Aims. Electromagnetic induction heating has demonstrated in vitro antibacterial efficacy over biofilms on metallic biomaterials, although no in vivo studies have been published. Assessment of side effects, including thermal necrosis of adjacent tissue, would determine transferability into clinical practice. Our goal was to assess bone necrosis and antibacterial efficacy of induction heating on biofilm-infected implants in an in vivo setting. Methods. Titanium-aluminium-vanadium (Ti6Al4V) screws were implanted in medial condyle of New Zealand giant rabbit knee. Study intervention consisted of induction heating of the screw head up to 70°C for 3.5 minutes after implantation using a portable device. Both knees were implanted, and induction heating was applied unilaterally keeping contralateral knee as paired control. Sterile screws were implanted in six rabbits, while the other six received screws coated with Staphylococcus aureus biofilm. Sacrifice and sample collection were performed 24, 48, or 96 hours postoperatively. Retrieved screws were sonicated, and adhered bacteria were estimated via drop-plate. Width of bone necrosis in retrieved femora was assessed through microscopic examination. Analysis was performed using non-parametric tests with significance fixed at p ≤ 0.05. Results. The width of necrosis margin in induction heating-treated knees ranged from 0 to 650 μm in the sterile-screw group, and 0 to 517 μm in the biofilm-infected group. No significant differences were found between paired knees. In rabbits implanted with sterile screws, no bacteria were detected. In rabbits implanted with infected screws, a significant bacterial load reduction with median 0.75 Log10 colony-forming units/ml was observed (p = 0.016). Conclusion. Induction heating was not associated with any demonstrable thermal bone necrosis in our rabbit knee model, and might reduce bacterial load in S. aureus biofilms on Ti6Al4V implants. Cite this article: Bone Joint Res 2024;13(12):695–702

Aims. Here we used a mature seven-day biofilm model of Staphylococcus aureus, exposed to antibiotics up to an additional seven days, to establish the effectiveness of either mechanical cleaning or antibiotics or non-contact induction heating, and which combinations could eradicate S. aureus in mature biofilms. Methods. Mature biofilms of S. aureus (ATCC 29213) were grown on titanium alloy (Ti6Al4V) coupons for seven days and were subjected to the following treatments or their combinations: antibiotics, mechanical cleaning, or heat shock by induction heating of 60°C for one minute. Experiments were repeated at least five times. Results. In the untreated biofilm, growth up to 1.8×10. 11. colony-forming units (CFU)/cm. 2. was observed. Treatment with ciprofloxacin, flucloxacillin, vancomycin, cefuroxime, and amoxicillin all with rifampicin gave 6.0 log, 6.1 log, 1.4 log, 4.8 log, and 3.6 log reduction in CFU/cm. 2. , respectively. Mechanical cleaning alone resulted in 4.9 log reduction and induction heating in 7.3 log reduction. There was an additional effect of ciprofloxacin, flucloxacillin, and induction heating when used in combinations. There was no additional effect for mechanical cleaning. No bacterial growth could be detected after induction heating followed by seven days of ciprofloxacin with rifampicin. Conclusion. Mechanical cleaning, antibiotics, and non-contact induction heating reduced the bacterial load of mature S. aureus biofilms with approximately 5 log or more as a single treatment. The effect of mechanical cleaning on mature S. aureus biofilms was limited when used in combination with antibiotics and/or induction heating. Cite this article: Bone Joint Res 2022;11(9):629–638

