The degeneration of the intervertebral disc (IVD) is characterized by proteolytic degradation of the extracellular matrix, and its repair requires the production of an extracellular matrix with a high proteoglycan-to-collagen ratio characteristic of a nucleus pulposus (NP)-like phenotype in vivo. At the moment, there is no medical treatment to reverse or even retard disc degeneration. The purpose of the present study was to determine if a low dose of short link N (sLN), a recently discovered fragment of the link N peptide, could behave in a manner similar to that of link N in restoring the proteoglycan content and proteoglycan-to-collagen ratio of the disc in a rabbit model of IVD degeneration, as an indication of its potential therapeutic benefit in reversing disc degeneration.

BACKGROUND:In vitro studies suggest that the multiple functions of decorin are related to both its core protein and its dermatan sulfate chain. To determine the contribution of the dermatan sulfate chain to the functional properties of decorin in vivo, a mutant mouse whose decorin lacked a dermatan sulfate chain was generated.RESULTS:Homozygous mice expressing only the decorin core protein developed and grew in a similar manner to wild type mice. In both embryonic and postnatal mice, all connective tissues studied, including cartilage, skin and cornea, appeared to be normal upon histological examination, and their collagen fibrils were of normal diameter and organization. In addition, abdominal skin wounds healed in an identical manner in the mutant and wild type mice.CONCLUSIONS:The absence of a dermatan sulfate chain on decorin does not appear to overtly influence its functional properties in vivo.

Objectives We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. Methods Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. Results Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. Significance PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.

Background: Aggrecan is the major non-collagenous component of the intervertebral disc. It is a large proteoglycan possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. Its abundance and unique molecular features provide the disc with its osmotic properties and ability to withstand compressive loads. Degradation and loss of aggrecan result in impairment of disc function and the onset of degeneration.Scope of review: This review summarizes current knowledge concerning the structure and function of aggrecan in the normal intervertebral disc and how and why these change in aging and degenerative disc disease. It also outlines how supplementation with aggrecan or a biomimetic may be of therapeutic value in treating the degenerate disc.Major conclusions: Aggrecan abundance reaches a plateau in the early twenties, declining thereafter due to proteolysis, mainly by matrix metalloproteinases and aggrecanases, though degradation of hyaluronan and non-enzymic glycation may also participate. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process as degradation products may accumulate in the disc for decades. The low turnover rate of the remaining aggrecan is an additional contributing factor, preventing protein renewal. It may be possible to retard the degenerative process by restoring the aggrecan content of the disc, or by supplementing with a bioimimetic possessing similar osmotic properties.General significance: This review provides a basis for scientists and clinicians to understand and appreciate the central role of aggrecan in the function, degeneration and repair of the intervertebral disc. (C) 2014 Elsevier B.V. All rights reserved.

Metabolic bone disorders in children frequently are heritable, but the expanding number of genes associated with these conditions makes it difficult to perform molecular diagnosis. In the present study, we therefore evaluated a semiconductor (SC)-based sequencing system for this purpose. A total of 65 DNA samples were analyzed comprising 24 samples from patients with 27 known pathogenic mutations, 6 samples from patients with prior negative Sanger sequencing, and 35 consecutive samples from patients with suspected heritable metabolic bone disorders who had not had prior molecular diagnosis. In the samples with known pathogenic mutations, 26 of 27 mutations were identified by SC sequencing. All single nucleotide variants were correctly identified, but a 7-nucleotide duplication in CYP27B1 was not detected. SC sequencing revealed two pathogenic mutations in the six samples where prior Sanger sequencing had failed to identify a mutation. Finally, pathogenic mutations were found in 27 samples of patients with unknown mutation status (15 in COL1A1, 9 in COL1A2, 1 in LEPRE1, 1 in LRP5, 1 in PHEX). Subsequent Sanger sequencing confirmed the mutations in all 27 samples. In conclusion, we found that SC sequencing is suitable for the diagnosis of heritable metabolic bone disorders in children.

