Aim. Antimicrobial peptides occur naturally in our intrinsic immune system. PLG0206 is a novel, engineered, 24-amino acid peptide which has broad-spectrum antimicrobial activity, including in biofilm and against multi-drug resistant pathogens (1,2). This is the first clinical study to evaluate the safety and tolerability of PLG0206 when administered via an irrigation solution in patients with periprosthetic joint infections (PJI) following total knee arthroplasty (TKA) during debridement, antibiotics, and implant retention (DAIR). Secondary objectives were to evaluate pharmacokinetics (PK), biomarkers and initial clinical efficacy at one year post-DAIR procedure. Method. This prospective, multicenter, open-label, interventional study assessed two dose levels of PLG0206. Fourteen patients underwent revision for PJI after TKA. At the end of debridement, they received a single intra-articular irrigation of PLG0206 into the wound cavity lasting 15 minutes at concentrations of 3 mg/mL (n=7) or 10 mg/mL (n=7). Patients received post-operative care and intravenous/oral antimicrobial therapy as per their institutional guidelines. Patients were monitored for safety and signs of relapse or persistent infection for 12 months post study drug administration and PK and blood biomarkers were assessed. Results. All patients completed their final study assessment at Day 365. Over the 1-year follow-up, only one recurrence (7%) was noted at Day 169 in the low-dose cohort. Following dosing, nine patients (64.3%) had limited systemic exposure; maximum plasma concentration occurred 1-hour post-administration and declined rapidly to undetectable levels by 24 hours following treatment in all patients. The incidence of drug related treatment-emergent adverse events (TEAEs) was low. Two patients, both in the higher dose cohort, experienced a transient drug related TEAE; one of hypertransaminasaemia and one of neuralgia. Both events were moderate in severity and resolved within two weeks of onset. Conclusions. A single 15-minute irrigation of PLG0206 into the wound cavity of patients undergoing a DAIR procedure for PJI following TKA, is safe and well tolerated by patients. This new antimicrobial peptide offers a promising therapeutic option in musculoskeletal infection. The initial clinical efficacy is encouraging but now needs to be investigated in a much larger clinical trial

Osteoarthritis (OA) is a multifactorial disease that affects millions of Canadians. Although, there is not one specific mechanism that causes OA, the biological outcome is cartilage degradation. The articular cartilage in joints is composed primarily of the proteoglycan aggrecan and type II collagen (Col II) which together provide cartilage with functional properties. In OA, the imbalance of the anabolic and catabolic activities of chondrocytes favors cartilage catalysis. The main inflammatory cytokine involved in cartilage degradation is interleukin (IL) 1β. It has previously been demonstrated that Link N, a 16 residue peptide derived from proteolytic cleavage of link protein, can stimulate matrix proteins in normal cartilage and intervertebral discs (IVDs). Recently, we showed that a shorter sequence of Link N (sLink N), consisting of the first 8 residues of the peptide, has the potential to increase synthesis of matrix proteins in IVD cells in vitro and stimulate repair in ex vivo IVD organ culture. There are currently no treatments that actively repair cartilage in OA joints. In the present study, we aimed to evaluate the potential of sLink N as a therapeutic agent in the repair of OA cartilage. OA cartilage was isolated from four donors undergoing total knee replacement (50–70 y). Cells were recovered from the cartilage of each knee by sequential digestion with Pronase followed by Collagenase, and expanded in PrimeGrowth culture medium (Wisent Bioproducts, Canada; Cat# 319–510-CL, −S1, and −S2). After 7 days in culture, cells were treated for 24h with sLink N (0.5, 5, 50, 500 or 5000 ng/ml) or sLink N in combination with IL-1β (1 ng/ml) to mimic an inflammatory milieu. Conditioned media was collected and measured for proteoglycan (GAG) release using the safranin O and for Col II synthesis by Western blotting. Human articular cartilage explants including cartilage with subchondral bone were prepared from the same donors using the PrimeGrowth Isolation kit (Wisent, Canada) and cultured for 21 days in presence of IL-1β (1ng/ml) and sLink N (0.5, 5, 50, 500 or 5000 ng/ml). Aggrecan and Col II were extracted with guanidine buffer and measured by Western blotting. Treatment of OA chondrocytes significantly increased the GAG and Col II synthesis. The EC50 dose-response of sLink N on GAG synthesis was 67 ± 41 nM [65 ± 40 ng/ml] and the GAG synthesis reached a maximum of 194 ± 30% with the highest dose above control. When chondrocytes were cultured in the presence of IL-1β, GAG synthesis was also elevated by sLink N above control. Treatment of OA cartilage explants with sLink N increased the content of aggrecan and Col II even in the presence of IL-1β. Our results suggest that sLink N is a growth factor supplement that can increase cartilage matrix protein synthesis, and a chondroprotective agent, by modulating the catabolic effects of IL-1β. sLink N is the first small-peptide to demonstrate potential in cartilage repair of OA joints

