondrocytes produced more H(2)O(2) under hypoxia than normoxia. H(2)O(2) addition to the chondrocyte culture also significantly increased BMP2 expression. SOD2 was also dramatically increased in the ischemic pig cartilage at 24h following surgery and in primary chondrocytes/cartilage explants culture under hypoxia. SOD2 protein addition to the chondrocyte culture significantly increased BMP2. Moreover, DFO significantly increased SOD2 while HIF1 silencing only partially reduced SOD2. These results suggest that the acute BMP2 response of chondrocytes to ischemic osteonecrosis is more dominantly through the H(2)O(2) production and only partly through the HIF1 pathway.

Two novel COL2A1 mutations associated with a Legg-Calvé-Perthes disease-like presentation.

BACKGROUND: Abnormal development and growth of the capital femoral epiphysis and acetabulum are associated with a wide variety of underlying etiologies, one of which is Legg-Calvé-Perthes disease. CASE DESCRIPTION: We report the cases of two children who presented with abnormal development of both hips and in whom novel mutations in the COL2A1 gene were found. These cases illustrate the importance of identifying individuals with a type II collagen abnormality, as it informs management, allows investigation for other complications, and provides the opportunity for accurate genetic counseling and consideration of other family members who might be at risk. LITERATURE REVIEW: The literature documents numerous private mutations in COL2A1 associated with diverse clinical phenotypes including bilateral hip dysplasia and premature osteoarthritis. Some of these mutations are associated with a joint-specific phenotype but few other skeletal or extraskeletal manifestations. Only careful clinical examination of children presenting with hip anomalies therefore will reveal additional findings that warrant an evaluation by a clinical geneticist. DNA mutation analysis may be useful for making a specific diagnosis and identifying other at-risk family members. PURPOSES AND CLINICAL RELEVANCE: The purpose of our report is to alert clinicians to the possibility that children who present with bilateral Perthes-like disease of the hip might have an underlying mutation in the gene encoding type II collagen. It is important to consider this in the differential diagnosis and workup of such children as it has specific prognostic, clinical, genetic counseling, and reproductive sequelae.

Serum prolidase activity and oxidative-antioxidative status in Legg-Calve-Perthes disease.

We aimed to find out that whether collagen turnover is altered in the context of Legg-Calve-Perthes disease (LCPD) by evaluating serum prolidase activity. We also investigated the correlation between collagen turnover and oxidative-antioxidative status in LCPD. Plasma prolidase activity, total oxidant status (TOS), total antioxidant capacity, and oxidative stress index (OSI) were determined for 39 patients with LCPD and 40 healthy controls. Serum prolidase activity, TOS, and OSI were higher, but TAC was lower in patients with LCPD compared with controls. Prolidase activity was positively correlated with TOS and OSI levels. Serum prolidase activity is significantly associated with LCPD.

A histological and ultrastructural study of femoral head cartilage in a new type II collagenopathy.

A new type II collagenopathy, caused by the p.Gly1170Ser mutation of COL2A1, which presents as premature hip osteoarthritis (OA), avascular necrosis of the femoral head (ANFH) or Legg-Calvé-Perthes (LCP) disease, was recently found in several families with an inherited disease of the hip joint. In this study, femoral head cartilage was harvested for histological and ultrastructural examination to determine the pre-existing generalised abnormalities of the mutant cartilage. The histological results showed that the hierarchical structure of the mutant cartilage and the embedded chondrocytes were markedly abnormal. The expression and distribution of type II collagen was non-uniform in sections of the mutant cartilage. Ultrastructural examination showed obvious abnormal chondrocytes and disarrangement of collagen fibres in the mutant cartilage. Furthermore, the predicted stability of type II collagen dramatically decreased with the substitution of serine for glycine. Our study demonstrated that the p.Gly1170Ser mutation of COL2A1 caused significant structural alterations in articular cartilage, which are responsible for the new type II collagenopathy.

RANKL inhibition: a novel strategy to decrease femoral head deformity after ischemic osteonecrosis.

