Sequencing identifies a distinct signature of circulating microRNAs in early radiographic knee osteoarthritis.

OBJECTIVE: MicroRNAs act locally and systemically to impact osteoarthritis (OA) pathophysiology, but comprehensive profiling of the circulating miRNome in early vs late stages of OA has yet to be conducted. Sequencing has emerged as the preferred method for microRNA profiling since it offers high sensitivity and specificity. Our objective was to sequence the miRNome in plasma from 91 patients with early [Kellgren-Lawrence (KL) grade 0 or 1 (n = 41)] or late [KL grade 3 or 4 (n = 50)] symptomatic radiographic knee OA to identify unique microRNA signatures in each disease state. DESIGN: MicroRNA libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the Illumina NextSeq 550. Counts were produced for microRNAs captured in miRBase and for novel microRNAs. Statistical, bioinformatics, and computational biology approaches were used to refine and interpret the final list of microRNAs. RESULTS: From 215 differentially expressed microRNAs (FDR < 0.01), 97 microRNAs showed an increase or decrease in expression in ≥85% of samples in the early OA group as compared to the median expression in the late OA group. Increasing this threshold to ≥95%, seven microRNAs were identified: hsa-miR-335-3p, hsa-miR-199a-5p, hsa-miR-671-3p, hsa-miR-1260b, hsa-miR-191-3p, hsa-miR-335-5p, and hsa-miR-543. Four novel microRNAs were present in ≥50% of early OA samples and had 27 predicted gene targets in common with the prioritized set of predicted gene targets from the 97 microRNAs, suggesting common underlying mechanisms. CONCLUSION: Sequencing of well-characterized patient cohorts produced unbiased profiling of the circulating miRNome and identified a unique panel of 11 microRNAs in early radiographic knee OA.

Effective repair of articular cartilage using human pluripotent stem cell-derived tissue.

In an effort to develop an effective source of clinically relevant cells and tissues for cartilage repair a directed differentiation method was used to generate articular chondrocytes and cartilage tissues from human embryonic stem cells (hESCs). It has previously been demonstrated that chondrocytes derived from hESCs retain a stable cartilage-forming phenotype following subcutaneous implantation in mice. In this report, the potential of hESC-derived articular-like cartilage to repair osteochondral defects created in the rat trochlea was evaluated. Articular cartilage-like tissues were generated from hESCs and implanted into the defects. After 6 and 12 weeks, the defects were evaluated histologically and immunohistochemically, and the quality of repair was assessed using a modified ICRS II scoring system. Following 6 and 12 weeks after implantation, hESC-derived cartilage tissues maintained their proteoglycan and type II collagen-rich matrix and scored significantly higher than control defects, which had been filled with fibrin glue alone. Implants were found to be well integrated with native host tissue at the basal and lateral surfaces, although implanted human cells and host cells remained regionally separated. A subset of implants underwent a process of remodeling similar to endochondral ossification, suggesting the potential for a single cartilaginous implant to promote the generation of new subchondral bone in addition to repair of the articular cartilage. The ability to create cartilage tissues with integrative and reparative properties from an unlimited and robust cell source represents a significant advance for cartilage repair that can be further developed in large animal models before clinical- setting application.

Neuropathic pain in end-stage hip and knee osteoarthritis: differential associations with patient-reported pain at rest and pain on activity.