C-type natriuretic peptide is the most abundant natriuretic peptide in the central nervous system. It has been implicated in neurogenesis and may have a significant role in spinal regeneration. We postulated that the spinal concentration of CNP would be reflected in the plasma concentrations of both CNP and the pro-hormone (NTproCNP) and this may be an indicator of repair potential in spinal injuries. Concurrent plasma and CSF concentrations of CNP forms were measured in 51 subjects undergoing spinal anaesthesia for elective total hip and knee replacement. Associations with CNP activity and metabolism in CSF were sought by measuring CSF levels of cGMP and neprilysin respectively. Elevated concentrations of NTproCNP (1045±359 pmol/L) were found in CSF and greatly exceeded those of CNP (7.9±3.2 pmol/L). The ratio of NTproCNP to CNP in CSF (145±55) was much higher than in plasma (31±27). A significant inverse relation was found between plasma and CSF CNP concentrations (r=−0.29, p<0.05). cGMP and neprilysin were unrelated to CNP levels in CSF. Despite markedly elevated levels of NTproCNP in CSF, it is unlikely that these contribute to systemic levels in healthy adults. Identifying NTproCNP as the dominant CNP form in CSF opens up the possibility of its use in future studies exploring CNP regulation within the CNS and possible applications in diagnosis and monitoring of healing in patients with spinal cord injury

Previously, we reported impaired biomechanical bone properties and inferior bone matrix quality in tachykinin1 (Tac1)-deficient mice lacking the sensory neuropeptide substance P (SP). Additionally, fracture callus development is affected by the absence of SP indicating a critical effect of sensory nerve fibers on bone health and regeneration. For α-calcitonin gene-related peptide (α-CGRP)-deficient mice, a profound distortion of bone microarchitecture has also been described. We hypothesize that SP and α-CGRP modulate inflammatory as well as pain-related processes and positively affect bone regeneration during impaired fracture healing under osteoporotic conditions. Therefore, this study investigates the effects of SP and α-CGRP on fracture healing and fracture-related pain processes under conditions of experimental osteoporosis using SP- and α-CGRP-deficient mice and WT controls. We ovariectomized female WT, Tac1−/− and α-CGRP−/− mice (age 10 weeks, all strains on C57Bl/6J background) and set intramedullary fixed femoral fractures in the left femora 28 days later. We analyzed pain threshold (Dynamic Plantar Aesthesiometer Test) and locomotion (recorded at day and night, each for 1 hour, EthoVision®XT, Noldus) at 5, 9, 13, 16 and 21 days after fracture. At each time point, fractured femora were prepared for histochemical analysis of callus tissue composition (alcian blue/sirius red staining). Pain threshold is significantly higher in Tac1−/− mice 13 days after fracture and tends to be higher after 21 days compared to WT controls. In contrast, touch sensibility was similar in α-CGRP−/− mice and WT controls but compared to Tac1−/− mice pain threshold was significantly lower in α-CGRP−/− mice 13 and 16 days and tends to be lower 21 days after fracture. Locomotion of Tac1−/− mice during daylight was by trend higher 9 days after fracture and significantly higher 16 days after fracture whereas nightly locomotion is reduced compared to WT mice. Analysis of locomotion during daylight or night revealed no differences between α-CGRP−/− and WT mice. During early fracture healing phase, 5 and 9 days after fracture, transition of mesenchymal to cartilaginous callus tissue tends to be faster in Tac1−/− mice compared to WT controls whereas no difference was observed during late stage of fracture healing, 13, 16 and 21 days after fracture. In contrast, callus tissue maturation seems to be similar in α-CGRP−/− and WT mice. Our data indicate different effects of SP and α-CGRP on fracture healing under conditions of experimental osteoporosis as a model for impaired bone tissue. Lack of α-CGRP seems to have no effects, but loss of SP affects locomotion throughout osteoporotic fracture healing and fracture-related pain processes during late phases of osteoporotic fracture healing. This indicates a modified role of SP during fracture healing under impaired versus healthy conditions, where SP changed early fracture-related pain processes and had no influence on callus tissue composition

Previous studies have described an age-dependent distortion of bone microarchitecture for α-CGRP-deficient mice (3). In addition, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from young wildtype (WT) mice when stimulated with α-CGRP whereas loss of α-CGRP showed only little effects on bone cell metabolism of cells isolated from young α-CGRP-deficient mice. We assume that aging processes differently affect bone cell metabolism in the absence and presence of α-CGRP. To further explore this hypothesis, we investigated and compared cell metabolism of osteoblasts and bone marrow derived macrophages (BMM)/osteoclast cultures isolated from young (8–12 weeks) and old (9 month) α-CGRP-deficient mice and age matched WT controls.

Isolation/differentiation of bone marrow macrophages (BMM, for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from young (8–12 weeks) and old (9 month) female α-CGRP−/− and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (crystal violet staining), proliferation (BrdU incorporation) and caspase 3/7-activity (apoptosis rate). Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and counting of multinucleated (≥ 3 nuclei), TRAP (tartrate resistant acid phosphatase) stained osteoclasts reflects osteoclast differentiation capacity.

