Konstantinos N. Malizos

Integrative MicroRNA and Proteomic Approaches Identify Novel Osteoarthritis Genes and Their Collaborative Metabolic and Inflammatory Networks

Background Osteoarthritis is a multifactorial disease characterized by destruction of the articular cartilage due to genetic, mechanical and environmental components affecting more than 100 million individuals all over the world. Despite the high prevalence of the disease, the absence of large-scale molecular studies limits our ability to understand the molecular pathobiology of osteoathritis and identify targets for drug development. Methodology/Principal Findings In this study we integrated genetic, bioinformatic and proteomic approaches in order to identify new genes and their collaborative networks involved in osteoarthritis pathogenesis. MicroRNA profiling of patient-derived osteoarthritic cartilage in comparison to normal cartilage, revealed a 16 microRNA osteoarthritis gene signature. Using reverse-phase protein arrays in the same tissues we detected 76 differentially expressed proteins between osteoarthritic and normal chondrocytes. Proteins such as SOX11, FGF23, KLF6, WWOX and GDF15 not implicated previously in the genesis of osteoarthritis were identified. Integration of microRNA and proteomic data with microRNA gene-target prediction algorithms, generated a potential “interactome” network consisting of 11 microRNAs and 58 proteins linked by 414 potential functional associations. Comparison of the molecular and clinical data, revealed specific microRNAs (miR-22, miR-103) and proteins (PPARA, BMP7, IL1B) to be highly correlated with Body Mass Index (BMI). Experimental validation revealed that miR-22 regulated PPARA and BMP7 expression and its inhibition blocked inflammatory and catabolic changes in osteoarthritic chondrocytes. Conclusions/Significance Our findings indicate that obesity and inflammation are related to osteoarthritis, a metabolic disease affected by microRNA deregulation. Gene network approaches provide new insights for elucidating the complexity of diseases such as osteoarthritis. The integration of microRNA, proteomic and clinical data provides a detailed picture of how a network state is correlated with disease and furthermore leads to the development of new treatments. This strategy will help to improve the understanding of the pathogenesis of multifactorial diseases such as osteoarthritis and provide possible novel therapeutic targets.

Repair and tissue engineering techniques for articular cartilage

Chondral and osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis, eventually leading to progressive total joint destruction. Although current progress suggests that biologic agents can delay the advancement of deterioration, such drugs are incapable of promoting tissue restoration. The limited ability of articular cartilage to regenerate renders joint arthroplasty an unavoidable surgical intervention. This Review describes current, widely used clinical repair techniques for resurfacing articular cartilage defects; short-term and long-term clinical outcomes of these techniques are discussed. Also reviewed is a developmental pipeline of acellular and cellular regenerative products and techniques that could revolutionize joint care over the next decade by promoting the development of functional articular cartilage. Acellular products typically consist of collagen or hyaluronic-acid-based materials, whereas cellular techniques use either primary cells or stem cells, with or without scaffolds. Central to these efforts is the prominent role that tissue engineering has in translating biological technology into clinical products; therefore, concomitant regulatory processes are also discussed.

Epigenetic regulation of leptin affects MMP-13 expression in osteoarthritic chondrocytes: possible molecular target for osteoarthritis therapeutic intervention

Objective: To investigate whether epigenetic mechanisms can regulate leptin’s expression and affect its downstream targets as metalloproteinases 3,9,13 in osteoarthritic chondrocytes. Methods: DNA methylation in leptin promoter was measured by DNA bisulfite sequencing, and mRNA expression levels were measured by real-time quantitative PCR in osteoarthritic as well as in normal cartilage. Osteoarthritic articular cartilage samples were obtained from two distinct locations of the knee (n = 15); from the main defective area of maximum load (advanced osteoarthritis (OA)) and from adjacent macroscopically intact regions (minimal OA). Using small interference RNA, we tested if leptin downregulation would affect matrix metalloproteinase (MMP)-13 activity. We also evaluated the effect of the demethylating agent, 5′-Aza-2-deoxycytidine (AZA) and of the histone deacetylase inhibitor trichostatin A (TSA) on leptin expression in chondrocyte cultures. Furthermore, we performed chromatin immunoprecipitation in leptin’s promoter area. Results: We found a correlation between leptin expression and DNA methylation and also that leptin controls MMP-13 activity in chondrocytes. Leptin’s downregulation with small interference RNA inhibited MMP-13 expression dramatically. After 5-AZA application in normal chondrocytes, leptin’s methylation was decreased, while its expression was upregulated, and MMP-13 was activated. Furthermore, TSA application in normal chondrocyte cultures increased leptin’s expression. Also, chromatin immunoprecipitation in leptin’s promoter after TSA treatment revealed that histone H3 lysines 9 and 14 were acetylated. Conclusion: We found that epigenetic mechanisms regulate leptin’s expression in chondrocytes affecting its downstream target MMP-13. Small interference RNA against leptin deactivated directly MMP-13, which was upregulated after leptin’s epigenetic reactivation, raising the issue of leptin’s therapeutic potential for osteoarthritis.