OBJECTIVE: We investigated whether pain at rest and pain on activity were differentially associated with neuropathic pain scores in individuals with end-stage hip and knee OA. DESIGN: Study participants were 843 patients with hip or knee OA scheduled for total joint arthroplasty. In pre-surgery questionnaires, measures of socio-demographics, health status, medication use, neuropathic pain (painDETECT), pain at rest and pain on activity (WOMAC pain items), depression (HADS) and pain catastrophizing (PCS) were collected. Multivariable linear regression models were estimated for men and women separately to examine the association between neuropathic pain scores (outcome) and study measures, entered in blocks. RESULTS: Sample mean age was 65.1 years (SD: 9.6); 57.1% were women. Mean painDETECT scores were significantly higher (P ≤Ö¹ 0.001) for women (11.2 ± 6.6 out of 38) than men (9.3 ± 7.0), with 35.6% of women and 27.7% of men meeting cut-offs for possible or likely neuropathic pain. In the final regression model for women, the coefficients for both types of pain were statistically significant, although the coefficient for pain at rest was 1.6 times greater than that for pain on activity. For men, only pain at rest was significantly associated with neuropathic pain scores. CONCLUSIONS: Findings support that possible neuropathic pain is experienced by a notable proportion of patients with end-stage hip and knee OA and is more strongly associated with pain at rest than pain on activity, particularly in men. Clinical presentation of pain at rest may warrant more thorough evaluation for potential neuropathic pain and have implications for appropriate pain management.

Controversies relating to the management of acromioclavicular joint dislocations.

The aim of this review is to address controversies in the management of dislocations of the acromioclavicular joint. Current evidence suggests that operative rather than non-operative treatment of Rockwood grade III dislocations results in better cosmetic and radiological results, similar functional outcomes and longer time off work. Early surgery results in better functional and radiological outcomes with a reduced risk of infection and loss of reduction compared with delayed surgery. Surgical options include acromioclavicular fixation, coracoclavicular fixation and coracoclavicular ligament reconstruction. Although non-controlled studies report promising results for arthroscopic coracoclavicular fixation, there are no comparative studies with open techniques to draw conclusions about the best surgical approach. Non-rigid coracoclavicular fixation with tendon graft or synthetic materials, or rigid acromioclavicular fixation with a hook plate, is preferable to fixation with coracoclavicular screws owing to significant risks of loosening and breakage. The evidence, although limited, also suggests that anatomical ligament reconstruction with autograft or certain synthetic grafts may have better outcomes than non-anatomical transfer of the coracoacromial ligament. It has been suggested that this is due to better restoration horizontal and vertical stability of the joint. Despite the large number of recently published studies, there remains a lack of high-quality evidence, making it difficult to draw firm conclusions regarding these controversial issues.

Orthopaedic Web Links (OWL): a way to find professional orthopaedic information on the internet.

BACKGROUND: Finding useful high-grade professional orthopaedic information on the Internet is often difficult. Orthopaedic Web Links (OWL) is a searchable database of vetted online orthopaedic resources. OWL uses a subject directory (OWL Directory) and a custom search engine (OWL Web) to provide a list of resources. The most effective way to find readily accessible, full text on-subject material suitable for education of an orthopaedic surgeon or trainee has not been defined. QUESTIONS/PURPOSES: We therefore (1) proposed a method for selecting topics and evaluating searches and (2) compared the search results from an orthopaedic-specific directory (OWL Directory), a custom search engine (OWL Web), and standard Google searches. METHODS: A scoring system for evaluation of the search results was developed for standardized comparison. Single words and sets of three words from randomly selected examination questions provided the search strings to compare the three strategies. RESULTS: For single keyword searches, the OWL Directory scored highest (16.4/50) of the three methods. For the three keywords searches, OWL Web had the highest mean score (26.0/50), followed by Google (22.8/50), and the OWL Directory (1.0/50). OWL Web searches had higher scores than Google searches, while returning 800 times fewer search results. CONCLUSION: The OWL Directory of orthopaedic subjects on the Internet provides a simple browsable category structure to find information. The OWL Web search engine scored higher than Google and resulted in a greater proportion of valid, on-subject, and accessible resources in the search results.

Revision of the humeral component for aseptic loosening in arthroplasty of the shoulder.