We counted reduced numbers of BMM from young α-CGRP−/− mice after initial seeding compared to young WT controls but we found no differences between old α-CGRP−/− mice and age-matched controls. Total BMM number was higher in old compared to young animals. Migration of osteoblast-like cells out of bone chips was comparable in both, young and old α-CGRP−/− and WT mice, but number of osteoblast-like cells was lower in old compared to young animals. Proliferation of old α-CGRP−/− BMM was higher when compared to age-matched WT whereas proliferation of old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation was lower. No differences in bone cell proliferation was detected between young α-CGRP−/− and age-machted WT mice. Caspase 3/7 activity of bone cells from young as well as old α-CGRP−/− mice was comparable to age-matched controls. Number of TRAP-positive multinucleated osteoclasts from young α-CGRP−/− mice was by trend higher compared to age-matched WT whereas no difference was observed in osteoclast cultures from old α-CGRP−/− mice and old WT. ALP activity, as a marker for bone formation activity, was comparable in young WT and α-CGRP−/− osteoblasts throughout all time points whereas ALP activity was strongly reduced in old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation compared to age-matched WT.

Our data indicate that loss of α-CGRP results in a reduction of bone formation rate in older individuals caused by lower proliferation and reduced activity of osteogenic cells but has no profound effects on bone resorption rate. We suggest that the osteopenic bone phenotype described in aged α-CGRP-deficient mice could be due to an increase of dysfunctional matured osteoblasts during aging resulting in impaired bone formation.

Aim. In the current study we aim to characterize the use of cationic host defense peptides (HDPs) as alternative antibacterial agents to include into novel antibacterial coatings for orthopedic implants. Staphyloccous aureus represent one the most challenging cause of infections to treat by traditional antibacterial therapies. Thanks to their lack of microbial resistance described so far, HDPs represent an attractive therapeutic alternative to antibiotics. Furthermore, HDPs have been showed to control infections via a dual function: direct antimicrobial activity and regulation of immune response. However, HDPs functions characterization and comparison is controversial, as changing test conditions or cell type used might yield different effects from the same peptide. Therefore, before moving towards the development of HDP-based coatings, we need to characterize and compare the immunomodulatory and antibacterial functions under the same conditions in vitro of 3 well-known cathelicidins: human LL-37, chicken CATH-2, and bovine-derived IDR-1018. Method. S. aureus, strain SH1000, was incubated with different concentrations of each HDP and bacterial growth was monitored overnight. Primary human monocytes were isolated from buffy coats using Ficoll-Paque density and CD14 microbeads, and differentiated for 7 days to macrophages. After 24h incubation in presence of LPS and HDPs, macrophages cytokines production was measured by ELISA. Macrophages cultured for 24h in presence of HDPs were infected with serum-opsonized S. aureus. 30 min and 24h after infection, bacterial phagocytosis and intracellular killing by macrophages were measured by flow cytometry and colony forming units (CFU) count respectively. Results. All HDPs efficiently inhibit macrophages LPS-mediated activation, as observed by a reduced production of TNF-α and IL-10. Despite a comparable anti-inflammatory action, only CATH-2 shows direct antibacterial properties at concentrations 10-times lower than those needed to stimulate immune cells. Although stimulation with HDPs fails to improve macrophages ability to kill intracellular S. aureus, IDR-1018 decreases the proportion of cells phagocytosing bacteria. Conclusions. In addition to a strong anti-inflammatory effect provided by all HDPs tested, CATH-2 has direct antibacterial effects while IDR-1018 reduces the proportion of macrophages infected by S. aureus. Use of these HDPs in combination with each other or with other conventional antibacterial agents could lead the way to the design of novel antibacterial coatings for orthopedic implants

Aims. Prosthetic joint infection (PJI) remains the most severe complication of arthroplasty. Failure of intensive, long-term antibiotic treatment for PJI often requires removal of the implant. Antibiotic failure is thought to be caused by biofilm and persister formation. Novel anti-biofilm and anti-persister strategies are urgently needed. Here, we investigated the effects of several antimicrobial peptides on the bacteria within antibiotic-treated biofilms in an in vitro mature biofilm model on abiotic surfaces. Methods. On polystyrene, a mature (7 day-old) methicillin-resistant Staphylococcus aureus (MRSA) biofilm was developed. Thereafter, bacteria in the biofilm were exposed to rifampicin and ciprofloxacin (both 10× >MIC) for three days. Surviving bacteria in the antibiotic-treated biofilm, presumed to include persisters, were exposed to increasing doses of the antimicrobial peptides SAAP-148, acyldepsipeptide 4 (ADEP4), LL-37 and pexiganan. SAAP-148 was further tested on antibiotic-treated mature biofilms on titanium/aluminium/niobium (TAN) discs and prosthetic joint liners. Results. Daily exposure of the mature biofilm for seven days with antibiotics resulted in a 4-log reduction of MRSA without elimination of the bacteria. The surviving bacteria within the biofilm were eliminated upon subsequent exposure to SAAP-148 and pexiganan but not with LL-37 ad ADEP4. Antibiotic treatment of mature biofilms on TAN discs followed by SAAP-148 also resulted in eradication of bacteria within the biofilm. SAAP-148 also fully eliminated bacteria within antibiotic-treated mature MRSA biofilms on an ex vivo liner of a prosthetic joint. Conclusions. A novel mature biofilm model has been developed in which the efficacy of antimicrobial peptides against bacteria, including persisters, residing within a biofilm was investigated. SAAP-148 and pexiganan were highly effective against the bacteria residing in antibiotic-exposed mature MRSA biofilms. This in vitro model system will be used to analyze the effects of novel antibiotic strategies and other anti-PJI agents

