Joint disease-specificity at the regulatory base-pair level.

Given the pleiotropic nature of coding sequences and that many loci exhibit multiple disease associations, it is within non-coding sequence that disease-specificity likely exists. Here, we focus on joint disorders, finding among replicated loci, that GDF5 exhibits over twenty distinct associations, and we identify causal variants for two of its strongest associations, hip dysplasia and knee osteoarthritis. By mapping regulatory regions in joint chondrocytes, we pinpoint two variants (rs4911178; rs6060369), on the same risk haplotype, which reside in anatomical site-specific enhancers. We show that both variants have clinical relevance, impacting disease by altering morphology. By modeling each variant in humanized mice, we observe joint-specific response, correlating with GDF5 expression. Thus, we uncouple separate regulatory variants on a common risk haplotype that cause joint-specific disease. By broadening our perspective, we finally find that patterns of modularity at GDF5 are also found at over three-quarters of loci with multiple GWAS disease associations.

Piezo2 expressed in proprioceptive neurons is essential for skeletal integrity.

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.

WISP-2, an upregulated gene in hip cartilage from the DDH model rats, induces chondrocyte apoptosis through PPARγ in vitro.

Chondrocyte apoptosis plays an important role in the developmental dysplasia of the hip (DDH) development. It has been found that WNT1 inducible signaling pathway protein 2 (WISP-2) and peroxisome proliferator-activated receptor γ (PPARγ) are involved in cell apoptosis. In this study, we performed the straight-leg swaddling DDH rat model and we found that cartilage degradation and chondrocyte apoptosis were remarkably increased in DDH rats in vivo. Moreover, we found that WISP-2 was upregulated in hip acetabular cartilage of DDH rats compared to control rats. Next, the effects of WISP-2 on chondrocyte apoptosis and its possible underlying mechanism were examined in vitro. The lentivirus-mediated gain- and loss-of-function experiments of WISP-2 and peroxisome proliferator-activated receptor γ (PPARγ) for cell viability and apoptosis were performed in primary rat chondrocytes. The results showed that the overexpression of WISP-2 induced chondrocyte apoptosis, and knockdown of WISP-2 could suppress the chondrocyte apoptosis induced by advanced glycation end products (AGE). Additionally, WISP-2 could negatively regulate the expression of PPARγ in chondrocytes. Moreover, the knockdown of PPARγ promoted chondrocyte apoptosis and overexpression of PPARγ abated the increased apoptosis and decreased cell viability of chondrocytes induced by WISP-2. This study demonstrated that WISP-2 might contribute to chondrocyte apoptosis of hip acetabular cartilage through regulating PPARγ expression and activation, which may play an important role in the development of DDH.

Early-Onset Osteoarthritis originates at the chondrocyte level in Hip Dysplasia.

Subjects with developmental dysplasia of the hip (DDH) often show early-onset osteoarthritis (OA); however, the molecular mechanisms underlying this pathology are not known. We investigated whether cellular changes in chondrocytes from OA cartilage can be detected in chondrocytes from DDH cartilage before histological manifestations of degeneration. We characterized undamaged and damaged articular cartilage from 22 participants having hip replacement surgery with and without DDH (9 DDH-OA, 12 OA-only, one femoral fracture). Tissue immunostaining revealed changes in damaged OA-only cartilage that was also found in undamaged DDH-OA cartilage. Chondrocytes in situ from both groups show: (i) thicker fibers of vimentin intermediate filaments, (ii) clusters of integrin α5ß1, (iii) positive MMP13 staining and (iv) a higher percentage of cells expressing the serine protease HtrA1. Further characterization of the extracellular matrix showed strong aggrecan and collagen II immunostaining in undamaged DDH cartilage, with no evidence of augmented cell death by activation of caspase 3. These findings suggest that early events in DDH cartilage originate at the chondrocyte level and that DDH cartilage may provide a novel opportunity to study these early changes for the development of therapeutic targets for OA.

3D-bioprinting a genetically inspired cartilage scaffold with GDF5-conjugated BMSC-laden hydrogel and polymer for cartilage repair.