Between 1976 and 2004, 38 revision arthroplasties (35 patients) were performed for aseptic loosening of the humeral component. The mean interval from primary arthroplasty to revision was 7.1 years (0.4 to 16.6). A total of 35 shoulders (32 patients) were available for review at a mean follow-up of seven years (2 to 19.3). Pre-operatively, 34 patients (97%) had moderate or severe pain; at final follow-up, 29 (83%) had no or only mild pain (p < 0.0001). The mean active abduction improved from 88 degrees to 107 degrees (p < 0.01); and the mean external rotation from 37 degrees to 46 degrees (p = 0.27). Excellent or satisfactory results were achieved in 25 patients (71%) according to the modified Neer rating system. Humeral components were cemented in 29, with ingrowth implants used in nine cases. There were 19 of standard length and 17 were longer (two were custom replacements and are not included). Bone grafting was required for defects in 11 humeri. Only two glenoid components were left unrevised. Intra-operative complications included cement extrusion in eight cases, fracture of the shaft of the humerus is two and of the tuberosity in four. There were four re-operations, one for recurrent humeral loosening, with 89% survival free of re-operations at ten years. Revision surgery for aseptic loosening of the humeral component provides reliable pain relief and modest improvement of movement, although there is a substantial risk of intra-operative complications. Revision to a total shoulder replacement gives better results than to a hemiarthroplasty.

Endothelin-1 down-regulates the expression of vascular endothelial growth factor-A associated with osteoprogenitor proliferation and differentiation.

Endothelin-1 (ET-1) is implicated in the signaling between vascular endothelial cells (VECs) and osteoblasts during bone development, remodeling and repair. Vascular endothelial growth factor (VEGF) also plays an important role in these intercellular interactions. Our objectives were to identify which specific VEGF isoforms were produced during osteoblastic proliferation and differentiation and to determine the effects of ET-1 on VEGF mRNA and protein production by osteoblastic cells. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and ELISA were used to evaluate VEGF mRNA isoform expression and protein synthesis at different stages of ET-1-induced osteoblastic differentiation in fetal rat calvaria (FRC) osteoblastic cells. Three VEGF mRNA isoforms were identified corresponding to VEGF(120), VEGF(164) and VEGF(188). Predominant isoforms VEGF(120) and VEGF(164) had a bimodal expression that increased in the early proliferation and late mineralization phases. ET-1 stimulated osteoblastic proliferation and differentiation, but surprisingly, ET-1 down-regulated VEGF mRNA and protein expression and sustained the down-regulation over time in long-term cultures. Time course studies showed that ET-1 inhibited VEGF mRNA expression after incubation for 3 h in 7- and 14-day FRC cell cultures. Similarly, ET-1 inhibited VEGF protein secretion by 5.8- and 2.8-fold in 7- and 14-day FRC cells, respectively. VEGF-A protein secretion was inhibited by ET-1 in a dose-dependent manner with a maximal effect at 10(-7) M. This study supports a novel inhibitory role for ET-1 on VEGF synthesis in osteoblastic cells as a feedback mechanism in the temporal and spatial coupling of angiogenesis to bone formation and resorption.

Endothelin-1 promotes osteoprogenitor proliferation and differentiation in fetal rat calvarial cell cultures.

Endothelin-1 (ET-1), a peptide produced by vascular endothelial cells, has been suggested to be one of the signaling factors between vascular and osteoblastic cells during bone growth and remodeling. The osteoinductive effects of ET-1 were tested on fetal rat calvaria which have the ability to form bone nodules in culture. ET-1 (10(-10) to 10(-6) M) dose-dependently increased cell proliferation. The effect of ET-1 (10(-8) M) on proliferation was greater than that of dexamethasone (Dex; 10(-8) M). ET-1 also increased the number of bone nodules by 146% over untreated cells, which coincided with a 3.1-fold increase in alkaline phosphatase activity. Limiting dilution assays showed that ET-1 treatment increased the number of osteoprogenitors (CFU-AP and CFU-OB) beyond what would be expected by a proliferative effect alone, indicating that ET-1 also stimulated osteoblast differentiation. Osteocalcin mRNA expression was upregulated as shown by Northern blot analysis. Using cDNA microarray analysis, ET-1 treatment resulted in an expression profile that included an upregulation of 163 genes and expressed sequence tags. Simultaneous addition of ET-1 and Dex to the medium further increased the number of bone nodules and alkaline phosphatase activity over either treatment alone. Our results show that ET-1 promotes both osteoblastic proliferation and differentiation and that the effects of ET-1 and Dex on differentiation are cooperative.

DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer.

Ku antigen is composed of 70 and 82 kDa subunits (Ku70 and Ku80, respectively) that together bind with high affinity to ends of double-stranded DNA and other DNA structures in vitro. When bound to DNA, the Ku 70/80 heterodimer enhances the kinase activity of the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs. Ku and DNA-PKcs are required for V(D)J recombination and DNA double-strand break repair in vivo and may also play a role in regulation of transcription. Ku is phosphorylated by DNA-PKcs in vitro, and cells that lack DNA-PKcs are deficient in Ku phosphorylation in vitro, suggesting that Ku may be a physiological target for DNA-PK. Here we have identified the sites of DNA-PK phosphorylation in human Ku protein. We find that Ku70 is phosphorylated at a single serine residue, serine 6, located in the putative transcriptional activation domain, and Ku80 is phosphorylated at serines 577 and 580 and at threonine 715. Interestingly, none of the phosphorylation sites identified in Ku correspond to the serine-glutamine consensus for DNA-PK phosphorylation, consistent with previous reports that DNA-PK can recognize additional phosphorylation motifs.

Localization and characterization of porcine patellar tendon xenograft antigens in a rabbit model of medial collateral ligament replacement.

BACKGROUND: Ligament injuries of the knee are common and, if severe, can predispose to joint pain, instability, reinjury, and, ultimately, osteoarthritis. Xenograft replacement of ligaments could have potential; however, a limited understanding of the immunology of ligament xenograft rejection has inhibited their use. The purpose of this study was to characterize the antigenic elements of a fresh porcine tendon xenograft in a rabbit model and to provide a better understanding of what would need to be done to either block or extract these antigenic elements. METHODS: Three experimental situations were evaluated in a pig to rabbit ligament transplantation model: subcutaneous implantation of fresh porcine patellar tendon (PPT), implantation of fresh PPT into a medial collateral ligament midsubstance gap, and replacement of the entire medial collateral ligament complex with either fresh or guanidinium hydrochloride-extracted PPT. Preimmune and immune sera were collected from rabbits and used to localize antigenic targets in PPT, meniscus, and cartilage with indirect immunofluorescence techniques. The reactivities of the same rabbit sera towards tissue extracts of PPT, meniscus, and cartilage by Western immunoblot analyses were used to characterize the antigenic components. RESULTS: Indirect immunofluorescence with preimmune rabbit sera on PPT showed staining of tendon fibroblasts. Immune sera from rabbits transplanted with xenografts stained regions of the extracellular matrix of PPT. Fresh PPT induced antibodies that consistently recognized six extracellular matrix components with molecular masses of >200 kDa, 180 kDa, 135 kDa, 108 kDa, 63 kDa, and 59 kDa. CONCLUSIONS: Our results suggest that naturally occurring rabbit anti-pig antibodies of the IgG isotype recognize immunogenic components on tendon fibroblasts, whereas induced rabbit anti-pig antibodies recognize a specific subset of six extracellular matrix components of PPT. PPT xenografts appeared to induce a similar humoral immune response irrespective of graft location. Finally, our results indicate that selective extraction of PPT xenograft components before implantation altered the induced rabbit anti-pig antibody response; however, such extraction did not change the ultimate fate of the transplant tissue.

A novel candidate tumor suppressor, ING1, is involved in the regulation of apoptosis.