Aims. Induction heating is a noninvasive, nonantibiotic treatment modality that can potentially be used to cause thermal damage to the bacterial biofilm on the metal implant surface. The purpose of this study was to determine the effectiveness of induction heating on killing Staphylococcus epidermidis from biofilm and to determine the possible synergistic effect of induction heating and antibiotics. Methods. S. epidermidis biofilms were grown on titanium alloy (Ti6Al4V) coupons for 24 hours (young biofilm) and seven days (mature biofilm). These coupons with biofilm were heated to temperatures of 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, and 90°C for 3.5 minutes and subsequently exposed to vancomycin and rifampicin at clinically relevant concentrations. Results. For the young biofilm, total eradication was observed at 65°C or higher for 3.5 minutes followed by 24 hours of vancomycin 10 mg/l and rifampicin 1 mg/l. For the mature biofilm, total eradication was observed at 60°C for 3.5 minutes followed by 24 hours of vancomycin 10 mg/l and rifampicin 1 mg/l. Total eradication was also observed at 60°C for 3.5 minutes followed by 24 hours of vancomycin 1 mg/l and rifampicin 1 mg/l followed by another thermal shock of 60°C for 3.5 minutes (two thermal shocks). Conclusion. Induction heating of Ti6Al4V coupons is effective in reducing bacterial load in vitro for S. epidermidis biofilms. Induction heating and antibiotics have a synergistic effect resulting in total eradication of the biofilm at 60°C or higher for clinically relevant concentrations of vancomycin and rifampicin. Cite this article:Bone Joint Res. 2020;9(4):192–199

Objectives. Prosthetic joint infection (PJI) is a devastating complication following total joint arthroplasty. Non-contact induction heating of metal implants is a new and emerging treatment for PJI. However, there may be concerns for potential tissue necrosis. It is thought that segmental induction heating can be used to control the thermal dose and to limit collateral thermal injury to the bone and surrounding tissues. The purpose of this study was to determine the thermal dose, for commonly used metal implants in orthopaedic surgery, at various distances from the heating centre (HC). Methods. Commonly used metal orthopaedic implants (hip stem, intramedullary nail, and locking compression plate (LCP)) were heated segmentally using an induction heater. The thermal dose was expressed in cumulative equivalent minutes at 43°C (CEM43) and measured with a thermal camera at several different distances from the HC. A value of 16 CEM43 was used as the threshold for thermal damage in bone. Results. Despite high thermal doses at the HC (7161 CEM43 to 66 640 CEM43), the thermal dose at various distances from the HC was lower than 16 CEM43 for the hip stem and nail. For the fracture plate without corresponding metal screws, doses higher than 16 CEM43 were measured up to 5 mm from the HC. Conclusion. Segmental induction heating concentrates the thermal dose at the targeted metal implant areas and minimizes collateral thermal injury by using the non-heated metal as a heat sink. Implant type and geometry are important factors to consider, as they influence dissipation of heat and associated collateral thermal injury. Cite this article: B. G. Pijls, I. M. J. G. Sanders, E. J. Kuijper, R. G. H. H. Nelissen. Segmental induction heating of orthopaedic metal implants. Bone Joint Res 2018;7:609–619. DOI: 10.1302/2046-3758.711.BJR-2018-0080.R1

Objectives. Infection of implants is a major problem in elective and trauma surgery. Heating is an effective way to reduce the bacterial load in food preparation, and studies on hyperthermia treatment for cancer have shown that it is possible to heat metal objects with pulsed electromagnetic fields selectively (PEMF), also known as induction heating. We therefore set out to answer the following research question: is non-contact induction heating of metallic implants effective in reducing bacterial load in vitro?. Methods. Titanium alloy cylinders (Ti6Al4V) were exposed to PEMF from an induction heater with maximum 2000 watts at 27 kHz after being contaminated with five different types of micro-organisms: Staphylococcus epidermidis; Staphylococcus aureus; Pseudomonas aeruginosa; spore-forming Bacillus cereus; and yeast Candida albicans. The cylinders were exposed to incremental target temperatures (35°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C) for up to 3.5 minutes. Results. There was an average linear heating rate of 0.39°C per second up to the target temperature, and thereafter the target temperature was maintained until the end of the experiment. At 60°C and higher (duration 3.5 minutes), there was a 6-log reduction or higher for every micro-organism tested. At 60°C, we found that the shortest duration of effective induction heating was 1.5 minutes. This resulted in a 5-log reduction or higher for every micro-organism tested. Conclusion. Non-contact induction heating of a titanium disk is effective in reducing bacterial load in vitro. These promising results can be further explored as a new treatment modality for infections of metal orthopaedic implants. Cite this article: B. G. Pijls, I. M. J. G. Sanders, E. J. Kuijper, R. G. H. H. Nelissen. Non-contact electromagnetic induction heating for eradicating bacteria and yeasts on biomaterials and possible relevance to orthopaedic implant infections: In vitro findings. Bone Joint Res 2017;6:323–330. DOI: 10.1302/2046-3758.65.BJR-2016-0308.R1