Rationale: Articular cartilage injury is extremely common in congenital joint dysplasia patients. Genetic studies have identified Growth differentiation factor 5 (GDF5) as a shared gene in joint dysplasia and OA progression across different populations. However, few studies have employed GDF5 in biological regeneration for articular cartilage repair. Methods & Results: In the present study, we report identified genetic association between GDF5 loci and hip joint dysplasia with genome-wide association study (GWAS). GWAS and replication studies in separate populations achieved significant signals for GDF5 loci. GDF5 expression was dysregulated with allelic differences in hip cartilage of DDH and upregulated in the repaired cartilage in a rabbit cartilage defect model. GDF5 in vitro enhanced chondrogenesis and migration of bone marrow stem cells (BMSCs), GDF5 was tested in ectopic cartilage generation with BMSCs by GDF5 in nude mice in vivo. Genetically inspired, we further generated functional knee articular cartilage construct for cartilage repair by 3d-bioprinting a GDF5-conjugated BMSC-laden scaffold. GDF5-conjugated scaffold showed better cartilage repairing effects compared to control. Meanwhile, transplantation of the 3D-bioprinted GDF5-conjugated BMSC-laden scaffold in rabbit knees conferred long-term chondroprotection. Conclusions: In conclusion, we report identified genetic association between GDF5 and DDH with combined GWAS and replications, which further inspired us to generate a ready-to-implant GDF5-conjugated BMSC-laden scaffold with one-step 3d-bioprinting for cartilage repair.

DNA hypermethylation of GDF5 in developmental dysplasia of the hip (DDH).

INTRODUCTION & OBJECTIVE: Developmental Dysplasia of the Hip (DDH) is one of the most common congenital skeletal anomalies. Body of evidence suggests that genetic variations in GDF5 are associated with susceptibility to DDH. DDH is a multifactorial disease and its etiology has not been entirely determined. Epigenetic changes such as DNA methylation could be linked to DDH. In this scheme, we hypothesized that changes in GDF5 DNA methylation could predispose a susceptible individual to DDH. METHODS: This study consisted of 45 DDH patients and 45 controls with healthy femoral neck cartilage, who underwent hemi-, or total arthroplasty for the femoral neck fracture. A cartilage sample of 1 cm in diameter and 1 mm in the thickness was obtained for DNA extraction. DNA was extracted and DNA methylation of GDF5 was evaluated by metabisulfite method. RESULTS: Methylation analysis showed that the promoter of GDF5 in cartilage samples from DDH patients was hypermethylated in comparison to healthy controls (p = .001). CONCLUSION: Our study showed that the methylation status of the GDF5 in patients with DDH is dysregulated. This dysregulation indicates that adjustment in the methylation might modify the expression of this gene. Since this gene plays an essential role in cartilage and bone development, thus reducing its expression can contribute to the pathogenesis of DDH. Further studies are needed to elucidate the role of GDF5 in this disease.

COMPARATIVE ANALYSIS OF WHOLE CARTILAGE TISSUE THERMOSTABILITY IN DISEASED PATIENTS VERSUS INJURED PATIENTS.

We conducted comparative thermodynamic analysis of femoral cartilages tissue of injured (healthy) patients and patients with congenital hip dislocation. It is shown, that temperature which corresponds to maximum of heat absorption peak of femoral cartilages tissue of diseased patient is on 6.4oC lower than heat absorption peak of femoral cartilages tissue of healthy patient. Heat absorbed during denaturation process in all these cases are close to each other with experimental error accuracy and corresponds to 52±2.6, 51±2.6 and 50±2.5 J/g of dried biomass accordingly. Analysis of the published data makes it possible to assert that the dominant heat absorption stage on DSC curves of tested fresh tissues samples is associated with melting of collagen fibers, hence the thermal stability of the collagen fibers in the patient's tissue is reduced relative to norm.

[Effects of transcutaneous electrical acupoint stimulation combined with general anesthesia on cerebral oxygen metabolism in elderly hip replacement patients during controlled hypotension].