CONTEXT:Homozygous mutations in SERPINF1 cause deficiency of pigment epithelium-derived factor (PEDF) and lead to osteogenesis imperfecta (OI) type VI, but it is not known whether heterozygous mutations in SERPINF1 cause a phenotype.OBJECTIVE:In the present study, we therefore assessed family members of individuals with OI type VI and compared the results of SERPINF1 mutation carriers with those of noncarriers of SERPINF1 mutations.SETTING:This study was conducted at a metabolic bone clinic of a pediatric orthopedic hospital.SUBJECTS:The study population comprised 29 family members (age range 8-89 y; 18 females, 11 males) of patients with a diagnosis of OI type VI. Eighteen individuals were heterozygous for SERPINF1 mutations, but the others did not carry a mutation.MAIN OUTCOME MEASURES:PEDF expression was assessed in skin fibroblasts from four heterozygous SERPINF1 mutation carriers. Skeletal characteristics and body composition were measured using dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Serum samples were used to quantify markers of bone metabolism, lipid status, and PEDF.RESULTS:Carriers of heterozygous stop or frame shift mutations in SERPINF1 had low SERPINF1 transcript levels. Mean PEDF serum concentrations were significantly lower in the carrier group than in the noncarriers (P = .04). However, no group differences were found with regard to areal bone density at the lumbar spine and total body, volumetric bone density at the radius and tibia, body composition, lipid status, and markers of bone metabolism.CONCLUSION:Heterozygous SERPINF1 mutation carriers had no detectable abnormalities in fat and bone, despite decreased PEDF expression.

Juvenile osteoporosis (JO) is characterized by bone fragility during development, low bone mass and absence of extraskeletal features. Heterozygous loss-of-function mutations in LRP5 have been found in a few patients, but bone tissue and bone material abnormalities associated with such mutations have not been determined. Here we report on a 6-year-old boy who presented with a history of seven low-energy long-bone fractures starting at 19months of age and absence of extraskeletal involvement. Spine radiographs revealed multiple vertebral compression fractures. Despite tall stature (95th percentile), lumbar spine areal bone mineral density was low (z-score=-3.2). Trabecular volumetric bone mineral density, measured by peripheral quantitative computed tomography at the distal radius, was low (z-score=-5.1), but cortical thickness at the radial diaphysis was normal. Iliac bone histomorphometry demonstrated low bone formation activity in trabecular but not in cortical bone. Quantitative backscattered electron imaging showed normal material bone density in trabecular bone, but elevated results in the cortex. Whole-exome sequencing revealed a heterozygous insertion of a nucleotide in exon 12 of LRP5. This mutation had previously been reported in another JO patient and had been shown to lead to nonsense-mediated decay. Thus, heterozygous loss-of-function mutations in LRP5 can be associated with a bone formation deficit that affects mostly the trabecular compartment and can result in bone fragility during the first years of life.

Intervertebral disc (IVD) degeneration is the most common cause of back pain. Presently there is no medical treatment, leaving surgery as the only offered option. Here we evaluate the potential of Link-N to promote extracellular matrix regeneration in human IVDs. Human disc cells cultured in alginate and intact human discs were exposed to a combination of Link-N and ³⁵SO₄ in the presence or absence of interleukin (IL)-1, and the effect on proteoglycan synthesis was evaluated. In addition, message levels of aggrecan, matrix metalloproteinase (MMP)-3, MMP-13, a Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS)-4 and ADAMTS-5 were evaluated in alginate cultures. Human disc cells responded in a dose dependent manner with maximal proteoglycan synthesis at 1 µg/mL Link-N. Link-N treatment also induced proteoglycan synthesis in intact human discs, and a prolonged effect was found up to one week after Link-N treatment. Message levels of proteinases were decreased by Link-N in the presence of IL-1. Thus, Link-N can promote proteoglycan synthesis and deplete proteinase expression in adult human discs. Link-N could therefore be a promising candidate for biologically-induced disc repair, and could provide an alternative to surgical intervention for early stage disc degeneration.

Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca2+ signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.

Osteogenesis imperfecta type VI (OI type VI) is a rare autosomal recessive disorder caused by mutations in the SERPINF1 gene that encodes pigment epithelium-derived factor (PEDF). Cystinosis is an autosomal recessive lysosomal transport disorder caused by mutations in the CTNS gene. Both SERPINF1 and CTNS are located on chromosome 17p13.3. We describe an individual presenting with both OI type VI and cystinosis. The patient was diagnosed with cystinosis at the age of 11 months and OI type VI on bone biopsy at the age of 8 years. He has sustained over 30 fractures during his lifetime, and at the age of 19 years entered end-stage renal disease and subsequent renal transplant. An Affymetrix 6.0 array was used to look for areas of loss of heterozygosity on chromosome 17. Sequencing of the SERPINF1 and CTNS genes was performed, followed by quantitative PCR and Western blot of PEDF to characterize the identified mutation. A 6.58 Mb region of homozygosity was identified on the Affymetrix 6.0 array, encompassing both the SERPINF1 and CTNS genes. Sequencing of the genes identified homozygosity for a known pathogenic CTNS mutation and for a novel in-frame duplication in SERPINF1. Skin fibroblasts produced a markedly reduced amount of SERPINF1 transcript and PEDF protein. This patient has the concurrent phenotype of two rare recessive diseases, cystinosis and OI type VI. We identified for the first time an in-frame duplication in SERPINF1 that is responsible for the OI type VI phenotype in this patient.