The main problem of infected orthopaedic implants is that the presence of microorganisms in an organized biofilm making them difficult accessible for antibiotics. This biofilm consists of a complex community of microorganisms embedded in an extracellular matrix that forms on surfaces such as an implant. Non-contact induction heating uses pulsed electromagnetic fields to induce so-called ‘eddy currents’ within metal objects which causes them to heat up. This heat causes thermal damage to the bacterial biofilm hence killing the bacteria on the metal implant. The purpose of this study is to determine the effectiveness of induction heating on killing Staphylococcus epidermidis in a biofilm. S. epidermidis biofilms were grown on Titanium alloy (Ti6Al4V) coupons and subsequently were heated with a custom-built induction heater to temperatures of 60°C, 70°C, 80°C and 90°C for 3.5 minutes. Temperature was controlled with an infra-red thermal sensor and micro-controller. We also included two control conditions without induction heating: C1 without induction heating and C2 with chlorhexidine 0.5% in 70% alcohol without induction heating. Experiments were repeated 5 times. In the C1 group (no induction heating), 1.3 * 10(7) colony forming units (CFU)/cm(−2) of S. epidermidis were observed. For 60°C, 70C, 80 C and 90C, a 3.9-log reduction, 5.3-log reduction, 5.5-log reduction and 6.1-log reduction in CFU/cm(−2) were observed, respectively. For the C2 (chlorhexidine) there was a 6.7-log reduction CFU/cm(-2). We concluded that induction heating of Titanium coupons is effective in reducing bacterial load in vitro for S. epidermidis biofilms

Using bone decalcilied with 0.6 N hydrochloric acid as an inducing agent, the inductive capacity of different soft tissue sites was investigated. Muscle and fascia regularly permitted the induction of bone, while spleen, liver and kidney suppressed bone induction. Bone formation could be induced in these organs if living autologous fascia was implanted together with the inducing agent; while bone formation was inhibited when living autologous spleen tissue was implanted with the inducing agent to normally favourable sites. The administration of systemic heparin and the diphosphonate ethane-1-hydroxyl, 1-diphosphonic acid (EHDP) suppressed bone induction. It is suggested that for bone induction to occur in soft tissues, three conditions must be present: 1) an inducing agent; 2) an osteogenic precursor cell; and 3) an environment which is permissive to osteogenesis. The presence of osteogenic inhibitors in spleen, liver and kidney is postulated

1. Isografts of articular cartilage of young rats, with mucoproteins labelled with . 35. S, extracellular fibrous proteins labelled with . 3. H-glycine, and nuclei labelled with . 3. H-thymidine, were transplanted into the anterior chamber of the eye. 2. Thin split-thickness transplants of the cells of the gliding surface of immature articular cartilage induced the formation of fibrous tissue. 3. Thick transplants and subsurface slices of immature articular cartilage, containing germinal cells of the epiphysial cartilage, induced the formation of new bone consistently within 4 weeks. 4. Full-thickness transplants in articular cartilage from senile rats induced only the formation of fibrous tissue. 5. Slices of growing cartilage, devitalised by cryolysis, or extraction of acid-soluble proteins, produced scanty deposits of bone or cartilage, or both, but only infrequently and generally after a lag phase extending from six to twelve weeks. 6. Reduction in the amount of mucoprotein in the cartilage matrix by papain, and suppression of the resynthesis of tissue proteins by cortisone, retarded but did not prevent bone induction. 7. Bone induction is the product of a series of interactions between inducing cells and responding cells by intracellular and intercellular reactions too complex to characterise in physico-chemical terms at this time