OBJECTIVE: To observe the protective effect of transcutaneous electrical acupoint stimulation (TEAS) on cerebral tissue in elderly hip replacement operation patients during general anesthesia under controlled hypotension. METHODS: Forty hip replacement operation patients were randomly divided into general anesthesia (GA) control group and TEAS + GA group (n = 20 in each group). Patients of the two groups during operation were treated with controlled hypotension for reducing blood loss. TEAS (2 Hz/100 Hz, 8-12 mA) was applied to bilateral Yuyao (EX-HN 4) and Fengchi (GB 20) and began 30 min before GA. General anesthesia was performed by intravenous injection of Midazolam, Diprivan, Fentanyl and Cis-atracurium, sevoflurane-inhaling, Remifentanil, etc., and the patient's mean arterial pressure was maintained to be about 70% of the normal level (controlled hypotension) by using venous administration of Remifentanil about 10 min after the operation. GEM Premier 3000 blood gas analyzer was used to analyze levels of the arterial oxygen (CaO2), internal jugular venous oxygen (CjvO2), arterio-venous oxygen content difference (Da-jvO2) and cerebral oxygen (CERO2) uptake rates of blood samples before controlled hypotension (T0), 20 min after controlled hypotension (T ). 40 min after controlled hypotension (T2) and 20 min after the end of controlled hypotension (T3). RESULTS: Self-comparison of each group showed that in comparison with pre-controlled hypotension, CjvO2 levels at the time-points of T, T2 and T3 were significantly increased in both GA control and TEAS+GA groups (P < 0.05), while Da-jvO2 and CERO2 uptake rate levels at the time-points of T,. T2 and T3 in these two groups were significantly decreased (P < 0.05). Comparison between two groups showed that CivO2 levels at the time-points of T2 and T3 were significantly higher in the TEAS + GA group than in the GA control group (P<0. 05). while Da-jvO2 and CERO2 uptake rate levels at the time-point of T2 were obviously lower in the TEAS + GA group than in the GA control group(P<0. 05), suggesting a reduction of cerebral oxygen consumption after TEAS. No significant differences were found between these two groups in blood CjvO2, Da-jvO2 and CERO2 uptake rate levels at the time-points of T0 and T1 (P > 0.05). CONCLUSION: TEAS can reduce cerebral oxygen uptake rate in elderly patients undergoing hip replacement during controlled hypotension, suggesting a protective effect of TEAS on patient's cerebral tissue.

[Expressions of bone morphogenetic protein 2 and runt-related transcription factor 2 and microarchitecture of trabecular bone periacetabula in adult patients with developmental dysplasia of hip].

OBJECTIVE: To explore the expressions of bone morphogenetic protein 2 (BMP-2) and runt-related transcription facotr 2 (Runx2) and microarchitecture of trabecular bone periacetabula in adult patients with developmental dysplasia of the hip (DDH). METHODS: Between March and September 2008, the trabecular bone periacetabulum was collected from 8 patients with DDH who were scheduled for total hip arthroplasty (aged 37-55 years, 3 males and 5 females, trial group) and from 8 patients with avascular necrosis of the femoral head (Ficat stage II) who were scheduled for hip resurfacing arthroplasty (aged 36-55 years, 3 males and 5 females, control group). The expressions of BMP-2 and Runx2 in the trabecular bone were determined by real-time quantitative PCR, and the microarchitecture was observed by micro-CT and the following parameters were determined: bone volume/total volume (BV/TV), connectivity density (Conn.Dens), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and structure model index (SMI). RESULTS: The expressions of BMP-2 and Runx2 were significantly lower in trial group than in control group (P < 0.05). The micro-CT showed sparse trabecular bone in trial group and dense trabecular bone in control group. BV/TV and Tb.N in trial group were significantly lower than those in control group, and SMI and Tb.Sp in trial group were significantly higher than those in control group (P < 0.05); there was no significant difference in Conn.Dens and Tb.Th between 2 groups (P > 0.05). CONCLUSION: The trabecular bone is in a low metabolism condition and its microarchitecture is tendency to be osteoporosis trabecualr bone in adult patients with DDH. It may be related with the acetabular component loosening after total hip arthroplasty.

[Expression of COL1a1 and COL3a1 in the capsule of children with developmental dislocation of the hip].