BACKGROUND Osteogenesis imperfecta (OI) type VI is a rare autosomal recessive bone fragility disorder that is caused by inactivating mutations in SERPINF1, the gene that encodes pigment-epithelium derived factor (PEDF). Determining PEDF serum levels might facilitate the diagnosis of OI type VI. OBJECTIVE The objective of the study was to assess whether lack of circulating PEDF is a specific marker of OI type VI and to evaluate whether PEDF serum levels are influenced by other metabolic bone diseases. MATERIALS AND METHODS Serum PEDF concentrations were measured in 12 patients with OI type VI (aged 2.7-31 yr) as well as in 96 children and adolescents with OI types I, III, and IV; in 26 young patients with hypophosphatemic rickets; and in 19 healthy controls. RESULTS Circulating PEDF was undetectable in all 12 patients with OI type VI but was measurable for the other 141 study participants. No significant differences in serum PEDF concentrations were found between the diagnostic groups other than OI type VI. Treatment with bisphosphonates (in OI types I, III, and IV) and with phosphate and calcitriol (in hypophosphatemic rickets) did not have a detectable influence on serum PEDF. In patients with OI types I, III, and IV, serum creatinine, body mass index z-score, and OI severity were significant predictors of PEDF serum levels. CONCLUSION Determining PEDF serum concentration helps to diagnose OI type VI but does not seem to provide information on the activity of bone turnover or mineralization.

Purpose Proteoglycans are important to the functioning of the intervertebral disc. In addition to aggrecan there are the small leucine-rich proteoglycans (SLRPs). These are less common but in other locations their functions include collagen organisation, sequestering growth factors and stimulating inflammation. We have performed a comparative analysis of the SLRP core protein species present in intervertebral discs with various pathologies.Methods Eighteen intervertebral discs from patients with scoliosis (n = 7, 19-53 years), degenerative disc disease (n = 6, 35-51 years) and herniations (n = 5, 33-58 years) were used in this study. Proteoglycans were dissociatively extracted from disc tissues and the SLRPs (biglycan, decorin, fibromodulin, keratocan and lumican) assessed by Western blotting following deglycosylation with chondroitinase ABC and keratanase.Results Intact SLRP core proteins and a number of core protein fragments were identified in most of the discs examined. Biglycan and fibromodulin were the most extensively fragmented. Keratocan generally occurred as two bands, one representing the intact core protein, the other a smaller fragment. The intact core protein of lumican was detected in all samples with fragmentation evident in only one of the older scoliotic discs. Decorin was less obvious in the disc samples and showed little fragmentation.Conclusion In this cohort of pathological intervertebral discs, fragmentation of certain SLRP core proteins was common, indicating that some SLRPs are extensively processed during the pathological process. Identification of specific SLRP fragments which correlate with disc pathology may not only help understand their aetiopathogeneses, but also provide biomarkers which can be used to monitor disease progression or to identify particular disc disorders.

The present work describes a novel bovine disc organ culture system with long-term maintenance of cell viability, in which degenerative changes can be induced as a prelude to studying repair. Discs were isolated with three different techniques: without endplates (NEP), with bony endplates (BEP) and with intact cartilage endplates (CEP). Swelling, deformation, and cell viability were evaluated in unloaded cultures. Degeneration was induced by a single trypsin injection into the center of the disc and the effect on cell viability and matrix degradation was followed. Trypsin-treated discs were exposed to TGFβ to evaluate the potential to study repair in this system. NEP isolated discs showed >75% maintained cell viability for up to 10 days but were severely deformed, BEP discs on the other hand maintained morphology but failed to retain cell viability having only 27% viable cells after 10 days. In CEP discs, both cell viability and morphology were maintained for at least 4 weeks where >75% of the cells were still viable. To mimic proteoglycan loss during disc degeneration, a single trypsin injection was administered to the center of the disc. This resulted in 60% loss of aggrecan, after 7 days, without affecting cell viability. When TGFβ was injected to validate that the system can be used to study a repair response following injection of a bio-active substance, proteoglycan synthesis nearly doubled compared to baseline synthesis. Trypsin-treated bovine CEP discs therefore provide a model system for studying repair of the degenerate disc, as morphology, cell viability and responsiveness to bio-active substances were maintained.