Free vascularized fibular grafts for reconstruction of skeletal defects.

Abstract Nourished by the peroneal vessels, the versatile free vascularized fibular graft can be transferred to reconstruct skeletal defects of the extremities. It may be combined with skin, fascia, muscle, and growth‐plate tissue to address the needs of the recipient site. It may be cut transversely and folded to reconstruct the length and width of tibial or femoral defects. The main indications for this graft are defects larger than 5 to 6 cm or with poor vascularity of the surrounding soft tissues. Detailed preoperative planning, experience in microvascular techniques, and careful postoperative follow‐up are necessary to minimize complications and improve outcome. The free vascularized fibular graft has been successfully applied as a reconstruction option in patients with traumatic or septic skeletal defect, after tumor resection, and has shown promise in patients with congenital pseudarthrosis.

Treatment of avascular necrosis of the femoral head with vascularized fibular transplant.

Two hundred twenty-eight hips in 187 patients with avascular necrosis of the femoral head were treated with vascularized fibular transplant from March 1989 to March 2000. The etiologic factors associated with the disease included corticosteroids in 84 patients (44%; 101 hips, trauma in 25 patients (13%; 29 hips), alcohol abuse in 24 patients (12%; 28 hips), and 41 hips (18%) were classified as idiopathic. Systemic disorders, including systemic lupus erythematosus, sickle cell anemia, inflammatory bowel disease, pregnancy, and dysbaric disease were observed in 12, nine, four, three, and one hip(s), respectively. Of the 228 hips operated on, 184 hips (152 patients) were assessed postoperatively with followup ranging from 1 to 10 years (mean, 4.7 years). Using the Steinberg classification system, 39 hips (21%) were in Stage II; 45 hips (25%) were in Stage III; 77 hips (42%) were in Stage IV; and 23 hips (12%) were in Stage V. Of the 184 hips treated, 101 (54%) remained stable postoperatively, whereas 69 (38%) had progression, and 14 hips (8%) were converted to total hip arthroplasty. Of the 69 hips that had progression, 44 (64%) did not progress until 6 to 10 years after the procedure, whereas 25 (36%) progressed within the first 5 years postoperatively. The best results were obtained in patients with Stage II osteonecrosis in whom 95% of the hips did not progress postoperatively. In contrast, only 39% of the hips in patients with Stage V osteonecrosis remained stable. Preoperative and postoperative clinical evaluation using the Harris hip score showed an increase from 85 to 96 points in hips with Stage II disease; from 74 to 91 points in hips with Stage III disease; from 69 to 85 points in hips with Stage IV disease; and from 61 to 76 in hips with Stage V disease. The current results show that the vascularized fibular graft is an excellent procedure for the precollapse stages and a valuable alternative for patients with Stages III, IV, and V of the disease.

Treatment of intra-articular fractures of the distal radius: FLUOROSCOPIC OR ARTHROSCOPIC REDUCTION?

In a randomised prospective study, 20 patients with intra-articular fractures of the distal radius underwent arthroscopically- and fluoroscopically-assisted reduction and external fixation plus percutaneous pinning. Another group of 20 patients with the same fracture characteristics underwent fluoroscopically-assisted reduction alone and external fixation plus percutaneous pinning. The patients were evaluated clinically and radiologically at follow-up of 24 months. The Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire and modified Mayo wrist score were used at 3, 9, 12 and 24 months postoperatively. In the arthroscopically- and fluoroscopically-assisted group, triangular fibrocartilage complex tears were found in 12 patients (60%), complete or incomplete scapholunate ligament tears in nine (45%), and lunotriquetral ligament tears in four (20%). They were treated either arthroscopically or by open operation. Patients who underwent arthroscopically- and fluoroscopically-assisted treatment had significantly better supination, extension and flexion at all time points than those who had fluoroscopically-assisted surgery. The mean DASH scores were similar for both groups at 24 months, whereas the difference in the mean modified Mayo wrist scores remained statistically significant. Although the groups are small, it is clear that the addition of arthroscopy to the fluoroscopically-assisted treatment of intra-articular distal radius fractures improves the outcome. Better treatment of associated intra-articular injuries might also have been a reason for the improved outcome.