Introduction: Osteomyelitis often causes functional impairment due to tissue destruction and the incidence of this condition appears to be increasing. Antimicrobial peptides (AP) are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Human beta-defensins (HBD) represent a major subclass of antimicrobial peptides and act as a first line defence through their broad spectrum of potent antimicrobial activity (1). The aim of the present in vitro and in vivo investigations was to study the expression and regulation of HBD-2 and -3 in the case of bacterial bone infection and to analyze the effects of immunosuppressive drugs on bone-derived AP-expression. Methods: Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (primary-, hFOB- and SAOS-2 cells) were assessed for the expression of HBD-2/-3 by RT-PCR, immunohistochemistry or ELISA. Regulation of HBD-2/-3 was studied after exposure to Staphylococcus aureus (SAS) or Pseudomonas aeruginosa (PAS), proinflammatory cytokines (IL-1, 10ng/ml) and immunosuppressive drugs (glucocorticoids, methotrexate) and was assayed by ELISA. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologues of HBD-2/-3 by real time RT-PCR and immunohistochemistry. Results: ELISA experiments demonstrated, that samples of infected bone produce higher levels of endogenous antibiotics such as HBD-2 when compared with samples of healthy bone. After exposure of osteoblasts to bacteria or proinflammatory cytokines a clear HBD-2/-3 induction was observed. Additional treatment with glucocorticoids or methotrexate prevented bacteria mediated HBD-2 induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional up-regulation of the murine HBD-homologues in bone after intra-osseous contamination of the tibia. Discussion: Our study firstly demonstrate that osteoblasts are able to produce anti-inflammatory peptides such as HBD-2 in vitro and in an animal model of staphylococcal osteomyelitis. We provide evidence for a new role of osteoblasts during infection of bone tissues, namely, the ability to produce antimicrobial peptides and modulating immune responses in inflammatory bone diseases. Immunosuppressive drugs such as glucocorticoids or methotrexate may increase the susceptibility to bone infection by decreasing AP-expression levels in case of microbial challenge. Novel approaches to management are required particularly in the era of multi-resistant bacterial strains. Current investigation will focus on the regulation of human β-Defensins in bone and may allow artificial amplification for prevention of bacterial bone infection in the future

1. Decalcified lyophilised rat bone matrix prepared by Urist's method acts as an inductor of cartilage and bone when implanted into animals of other species, namely mice, rabbits and gerbils. Induction in rabbits and gerbils was very much weaker than in the mouse. 2. The site of implantation affected the outcome; intramuscular implants induced cartilage and bone more strongly and regularly than subcutaneous or intraperitoneal implants. 3. Rabbit transitional epithelium, growing in cortisone-treated gerbils, caused bone induction, but in general, results with this species suggest that it responds poorly to bone-inducing stimuli. 4. Cortisone, used as an immunosuppressant, did not inhibit bone and cartilage induction

Extraskeletal bone formation can be induced in rodents by implantation of demineralised bone matrix and such implantation has been used to treat bone defects in man, but it is uncertain if induction or merely conduction occurs. We studied bone induction in primates by excising segments of the fibulae of adult squirrel monkeys, defatting and demineralising them before reimplanting them into the quadriceps of the same animal. As a control experiment, rat matrix was prepared in exactly the same way and implanted in rats. After six weeks the implants were harvested and either ashed and analysed for calcium content or prepared for histology. In the rats, the calcium content indicated that about 20% of the original matrix had been replaced by new bone. In the monkeys the calcium content was about the same as that in normal body fluid and no bone was seen in histological sections. This result casts doubt on the use of demineralised human bone matrix as a bone inductor, although it may function by other mechanisms