OBJECTIVE: The etiology of developmental dislocation of the hip (DDH) remains uncertain, but some research has shown that this disorder is closely related to hip joint laxity. This study examined the expression of collagens type I and III mRNA and protein in the hip capsule of children with DDH in order to investigate the roles of collagens type I and III in hip joint laxity. METHODS: Nine children with DDH and nine age and gender-matched normal children (control group) were enrolled. Semiquantitative RT-PCR method was used to detect mRNA expression of COL1a1 and COL3a1 in the hip capsule. Western-Blot method was used to detect protein expression of COL1a1 and COL3a1 in the hip capsule. The quantitative analysis of the COL1a1 and COL3a1 was performed by professional image software and the results were analyzed with standard statistical methods. RESULTS: mRNA and protein expression of COL1a1 in the DDH group was significantly lower than that in the control group (P<0.01). Compared with the control group, COL1a3 mRNA expression in the DDH group decreased significantly (P<0.01), but COL1a3 protein expression was not significantly different. CONCLUSIONS: The decreased collagen I mRNA and protein expression in the hip capsule might contribute to hip joint laxity in children with DDH. Collagen type III may not be associated with hip joint laxity in DDH.

Bone metabolism after total hip revision surgery with impacted grafting: evaluation using H2 15O and [18F]fluoride PET; a pilot study.

PURPOSE: To evaluate bone blood flow and bone formation in patients after total hip revision surgery with impacted bone grafting using H2 15O and [18F]fluoride positron emission tomography (PET). PROCEDURES: To asses bone blood flow and bone metabolism in bone allograft after impaction grafting, four patients treated with total hip revision surgery were enrolled prospectively in this study. Six patients scheduled for primary hip arthroplasties were included as a control group. The study protocol consisted of three H2 15O and [18F]fluoride PET scans in each patient. RESULTS: Bone blood flow increased significantly compared to the preoperative state in patients treated for primary hip arthroplasty. In patients undergoing revision surgery, bone blood flow was twofold to threefold higher compared to the preoperative state, but did not reach significance. Bone metabolism in patients undergoing revision was threefold higher 2 weeks postoperatively compared to the primary hip group. We found a significant correlation between Ki and bone blood flow. CONCLUSIONS: Allogeneic bone grafts induce a higher rate of local periprosthetic bone formation compared to periprosthetic bone formation after a primary total hip placement. In vivo coupling between bone blood flow and bone metabolism suggests that bone metabolism in allogeneic bone grafts may partly rely on bone blood flow adaptations.

Cartilaginous differentiation in the joint capsule.

The proliferation and differentiation of cells are greatly influenced by their environment. Many growth factors and cytokines are reported to be environmental factors that affect the proliferation and differentiation of cells. Mechanical stress is also considered to influence these physiological reactions. The joint capsule, which is a part of the joint tissue, plays a very important role in the stability of the joint and in maintaining the intracapsular phenomenon. In patients with dislocated hip arthropathy, this capsule is involved in the weightbearing function by forming a sliding surface between the capsule and the femoral head articular cartilage. The surface of the tissue macroscopically shows cartilaginous change, which indicates cartilaginous differentiation caused by mechanical stress. We examined the cartilage-specific proteoglycan component, which is composed of cartilaginou matrix at the differentiation site. We investigated proteoglycan production, molecular size, and the gene expression of cartilaginous substrate. At the inner layer of the weightbearing area of the joint capsule, proteoglycan production was significantly higher than that of other noncartilaginous tissue. We also identified the gene expression of cartilaginous proteoglycan using the reverse transcription polymerase chain reaction (RT-PCR) method.

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.

In vitro synthesis of immunoglobulin by rheumatoid synovial membrane.

A technique for the in vitro culture of rheumatoid synovial tissue with (14)C-amino acids and isolation and quantitation of the newly synthesized immunoglobulins has been developed. This technique has been used to compare immunoglobulin synthesis of 12 rheumatoid synovia with that of synovia from nonarthritic patients and with that of normal human lymph nodes and spleen. In addition, the spleen of a patient with Felty's syndrome has also been examined. Immunoglobulin synthesis in rheumatoid synovia has been shown to be quantitatively and qualitatively similar to that of normal human spleen and lymph nodes although somewhat less active than the Felty's syndrome spleen examined. 79% of the immunoglobulin produced in rheumatoid synovia was of the IgG type, whereas IgM comprised 10% and IgA, 11% of the total. Less than 10% of the IgM synthesized was found to be rheumatoid factor. A fraction containing approximately 90% of its radioactivity in the form of IgG has been obtained for further studies.