Background. Staphylococcus epidermidis is one of the main organisms associated with prosthetic joint infections. One of the major pathogenic attributes of this organism is the ability to form biofilms, making it extremely resistant to currently available antimicrobial therapies. There is, therefore, an urgent requirement for novel agents that are effective against this organism. Antimicrobial peptides represent a novel group of agents that show good activity towards biofilm-forming S. epidermidis. Antimicrobial peptides are particularly interesting due to their multiple modes of action which are thought to reduce the rate of resistance development to the agents. Aim. The aim of the current study was to assess the effect of antimicrobial peptides gallidermin and novel peptide NI01 on the growth of reference and clinical isolates of S. epidermidis on polymethylmethacrylate (PMMA) cement following incorporation of the peptides. Methods. To do this a novel biofilm flow-through model was used to determine the effect of the peptides on growth of the strains, allowing the assessment of planktonic and biofilm cells. The antimicrobial peptides were incorporated into 4mm diameter PMMA beads (1% w/w) created using a specially developed poly (tetrafluoroethylene) (PTFE) mould. Results. 10 beads were used per housing unit for each of the different treatments. Each of the 10 beads was inoculated with 6 × 10. 8. cfu/ml of S. epidermidis reference strain RP62a and clinical isolates 53 and 156. The growth of the strains was then monitored over a period of five days and the effect of the peptides assessed. Results identified both gallidermin and NI01 caused a significant decrease in the growth of S. epidermidis reference strain RP62a and clinical isolates 53 and 156 (P=< 0.05). Conclusion. This study identifies the effectiveness of antimicrobial peptides gallidermin and novel antimicrobial peptide NI01 against biofilm forming S. epidermidis and highlights their potential use in the orthopaedic setting

Osteomyelitis caused by resistant bacterial strains can be dealt with antimicrobial agents which have a different mode of action compared to antibiotics. A very promising appears to be antimicrobial peptides (AMPs). We found and verified in vitro experiments that one of the most effective and least toxic antimicrobial peptides are contained in the wild bee venom. The aim of this study was to verify the efficacy of topically applied, synthetically prepared antimicrobial peptide (Hal 2/27) with carrier, originally isolated from the venom of the wild bee in experiments on laboratory rats. It was used 18 rats, which were indicated osteomyelitis of the left femurs. After a week of six rats were injected calcium phosphate carrier with AMP Hal 2/27, six rats received only a carrier without AMP and six other rats remained without further intervention. After a week, rats were sacrificed and X-ray was performed in all rats limbs. Rats who received carrier with AMP Hal 2/27 had less X-ray evidence of osteomyelitis of femurs compared to rats after administration of the carrier without AMP. Topical application of a new synthetic antimicrobial peptides isolated from wild bee venom (Hal 2/27) using local carriers seems to be a promising way to treat and prevent infectious complications in orthopedics and traumatology. Internal grant of University Hospital Motol, Advanced Therapies, NO: 9777 and Internal grant of University Hospital Motol, NO 6010

Introduction. Pin-tract infections are a common problem in orthopaedic surgery, which limits the time an external fixator or Taylor spatial frame can be applied to a patient. The purpose of our study is to evaluate the ability of a novel implant surface coating — cationic steroid antibiotic (CSA)-44 — to delay or prevent the onset of these infections. This coating mimics endogenous antimicrobial peptides of the innate immune system and has been shown to effectively eradicate biofilms as well as prevent infection and stimulate healing of open, contaminated fractures. Methods. Surgeries were performed on 20 animals (outbred; Sprague-Dawley strain rats). Each animal received both CSA-coated and standard-of-care titanium pins, with pins randomized to the fifth or sixth vertebrae prior to surgeries. Animals were also randomized to either “Imaging” (imaging analysis) or “Infection” (microbiological analysis) cohorts. Surgeons were blinded to pin types and analyses cohorts. Digital images of pin sites were collected weekly over 12 weeks, and then graded by two orthopaedic surgery residents according to an established Likert scale. Graders were blinded to animal numbers, pin types, and timepoints (Figure 1). For the infection analysis cohort, four specimens per site were subjected to microbiological analysis from each site (i.e. pin, superficial skin swab, deep skin swab, sonicated bone). Each specimen was processed on three different microbiological plates (i.e. BAP, CAN, MAC) using standardized techniques. Imaging analysis was performed by dissecting vertebrae en bloc with pin retained, followed by fixation in 10% neutral buffered formalin for 72 hours. Following a graded ethanol series and storage in 70% ethanol, specimens were scanned with microcomputed tomography (µCT). Statistical analyses were performed to compare pin site appearance (chi-square testing) as well as total bacterial colony counts within each plate cohort and imaging data (Kruskal-Wallis testing); for all tests, significance was set at α=0.05. Results. Weekly digital images of each pin site were collected, graded, and then averaged (Figure 2). Statistical analysis showed no significant difference in pin appearance between the control and CSA pin cohorts at any timepoints. For the infection analysis cohort, bacterial colonies were counted on BAP, CAN, and MAC plates, followed by bacteria species identification (Figure3). Statistical analysis showed no significant difference in total bacterial colony counts between the control and CSA pin cohorts in any of the plate groups. For the imaging cohort, post-processing and subsequent data and statistical analyses are ongoing. Discussion. No significant differences were found between the control and CSA pin cohorts, with respect to pin appearance during the 12-week study or total bacterial colony counts on three plates, indicating that the control and CSA pins performed equivalently. Imaging analysis is ongoing. Although the environmentally-acquired infection model in an outbred rat strain was used to replicate the onset of pin tract infections in human populations, many animals showed Grade 1 or 2 pin site appearances at the 12-week endpoint. A follow-on study is underway using a direct bacterial seeding model. For any figures or tables, please contact the authors directly