Study Design. The response of cells from the annulus fibrosus (AF) and nucleus pulposus (NP) to varying oxygen (O-2) concentrations was examined when cultured in alginate.Objective. To study the effect of O-2 concentration on AF and NP cells.Summary of Background Data. AF and NP cells possess different metabolic profiles in situ. However, it is not clear whether this difference is maintained in in vitro culture conditions. AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply and O-2 levels. In vivo, O-2 levels vary from 1% to 5% within the intervertebral disc, and there is evidence that disc cell metabolism can vary with O-2 concentrations.Methods. An alginate scaffold was seeded with bovine AF or NP cells and maintained in culture for up to 18 days under different O-2 concentrations. The sulfated glycosaminoglycan (GAG) content in the culture medium and the expression of aggrecan, type I (COL1A2) and II (COL2A1) collagen genes were analyzed at day 9 and day 18.Results. In both NP and AF cells cultured either in normoxia (21% O-2) or in hypoxia (5% and 1% O-2), the GAG content of the culture medium increased with time, though the rate of increase was diminished in 5% O-2. With a decrease in O-2 levels, the expression of aggrecan mRNA increased in NP cells. There was little effect of O-2 on aggrecan mRNA level in AF cells. However, there was a slight decrease with time. Interestingly, aggrecan mRNA levels did not reflect GAG release for either NP or AF cells. There was no effect with time or O-2 levels on COL2A1 message in NP cells. The highest Aggrecan/COL2 message ratio for NP cells was with 1% O-2, suggesting this to be the best condition for maintaining the NP phenotype. COL1A2 gene expression in NP and AF cells increased with time, but showed little change with O-2 levels in NP cells. The highest COL2/COL1 ratio in NP cells was also observed with 1% O-2. Finally, NP cells tended to remain localized in the alginate beads, whereas AF cells tended to migrate from the beads.Conclusion. Both NP and AF cells showed little change in GAG production with O-2 levels ranging from 1% to 21%. Disc cell metabolism is not impaired at low O-2 concentrations, which appear beneficial to matrix composition. Furthermore, low oxygen may promote a gelatinous NP matrix, whereas increased oxygen levels may promote a fibrous matrix.

Cranial base abnormalities are an important complication of osteogenesis imperfecta (OI), a hereditary bone fragility disorder that in most patients is caused by mutations affecting collagen type I. To elucidate which clinical characteristics are associated with the occurrence of cranial base abnormalities in OI, we compared cephalometric results of 187 OI patients (median age 12.0 years, range 3.4 to 47 years; 96 female) with those of 191 healthy subjects and related findings to clinical descriptors of the disease. Overall, 41 patients (22%) had at least one unambiguously abnormal skull base measure. Multivariate logistic regression analysis in patients with OI types I, III, and IV (n = 169) revealed that height Z-score [odds ratio (OR) = 0.53, 95% confidence interval (CI) 0.43-0.66, p < .001]--but not age, gender, scleral hue, lumbar spine areal bone mineral density, or a history of bisphosphonate treatment--was a significant independent determinant of skull base abnormalities. Among patients with a height Z-score below -3, 48% had a skull base abnormality regardless of whether they had received bisphosphonate treatment in the first year of life or not. Genotype-phenotype correlations were evaluated in patients with detectable mutations in COL1A1 or COL1A2, the genes coding for collagen type I (n = 140). Skull base abnormalities were present in 6% of patients with haploinsufficiency (frameshift or nonsense) mutations, in 43% of patients with helical glycine substitutions caused by COL1A1 mutations, in 32% of patients with helical glycine substitutions owing to COL1A2 mutations, and in 17% of patients with splice-site mutations affecting either COL1A1 or COL1A2. However, multivariate logistic regression analysis showed that height Z-score but not the type of collagen type I mutation was independently associated with the prevalence of skull base abnormalities. In conclusion, this study shows that clinical severity of OI, as expressed by the height Z-score, was the strongest predictor of skull base abnormalities. We did not find evidence for the hypothesis that bisphosphonate treatment protects against skull base abnormalities.

Consistent with EBSJ's commitment to fostering quality research, we are pleased to feature some of the most highly rated abstracts from the 9th Annual AOSpine North America Fellows Forum in Banff, Canada. Enhancing the quality of evidence in spine care means acknowledging and supporting the efforts of young researchers within our AOSpine North America network. We look forward to seeing more from these promising researchers in the future.