UNLABELLED: A novel therapeutic strategy to decrease the development of femoral head deformity after ischemic osteonecrosis was studied in a large animal model of total head infarction. RANKL inhibition through exogenous osteoprotegerin administration significantly decreased pathologic bone resorption and deformity during repair of the infarcted head. INTRODUCTION: Legg-Calvé-Perthes disease (LCPD) is a juvenile form of osteonecrosis of the femoral head that can produce permanent femoral head deformity (FHD) and premature osteoarthritis. The development of FHD in LCPD is closely associated with the repair process, characterized by a predominance of bone resorption in its early stage that produces a fragmented appearance and collapse of the femoral head. We present here a novel strategy to preserve the femoral head structure after ischemic osteonecrosis based on inhibition of interaction between RANK and RANKL using exogenous administration of osteoprotegerin (OPG-Fc) in a large animal model of ischemic osteonecrosis. MATERIALS AND METHODS: Ischemic osteonecrosis was surgically induced in 18 male piglets by placing a ligature tightly around the right femoral neck to disrupt the blood flow to the right femoral head. Two weeks after the induction of total head infarction, OPG-Fc or saline was administered subcutaneously to nine animals per group for 6 weeks. The contralateral, normal (left) femoral heads from the animals treated with saline served as normal, nondisease controls. All animals were killed at 8 weeks when severe FHD has been previously shown to occur because of the repair process dominated by osteoclastic bone resorption. Radiographic, histomorphometric, and immunohistochemical assessments were performed. RESULTS: Radiographic assessment showed significantly better preservation of the femoral head structure in the OPG-Fc group compared with the saline group. Epiphyseal quotient (the ratio of epiphyseal height to diameter) was significantly higher in the OPG-Fc group (0.41 +/- 0.09) compared with the saline group (0.24 +/- 0.08, p < 0.001). Histomorphometric assessment revealed a significant reduction in the number of osteoclasts present in the OPG-Fc group (5.9 +/- 5.3mm(-2)) compared with the saline group (39.6 +/- 13.8 mm(-2), p < 0.001). Trabecular bone volume, number, and separation were significantly better preserved in the OPG-Fc group compared with the saline group (p < 0.001). No significant difference in femoral length was observed between the OPG-Fc and saline groups. Immunostaining revealed the presence of OPG-Fc only within the blood vessels, with no apparent staining of bone matrix or trabecular bone surfaces. CONCLUSIONS: To our knowledge, this is the first study to show that RANKL inhibition decreases bone resorption and FHD after ischemic osteonecrosis. Because RANKL inhibitors do not bind to bone, their effects on resorption are reversible as the drug is cleared from circulation. The reversible nature of RANKL inhibitors is very appealing for treating pediatric bone diseases such as LCPD, where the resorptive stage of the disease lasts for 1-2 years.

Children with acute Perthes' disease have asymmetrical lower leg growth and abnormal collagen turnover.

BACKGROUND: Abnormalities in distal growth and low levels of insulin-like growth factor (IGF)-I have been reported in children with Perthes' disease. Our aim was to establish whether the acute phase of Perthes' disease is associated with abnormalities of growth, of bone or of collagen turnover. METHODS: We performed a cross-sectional study of 15 children (3-11 years of age, 13 boys) at acute presentation and a longitudinal cohort study of 9 children. We measured (1) the lengths of both lower legs (by knemometry) at weeks 1, 2, 6 and 12, (2) height and weight at presentation and at the second-year follow-up, and (3) levels of IGF-I, IGFBP-3, collagen markers and bone alkaline phosphatase at weeks 1 and 12, and in year 2. RESULTS: Height SD scores were normal at presentation but declined thereafter. Lower leg growth was not impaired at presentation but was asymmetrical, ceased during weeks 2-6, and then resumed symmetrically. Patients had persistently low IGF-I, low soft tissue collagen synthesis and enhanced collagen breakdown compared with age- and sex-related reference data. Markers of bone formation increased during follow-up. INTERPRETATION: Acute changes in lower leg growth reflected differential weight bearing, then immobilization and remobilization. Persistently low IGF-I may have contributed to low soft tissue collagen synthesis and growth. Changes in bone formation markers most likely reflected bone healing.

[Mechanism of early metabolism disorders and immunological reactivity in children with Perthes disease]. / O mekhanizme rannikh narushenii obmena i immunologicheskoi reaktivnosti u detei s bolezn'iu Pertesa.