Osteoarthritis (OA) is a chronic degenerative joint disease with cartilage degeneration, subchondral bone sclerosis, synovial inflammation and osteophyte formation. Sensory nerves play an important role in bone metabolism and in the progression of inflammation. This study explored the effects of capsaicin-induced sensory nerve denervation on OA progression in mice. This study was approved by the Institutional Animal Care and Use Committee. OA was induced via destabilization of the medial meniscus (DMM). Sensory denervation was induced by subcutaneous injection of capsaicin (90mg/kg) one week prior to DMM. One week after capsaicin injection, sensory denervation in the tibia was confirmed by immunofluorescent staining with calcitonin gene-related peptide (CGRP)-specific antibodies. Four weeks after DMM, micro-CT scans, histological analysis and RT-PCR tests were performed to evaluate OA progression. Statistical analysis was performed using SPSS 13. P values of less than 0.05 were considered statistically significant. Subcutaneous injection of capsaicin successfully induced tibial sensory denervation (n=3), which aggravated OA by increasing subchondral bone resorption. The Osteoarthritis Research Society International (OARSI) score of the capsaicin+DMM group (n=8) (11.81±2.92) was significantly higher (P=0.003) than the score of the vehicle+DMM group (n=8) (8.31±1.80). The BV/TV of the tibial subchondral bone in the capsaicin+DMM group (n=8) was 55.67%±3.08, which was significantly lower (P < 0 .001) than in the vehicle+DMM group (n=8) (86.22%±1.92). In addition, the level of expression of somatostatin in the capsaicin+DMM group (n=8) was lower than in the vehicle+DMM group (n=8) (P=0.007). Capsaicin-induced sensory denervation increased tibial subchondral bone resorption, reduced the expression of somatostatin and eventually exacerbated the existing cartilage degeneration in mice. Despite capsaicin is often used clinically to relieve OA pain, its safety is still controversial according to the OARSI guidelines for the non-surgical management of knee osteoarthritis. The findings of our study suggest that application of capsaicin, although effective in relieving pain, may accelerate the progression of existing OA

INTRODUCTION. Total joint arthroplasty continues to gain acceptance as the standard of care for the treatment of severe degenerative joint disease, and is considered one of the most successful surgical interventions in the history of medicine. However, infection of these implants, called Periprosthetic Joint Infection (PJI), remains one of the biggest challenges facing orthopaedics today. PJI can lead to additional surgeries, revision, fusion and amputation. Diagnosis of PJI. It is important to accurately diagnose PJI because its management differs from that of other causes of arthroplasty failure. In acute infection, the local signs and symptoms (e.g., severe pain, swelling, erythema, and warmth at the infected joint) of inflammation are generally present. On the other hand, chronic infection usually has a more subtle presentation, with pain alone, and is often accompanied by loosening of the prosthesis at the bone-implant interface. The diagnosis of PJI has proven quite challenging, as both acute and chronic infections can be difficult to differentiate from other forms of inflammation. The reported literature on the diagnosis of PJI has focused on evaluated laboratory tests that were never developed specifically for the diagnosis of PJI. These include the erythrocyte sedimentation rate (ESR), the serum C-reactive protein (CRP), the synovial fluid white blood cell count and the leukocyte differential. Because these tests were not made for the purpose of diagnosing PJI, it has been the responsibility of the orthopaedic community to evaluate and recommend their interpretation. This has resulted in significant confusion regarding the appropriate thresholds and optimal combination of these tests. These difficulties were the motivation for the development of a specific test for the detection of PJI. The Synovasure® Test for Periprosthetic Joint Infection (PJI). The promising diagnostic capabilities of synovial fluid biomarkers for PJI have already been reported in the literature. These biomarkers include inflammatory proteins, cytokines, and microbicidal peptides / proteins that are known to be involved in the host response to infection. Studies have demonstrated that the alpha-defensin microbicidal peptide present in human neutrophils is an ideal biomarker for PJI due to the distinct separation it achieves between positive and negative results. A specific test allowing to measure the concentration of the alpha-defensin in the synovial fluid has been developed. The specificity and the sensitivity of this test for the detection of a PJI are respectively 96% and 97%. This test has been proven to have also a high reproducibility, its results not being influenced by antibiotics. DISCUSSION. A lateral flow version of this test (Synovasure PJI, distributed exclusively in Europe by Zimmer GmbH) has been recently developed. It allows reading the results in 10 minutes and it doesn't require any laboratories for its interpretation. Currently, this test device is in clinical evaluation in more than 200 European hospitals. CONCLUSIONS. In case that the clinical evaluation of this test device is positive, this method will be a new paradigm for the diagnosis of periprosthetic joint infection

Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. There are currently no therapies that reverse or repair cartilage degradation in OA patients. Link N (DHLSDNYTLDHDRAIH) is a naturally occurring peptide that has been shown to increase both collagen and proteoglycan synthesis in chondrocytes and intervertebral disc cells [1,2]. Recent evidence indicates that Link N activates Smad1/5 signaling in cultured rabbit IVD cells presumably by interacting with the bone morphogenetic protein (BMP) type II receptor [3], however, whether a similar mechanism exists in chondrocytes remains unknown. In this study we determined whether Link N can stimulate matrix production and reverse degradation of human OA cartilage under inflammatory conditions. OA cartilage was obtained from donors undergoing total knee arthroplasty with informed consent. OA cartilage/bone explants and OA chondrocytes were prepared from each donor. Cells were prepared in alginate beads (2×106 cells/mL) for gene expression analysis using qPCR. Cells and cartilage explants were exposed to IL-1β (10ng/ml), human Link N (hLN) (1μg/ml) or co-incubated with IL-1β+hLN for 7 and 21 days, respectively. Media was supplemented every three days. Cartilage/bone explants were measured for total glycosaminoglycan (GAG) content (retained and released) using the dimethylmethylene blue (DMMB) assay. Western blotting was performed to determine aggrecan and collagen expression in cartilage tissue. To determine NFκB activation, Western blotting was performed for detection of P-p65 in chondrocytes cultured in 2D following 10 min exposure of IL-1β in the presence of 10, 100, or 1000 ng/mL hLN. Link N significantly decreased in a dose-dependent manner IL-1β-induced NFκB activation in chondrocytes. Gene expression profiling of matrix proteins indicated that there was a trend towards increased aggrecan and decreased collagen type I expression following hLN and IL-1β co-incubation. HLN significantly decreased the IL-1β-induced expression of catabolic enzymes MMP3 and MMP13, and the neuronal growth factor NGF (p < 0 .0001, n=3). In OA cartilage/bone explants, hLN reversed the loss of proteoglycan in cartilage tissue and significantly increased its synthesis whilst in the presence of IL-1β. Link N stimulated proteoglycan synthesis and decreased MMP expression in OA chondrocytes under inflammatory conditions. One mechanism for Link N in preserving matrix protein synthesis may, in part, be due to its ability in rapidly suppressing IL-1β-induced activation of NF-κB. Further work is needed to determine whether Link N directly inhibits the IL-1β receptor or interferes with NFκB activation through an independent pathway(s)

Introduction. In revision surgery, detection of periprosthetic joint infection is of prime importance. Valuable preoperative and intraoperative diagnostic tests and tools are necessary. The classical standard procedures are puncture and bacteriology examination, frozen section intraoperative and powerfield micro analysis. Since autumn 2014 a new device for detection of periprosthetic joint infection is available, named Synovasure. It is a fast test for the detection of Alpha defensing, which plays a major role in the antimicrobial defence and only occurs in inflammatory processes. „The alpha-defensin test is an immunoassay that measures the concentration of the alpha-defensin peptide in human synovialfluid. A-Defensin is an antimicrobial peptide that is secreted into the synovial fluid by human cells in response to pathogenic presence” (Deirmengian C et al., CORR 2014). Summarized, the evidence of Alpha defensin indicates infection. It is produced by CD Diagnostics (Wynnewood, PA, USA) and merchandised by Zimmer (Warsaw, IL, USA). We are using Synovasure in daily routine at our department since September 2014. The aim of this conducted study is to present our first clinical experience and to report our results of the first 54 cases. Material and Methods. At our department Synovasure is standardly used in hip and knee revision surgery. Additionally an intraoperative frozen section and a standard bacteriology were performed. The explanted endprosthesis were sent to examination by sonification in order to gain culture of the sonification fluid and were further examined by Multiplex PCR. A pathologist with more than 15 years of experience conducted the frozen section. The results of Synovasure were matched with all above examinations in order to describe specifity and sensitivity of it. Results. A negative Synovasure Test during surgery and a negative PCR were observed in 3 patients, however, the bacterial culture was positive (after 14 days of breeding) as well as the Multiplex PCR. One patient had a negative frozen section and a negative culture but a positive PCR. Another patient with a high CRP level, all clinical signs of infection and a positive Synovasure Test, had 6 negative cultures. This patient suffered from a Metallosis as well, due to a broken PE inlay of the TKA, which supports the previously stated that Metallosis may interfere this new tool. Unfortunately in this patient neither a frozen section nor a PCR are available. One patient, who had explanation due to infection, underwent reimplantation. During surgery the Synovasure Test and the frozen section were negative (Synovial Fluid), but postoperatively a positive culture and a positive histological report for infection were assessed. Furthermore, a total of 5 tests showed an application error and the test did not show any control line. Conclusion. In conclusion Synovasure helps to detect perprosthetic joint infection in an easy and fast manner. It is simple to integrate into daily routine, nevertheless all standardized examinations for infection need to be conducted