We investigated bone induction by bone morphogenetic protein in primates, comparing it with that seen in rodents. Twelve Millipore diffusion chambers containing 5 mg of semipurified bone morphogenetic protein were implanted into the dorsal muscles of 12 young, adult crab-eating monkeys (Macaca fascicularis) and were retrieved six weeks later. In six of nine unbroken chambers, new bone with haematopoietic marrow had been induced on the host-side surface of the filter. The incidence of trans-filter bone induction in the monkeys was almost equal to that observed in mice, and the new bone yield was approximately half as much as in mice. Our results show that bone morphogenetic protein can induce new bone formation in primates within six weeks, and support the hope that it will be useful as a substitute for bone graft in man

Introduction. Various anti-infective agents can be added to the surface of orthopaedic implants to actively kill bacteria and prevent infection. Silver (Ag) is a commonly used agent in various anti-infective applications. Silver disrupts bacterial membranes and binds to bacterial DNA and to the sulfhydryl groups of metabolic enzymes in the bacterial electron transport chain, thus inactivating bacterial replication and key metabolic processes. Recently we are implanting Silver coated megaprosthesis for the treatment of post-traumatic septic non unions/bone defects and for infected hip or knee prosthesis revision. We treat these complications utilizing a two steps procedure: 1° step: devices removal, resection, debridment and antibiotic spacer implantation; 2° step: spacer removal and megaprosthesis implantation. This technique produce a reactive pseudosynovial membrane, well known in traumatology (Masquelet technique), following the Chamber Induction Technique principles. This chamber creates the perfect environment in which implant the prosthesis with safety. We are nowadays investigating if this membrane could optimize the Silver antimicrobical effects reducing the Silver ions dispersion and reducing toxicity on the human body. Objectives. The aim of this study is to perform a review of the literature about Silver coated implants in Orthopaedics and Trauma and to analyze our cases treated with this implants in order to measure their efficacy and the ion dispersion in urine and blood. Methods. We performed a literature review using the universally validated search engines in the biomedical field: PubMed / Medline, Google Scholar, Scopus, EMBASE. The keywords used were: “Silver”, “Silver coating”, “Silver surface”, “were crossed with “Prosthesis”, “Megaprosthesis”, “Infection”, “Sepsis”, “Revision”. We also analized all our patients treated with Silver coated implants measuring Silver dose in blood and urine before implantation, 1 day after implantation and then after 15 days, 3,6,12,24,36 months. Results. The search led to 468 items, of these were considered only article in English with full text available. We found 1 in vitro study, 1 animal study and 2 human studies. The animal study showed a reduction in periprosthetic infection from 47% to 7%, 1 human study in Oncology application of megaprosthesis showed a reduction of septic complications from 17,6% to 5,9%. Te other human study demonstrated that Silver surface implants don't have toxicity cause the blood level of silver Ions were only 56,4 parts per billion. The analysis of our casuistry is giving good results with low level of Silver in the blood and urine, lower concentrations are observed in patients treated with the 2 steps-CIT technique. Conclusions. The use of silver-coated prosthesis can reduce the infection rate in the medium-long term with no toxicity for the patients. Further studies with longer term follow-up periods and larger numbers of patients are warranted in order to confirm these encouraging results most of all in the patients treated with the 2 steps procedure in order to better understand the role of the membrane and of the Chamber Induction Technique in Silver ions dispersions

Previous research has shown catabolic cell signalling induced by TNF-α and IL-1β within intervertebral (IVD) cells. However, these studies have investigated this in 2D monolayer cultures, and under hyper-physiological doses. Thus, we aim to revisit the catabolic responses of bovine IVD cells in vitro in 3D culture under increasing doses of TNF-α or IL-1β stimulation at three different timepoints.