We have recently cloned a novel growth inhibitor and candidate tumor suppressor called p33ING1 (I. Garkavtsev et al., Nature Genet., 14: 415-420, 1996). Because some tumor suppressors participate in the regulation of apoptosis, we hypothesized that the ING1 gene may also play a role in this process. Our results show that p33ING1 levels increase upon the induction of apoptosis in P19 teratocarcinoma cells by serum deprivation. Elevated expression of ING1 in P19 and rodent fibroblast cells containing a tetracycline-controlled human c-myc gene enhanced the extent of serum starvation-induced apoptosis. This suggests that the pathway by which ING1 modulates cell death is synergistic with Myc-dependent apoptosis. Conversely, constitutive expression of an antisense construct of INGI conferred protection against apoptosis in these cells. These data support the idea that loss of proper ING1 function may facilitate tumorigenesis, in part, by reducing the cell's sensitivity to apoptosis.

Specific monoclonal antibody raised against the p33ING1 tumor suppressor.

An IgG1 mouse monoclonal antibody (CAb1) was produced against human recombinant p33ING1. The antibody is able to recognize native and denatured antigen in ELISA and Western blot protocols, respectively. CAb1 can be used to specifically detect the p33ING1 protein in dot blot and Western immunoblot protocols of both human and mouse cell lysates. In addition, this antibody is also useful for cellular localization of native and ectopically overexpressed p33ING1 protein by indirect immunofluorescence.

Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts.

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point in tissue remodeling. Previous studies have demonstrated elevated expression of the MMPs collagenase and stromelysin-1 by aged human diploid fibroblasts compared to early-passage cultures. We show here that aging cells display an altered response to transforming growth factor-beta 1 (TGF beta 1) that selectively affects MMP mRNA expression. In both young and old cells, phorbol myristoyl-13 acetate (PMA) induced the expression of transcripts of collagenase, stromelysin-1, gelatinase-B, TIMP-1, and TIMP-3. In young cells, TGF beta 1 reciprocally modulated PMA-induced MMP and TIMP gene expression leading to reduced levels of transcripts for the MMPs and augmented accumulation of TIMP-1 and TIMP-3 mRNAs. However, repressing effects of TGF beta 1 on collagenase, stromelysin-1, and gelatinase-B RNA expression were not apparent in old cells, though induction of the TIMP genes was unimpaired. By electrophoretic mobility shift analysis the nuclear transcription factors AP1 and serum response factor (SRF) showed reduced levels of DNA binding activities in old fibroblasts compared to young cells. A probe for the TGF beta-inhibitory element (TIE) gave equivalent levels of complexes with nuclear extracts from both types of cells, though of different mobilities. We conclude that the effects of TGF beta 1 on MMP and TIMP gene expression involve different cellular intermediaries, and suggest that altered composition or modification of TIE binding factors in aging cells may underlie the failure of TGF beta 1-mediated transcription repression. This mechanism may contribute to elevated constitutive expression of MMPs in old cells and to the connective tissue deterioration that accompanies the aging process.

Increased activity of p53 in senescing fibroblasts.

The p53 tumor-suppressor protein binds DNA and activates the expression of a 21-kDa protein that inhibits both the activity of cyclin-dependent kinases and the function of proliferating cell nuclear antigen. Since p21 expression has been reported to increase 10- to 20-fold as human diploid fibroblasts lose the ability to replicate, we examined the expression and activity of p53 during replicative aging. Similar levels of total p53 mRNA and protein were expressed in low-passage (young) and high-passage (old) cells but both DNA binding activity in vitro and transcriptional activity of p53 in vivo were increased severalfold in high-passage cells. While the basis of increased p53 activity is presently unclear, it is not correlated with differential phosphorylation or changes in p53-mouse double minute 2 gene product interactions. These results provide evidence for the activation of a protein involved in the control of cell cycle checkpoints during cellular aging, in the absence of increased expression.