Submitted in the paper are data to the effect that parameters of certain biochemical indices for blood plasma and cell link of the immune system could allow some judgement about the character of the pathological process in children with Perthes disease. Autoimmune inflammation caused by foci of chronic infection in the organism aggravates the course of aseptic necrosis in the proximal portion of the thighbone or else is a trigger mechanism of its development.

Increased levels of proteoglycan fragments and stromelysin in hip joint fluid in Legg-Calvé-Perthes disease.

The concentrations of proteoglycan fragments and stromelysin were analyzed in joint aspirates in 19 children with Legg-Calvé-Perthes disease. We found increased levels of proteoglycan fragments and stromelysin consistent with the presence of synovitis, an important clinical and possibly also prognostic factor in these children.

Normal somatomedin-C activity measured by radioimmunoassay in Perthes' disease.

In recent years, the association between somatomedin and Perthes' disease has been investigated. Somatomedin activity measured by different methods (i.e., bioassay and radioreceptor assay) has generated variable results. The purpose of this study was to examine plasma somatomedin-C activity in Perthes' disease using radioimmunoassay. Somatomedin-C activity in affected boys and girls between six and 11 years of age was normal compared to the standard data on normal children. It is difficult to prove a functional pituitary somatomedin target axis. Therefore, caution is advisable before hypothesizing an etiology of Perthes' disease on the basis of results of plasma somatomedin concentration.

[99mTc-fosfon distribution in the hip joints and femoral arteries in femur head osteochondropathy in children]. / Raspredelenie 99mTc-fosfona v tazobedrennykh sustavakh i bedrennykh arteriiakh pri osteokhondropatii golovki bedra u detei.

A study was made of 99mTc-fosfon distribution in the hip joint of children in health and Perthes' disease. The drug distribution asymmetry in the hip joints did not exceed +/- 10%. The 1st stage of disease was characterized by the absence of x-ray signs of head necrosis, a decrease in drug accumulation at the upper pole of the head up to 90% and less. The susceptibility of the test was 0.95, specificity--1, accuracy--0.97. A low level of drug accumulation at the upper pole of the head was retained at the 2nd and 3rd stages. At the 4th stage drug accumulation was increased. Changes in the relative level of drug accumulation at the upper pole of the head were correlated with changes in the indices characterizing the first passage of the drug in the femoral artery of the affected limb: a decrease in the volumetric blood flow at the 1st stage and its regeneration to values of the blood flow in the lateral artery at the 4th stage. The susceptibility of the test was 0.95, but its specificity and accuracy were lower. The combination of gamma topography of the hip joints with gamma chronometry of the first passage of the drug in the femoral arteries using the quantitative criteria of distribution asymmetry expanded and improved the potentialities of radionuclide diagnosis of Perthes' disease.

Transient synovitis of the hip in the child: increased levels of proteoglycan fragments in joint fluid.

The levels of proteoglycan antigen were measured in joint aspirates from the hip of children with transient synovitis, septic arthritis, Legg-Calvé-Perthes' disease and congenital and traumatic dislocation. Significantly increased levels were found in children with transient synovitis and septic arthritis as compared with other conditions. We propose that the proteoglycan antigens in the joint fluid were released from the articular cartilage in a partially degraded form as a result of an increased rate of proteolytic degradation. In transient synovitis, the source of proteolytic activity may be chondrocytes activated by factors released by synovial cells. The release of joint proteoglycan may cause a temporary increase in deformability of the hip cartilage of the child that could be an important pathogenetic mechanism in some of the sequelae of these diseases.

An assessment of endocrine function in boys with Perthes' disease.

The frequent association of short stature and retardation of skeletal maturation in Perthes' disease has prompted an investigation of growth-related hormones in this condition. A group of 18 prepubertal boys, aged five to 11 years, who either had a bone age two years or more less than their chronologic age or whose heights were less than the third centile, were studied. Their serum growth hormone (Se GH) response to insulin-induced hypoglycemia was significantly reduced, compared with a control group of short boys. They also demonstrated a tendency to elevated levels of somatomedin activity, measured by chick cartilage bioassay. Thyroid function was normal. These findings suggest a defect in the pituitary-somatomedin-target tissue axis in Perthes' disease.