Osteoarthritis (OA) is the fastest growing global health problem, with a total joint replacement being the only effective treatment for patients with end stage OA. Many groups are examining the use of bone marrow or adipose derived mesenchymal stem cells (MSCs) to repair cartilage, or modulate inflammation to promote healing, however, little efficacy in promoting cartilage repair, or reducing patient symptoms over temporary treatments such as micro-fracture has been observed. There is a growing body of literature demonstrating that MSCs derived from the synovial lining of the joint are superior in terms of chondrogenic differentiation and while improvements in clinical outcome measures have been observed with synovial MSCs, results from clinical studies are still highly variable. Based on our results, we believe this variability in clinical studies with MSCs results in part from the isolation, expansion and re-injection of distinct MSCs subtypes in normal vs. OA tissues, each with differing regenerating potential. However, it remains unknown if this heterogeneity is natural (e.g. multiple MSC subtypes present) or if MSCs are influenced by factors in vivo (disease state/stage). Therefore, in this study, we undertook an ‘omics’ screening approach on MSCs from normal and OA knee synovial tissue. Specifically, we characterized their global proteome and genomic expression patterns to determine if multiple MSC from normal and OA joints are distinct at the protein/gene expression level and/if so, what proteins/genes are differentially expressed between MSCs derived from normal and OA synovial tissue. Synovium tissue was collected from OA patients undergoing joint replacement and normal cadaveric knees. The in vitro adipogenic, chondrogenic and osteogenic differentiation potential of the MSCs was analyzed via qPCR and histology. Fully characterized MSC populations where then analyzed through an unbiased shotgun proteomics, and microarray analysis. Synovial MSCs isolated from both OA and normal knees demonstrated similar multipotent differentiation capacity. Likewise, both OA and normal MSCs display the typical MSCs cell surface marker profile in vitro (CD90+, CD44+, CD73+, CD105+). Using shotgun proteomics, 7720 unique peptides corresponding to 2183 proteins were identified and quantified between normal and OA MSCs. Of these 2183 proteins, 994 were equally expressed in normal and OA, MSCs, 324 were upregulated in OA MSCs (with 50 proteins exclusively expressed in OA MSCs), 630 proteins were upregulated in normal MSCs (with 16 proteins exclusively expressed in normal MSCs). Microarray analysis of normal and OA MSCs demonstrated a similar result in where, 967 genes were differentially expressed between normal and OA MSCs, with 423 genes upregulated in OA, and 544 genes upregulated in normal MSCs. In this project, we have demonstrated that although normal and OA synovial derived MSCs demonstrate similar multipotent differentiation potential and cell surface markers expression, these cells demonstrated significant differences at the molecular level (protein and gene expression). Further research is required to determine if these differences influence functional differences in vitro and/or in vivo and what drives this dramatic change in the regulatory pathways within normal vs. OA synovial MSCs

Purpose. Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response. Method. Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism. Results. Cell viability in CEP isolated discs was evaluated after four weeks and four months of organ culture under high and physiological nutritional state. Previous studies have shown that high glucose levels are needed to maintain cell viability in organ culture, but in our model 96–98% live cells were present throughout the disc independent of FBS and glucose levels and the duration of culture tested. Western blot probing for the G1 domain of aggrecan showed no difference with the change of nutritional state across all regions indicating that low nutritional state had no detrimental effect on disc metabolism. Conclusion. We have developed a novel technique for isolation and culturing of intact IVDs. The described CEP system maintained sufficient nutrient supply and high cell survival in all regions of the disc for up to four months of culture also under physiological culturing condition. As the CEP system maintains high cell viability in long term cultures, it is a suitable model in which the regenerative effect of various bioactive peptides can be studied. The availability of an intact disc organ culture system has considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, so making any response to catabolic or anabolic agents more physiologically relevant

Purpose. Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response. Method. Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism. Results. Cell viability in CEP isolated discs was evaluated after four weeks and four months of organ culture under high and physiological nutritional state. Previous studies have shown that high glucose levels are needed to maintain cell viability in organ culture, but in our model 96–98% live cells were present throughout the disc independent of FBS and glucose levels and the duration of culture tested. Western blot probing for the G1 domain of aggrecan showed no difference with the change of nutritional state across all regions indicating that low nutritional state had no detrimental effect on disc metabolism. Conclusion. We have developed a novel technique for isolation and culturing of intact IVDs. The described CEP system maintained sufficient nutrient supply and high cell survival in all regions of the disc for up to four months of culture also under physiological culturing condition. As the CEP system maintains high cell viability in long term cultures, it is a suitable model in which the regenerative effect of various bioactive peptides can be studied. The availability of an intact disc organ culture system has considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, so making any response to catabolic or anabolic agents more physiologically relevant