LONGSTANDING NONUNIONS OF SCAPHOID FRACTURES WITH BONE LOSS: SUCCESSFUL RECONSTRUCTION WITH VASCULARIZED BONE GRAFTS

Vascularized bone graft from the dorsum of distal radius was used to treat 22 nonunions of scaphoid fractures, with a mean delay of 4 years from the initial injury. Four of the eight patients with associated scapho-styloid arthritis also underwent a closing wedge osteotomy of the distal radius. Follow-up time ranged from 14 to 74 months. Union was accomplished in 6 to 12 weeks and wrist range of motion and grip strength improved postoperatively in all patients. Complete absence of pain was noted in 16 patients and the other six, all of whom had arthritic wrist changes or carpal collapse, only experienced pain with strenuous activities. The simple graft dissection, the avoidance of anastomoses and the lack of donor site morbidity are additional advantages to this surgical technique, which is successful even in cases with an avascular proximal pole.

New Sequence Variants in HLA Class II/III Region Associated with Susceptibility to Knee Osteoarthritis Identified by Genome-Wide Association Study

Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of ∼4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43×10−8 for rs7775228 and 6.73×10−8 for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA.

Diagnostic Accuracy of a New Clinical Test (the Thessaly Test) for Early Detection of Meniscal Tears

BACKGROUND Clinical tests used for the detection of meniscal tears in the knee do not present acceptable diagnostic sensitivity and specificity values. Diagnostic accuracy is improved by arthroscopic evaluation or magnetic resonance imaging studies. The objective of this study was to evaluate the diagnostic accuracy of a new dynamic clinical examination test for the detection of meniscal tears. METHODS Two hundred and thirteen symptomatic patients with knee injuries who were examined clinically, had magnetic resonance imaging studies performed, and underwent arthroscopic surgery and 197 asymptomatic volunteers who were examined clinically and had magnetic resonance imaging studies done of their normal knees were included in this study. For clinical examination, the medial and lateral joint-line tenderness test, the McMurray test, the Apley compression and distraction test, the Thessaly test at 5 degrees of knee flexion, and the Thessaly test at 20 degrees of knee flexion were used. For all clinical tests, the sensitivity, specificity, false-positive, false-negative, and diagnostic accuracy rates were calculated and compared with the arthroscopic and magnetic resonance imaging data for the test subjects and the magnetic resonance imaging data for the control population. RESULTS The Thessaly test at 20 degrees of knee flexion had a high diagnostic accuracy rate of 94% in the detection of tears of the medial meniscus and 96% in the detection of tears of the lateral meniscus, and it had a low rate of false-positive and false-negative recordings. Other traditional clinical examination tests, with the exception of joint-line tenderness, which presented a diagnostic accuracy rate of 89% in the detection of lateral meniscal tears, showed inferior rates. CONCLUSIONS The Thessaly test at 20 degrees of knee flexion can be used effectively as a first-line clinical screening test for meniscal tears, reducing the need for and the cost of modern magnetic resonance imaging methods.

Three-dimensional finite element modeling of guided ultrasound wave propagation in intact and healing long bones

The use of guided waves has recently drawn significant interest in the ultrasonic characterization of bone aiming at supplementing the information provided by traditional velocity measurements. This work presents a three-dimensional finite element study of guided wave propagation in intact and healing bones. A model of the fracture callus was constructed and the healing course was simulated as a three-stage process. The dispersion of guided modes generated by a broadband 1-MHz excitation was represented in the time-frequency domain. Wave propagation in the intact bone model was first investigated and comparisons were then made with a simplified geometry using analytical dispersion curves of the tube modes. Then, the effect of callus consolidation on the propagation characteristics was examined. It was shown that the dispersion of guided waves was significantly influenced by the irregularity and anisotropy of the bone. Also, guided waves were sensitive to material and geometrical changes that take place during healing. Conversely, when the first-arriving signal at the receiver corresponded to a nondispersive lateral wave, its propagation velocity was almost unaffected by the elastic symmetry and geometry of the bone and also could not characterize the callus tissue throughout its thickness. In conclusion, guided waves can enhance the capabilities of ultrasonic evaluation.