The number of shoulder arthroplasty procedures performed in the United States continues to rise. Currently, the number of procedures performed per year ranges from 55,000–80,000 and is expected to increase more than 300% in the coming years. Periprosthetic joint infection (PJI) is one of the most serious complications associated with arthroplasty surgery, leading to poor outcomes, increased cost, and technically difficult revision surgery. The incidence of infection following primary shoulder arthroplasty has been reported between 0.7% and 4%, representing 2.9–4.6% of all complications. Prosthetic shoulder joint infections are unlike prosthetic joint infections of the hip and knee. Shoulder PJIs are primarily indolent in nature and difficult to diagnose using traditional methods that have been shown to be accurate for periprosthetic infections of the hip and knee. The majority of infected revision shoulder arthroplasties are associated with growth of Propionibacterium acnes (P. Acnes). This slow-growing, anaerobic organism requires longer than normal incubation times for culture (7–21 days), and typically demonstrates a subtle, non-specific clinical presentation that can make the presence of infection difficult to identify. In the reported literature, P. Acnes accounts for about 70% of cases with positive cultures associated with revision for treatment of a painful shoulder arthroplasty and due to the bacteria's slow growing nature and virulence profile, the rate of infection following shoulder arthroplasty may often be underestimated. A more recent and promising tool for evaluation of periprosthetic infection has been analysis of synovial fluid. Synovial fluid biomarkers have been identified as part of the innate response to pathogens, and include pro-inflammatory cytokines and anti-microbial peptides, and marker levels have shown promise for improved diagnostic efficacy in hip and knee PJI. Currently, no highly predictive clinical test for diagnosis of PJI in the shoulder exists, however, several of these synovial biomarkers have recently been analyzed for their diagnostic capacity in the setting of periprosthetic shoulder infection. Synovial fluid cytokine analysis shows the potential to improve diagnosis of infection in revision shoulder arthroplasty. This information can help to guide decision-making in the management of PJI of the shoulder, including the decision to perform a single- vs. two-stage revision surgery, and the need for post-operative antibiotics following an unexpected positive culture result after revision surgery. However, there are still challenges to broader use of these synovial biomarkers. Synovial α-defensin (Synovsure, CD Diagnostic) is the only marker currently available as a commercial test, and no point-of-care test is currently available for any of the biomarkers to allow for intraoperative decision-making. While a preoperative synovial aspirate is possible to send for α-defensin analysis currently, with results back in approximately 24 hours, dry fluid aspirations are frequent in the shoulder because of the predominance of indolent pathogens and may limit utility of the test. In summary, indolent infection associated with P. acnes is a common cause for the painful total shoulder arthroplasty. Pre-operative diagnosis of infection is difficult as a result of the poor diagnostic accuracy of traditional methods of testing. Synovial biomarker testing may ultimately improve our ability to more accurately diagnosis and treat prosthetic shoulder joint infections

While advances in laboratory and imaging modalities facilitate the diagnosis of periprosthetic joint infection (PJI), clinical suspicion and a thorough history and physical remain the basis of evaluation. If clinical suspicion is high, the evaluation should be more vigorous, and vice versa. The erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are inexpensive as well as ubiquitous, and should be obtained as a preliminary screening tool. These tests have been found to be cost-effective and highly sensitive. If both tests are negative, there is a low risk of periprosthetic joint infection (i.e., good negative predictive value). Positive results on both tests, in contrast, are not as specific but again raise suspicion. When either the ESR or CRP is elevated, or if the clinical suspicion for infection is high, aspiration of the knee joint is suggested. Synovial fluid should be sent for a synovial fluid white blood cell count (WBC), differential and culture. Given the ability to get three data points from one intervention, arthrocentesis, is the best single maneuver the physician can perform to rule in or out PJI. The synovial fluid WBC count has demonstrated in multiple studies excellent specificity and sensitivity in the diagnosis of infection. Based on multiple recent studies, the proceedings of the International Consensus on PJI recommend cut-offs for the synovial fluid WBC count as >3000 cells/mL and > 80% neutrophils for the differential. Synovial fluid biomarkers represent an expanding area of clinical interests based on the unique cascade of gene expression that occurs in white blood cells in response to pathogens. Deirmegian et al. described the unique gene expression and biomarker production by neutrophils in response to bacteria that are detectable in synovial fluid. Specifically, alpha-defensin is one such antimicrobial peptide. Along with synovial CRP, alpha-defensin can be measured in a currently commercially-available immunoassays. The diagnosis of PJI can be difficult to make in spite of the variety of tests available. That being said, the diagnosis is easily made in our experience in 90% of patients by getting an ESR and CRP followed by selective aspiration of the joint if these values are elevated or if the clinical suspicion is high. Synovial fluid obtained should be sent for a synovial fluid WBC count, differential and cultures

Peri-prosthetic joint infection (PJI) can be both a diagnostic and therapeutic challenge in shoulder arthroplasty, due to the indolent nature of the common infecting organisms. Proprionobacterium acnes (P. acnes) is the most common pathogen cultured in revision shoulder arthroplasty. It is a slow growing, anaerobic organism – requires longer incubation period (7–21 days). Coagulase-negative Staphylococcus species (CNSS) is also a common organism responsible for PJI. Established diagnostic tests for hip and knee PJI are often negative in the shoulder despite post-operative growth of intra-operative cultures. Pre-operative synovial aspiration often low volume due to indolent pathogens and successful aspiration is often reported to be 50% or less with Dilisio et al, JBJS 2014: reporting 16.7% sensitivity, 100% specificity. Variable culture length for P. acnes culture protocols are reported from 7–28 days with most groups recommending 14 days. From our research, we demonstrated time to culture growth was significantly shorter in probable true positive culture group (median, 5 vs. 9 days, p=0.002). Frozen section analysis may help intra-operative decision-making (one- vs. two-stage reimplantation) yet the reported sensitivity and specificity in shoulder arthroplasty is far less than in hip and knee arthroplasty. Synovial fluid biomarkers have been identified as part of the innate response to pathogens include pro-inflammatory cytokines and antimicrobial peptides. In a series of prospective studies of revision shoulder arthroplasty, synovial fluid analysis reported by Frangiamore et al, JBJS 2015: IL-6, Frangiamore et al, JSES 2015: α-defensin (Synovasure. TM. ), Frangiamore et al, AAOS 2015: Broader cytokine analysis it was demonstrated that these markers are much more predictive of infection than synovial fluid cultures, frozen section or serum markers