Primary bovine nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated and expanded for two weeks. Subsequently, NP and AF cells were encapsulated in 1.2% alginate beads (4 × 106 cells/ml) and cultured for two weeks for phenotype recovery. Re-differentiated cells were stimulated with 0.1, 1 and 10 ng/ml TNF-α or with 0.01, 0.1 and 10 ng/ml IL-1β for one week. Beads were collected on the stimulation day (Day 0) and on Day 1 and 7 after stimulation.

A dose-dependent upregulation of catabolic markers was observed in both cell types after one day of TNF-α or IL-1β stimulation. 10 ng/ml TNF-α stimulation induced a significant upregulation (p<0.05) of ADAMTS4, MMP3 and MMP13 in AF cells after one day of stimulation. Similarly, MMP3 upregulation showed a strong trend (p=0.0643) in NP cells. However, no effects on expression were seen after seven days. In addition, no significant difference between treatments in COL2, COL1 and ACAN expression was observed, and cell viability was not reduced at any time point, regardless of the treatment.

We demonstrate a dose-dependent upregulation of catabolic markers in NP and AF cells under TNF-α or IL-1β stimulation, with a significant upregulation of ADAMTS4, MMP3 and MMP13 genes in AF cells after one day of treatment. Notably, after seven days of treatment, the dose-dependent effects were no longer observed possibly due to an adaptation mechanism of IVD cells to counter the metabolic shift.

Tumour volume reduction (i.e. response), assessed following induction chemotherapy, has been identified as a prognostic factor for localized embryonal rhabdomyosarcoma (RME) in the CWS studies. In combination with other risk factors, it has been used to stratify secondary local and systemic treatment. It is however unclear whether the poor outcome of non-responders is due to insufficient local and/or systemic post-induction treatment. We analyzed post-induction therapy of RME-patients < 21 years with unresected localized tumours (IRS-III) and poor response (NR, i.e. < 33% tumour volume reduction) treated 1980–2005 in five consecutive CWS-trials. The NR were reviewed and subclassified (Objective Response (OR; i.e.< 33%–0%) vs. Stable Disease/Progression (PD; i.e. no reduction)). From 758 IRS-III RME-patients, 59 were NR (n=34 OR, n=25 PD). Induction for NR included dactinomycin, vincristine, alkylators ± anthracyclines in all patients. There were no significant differences in comparison of the control group and NR with regard to age, size, TN-classification, apart from site (p=0.04), and no differences regarding these parameters between OR and PD. Twenty-four NR received continued induction chemotherapy, n=32 other combinations, and n=3 no further chemotherapy following response assessment. Four patients were treated with additional high-dose chemotherapy. Fourty-two NR were irradiated with a median dose of 48Gy (control group: 45Gy). In 20 NR, the tumours were completely resected. As of 9/2008, with a median follow-up of 4.5 years (range: 0.9–12.1) for NR survivors, 34 NR are alive in CR. Reasons for the 25 deaths were: local/combined failure (n=21), systemic failure (n=1), and other reasons (n= 3). 5-yrs-OS was 71±4% for the control group, 78±15% for OR, but only 43±15% for PD (p< 0.01). Response is an important surrogate marker of outcome, but per se associated with a poor prognosis only in tumours without any volume regression to induction chemotherapy. Ineffective local control drives mortality in these patients

Osteoarthritis (OA) is characterised by progressive erosion of articular cartilage, which, once started, cannot be halted. The breakdown of cartilage is mediated by proteases, including MMP-3 and -13. These may initially be derived from the synovium but are also produced by OA chondrocytes, particularly in later stages of the disease. In normal articular chondrocytes, the proteases are not expressed and it has previously been shown that this is due, in part, to silencing by epigenetic mechanisms, in particular DNA methylation at so-called CpG sites (Arthritis & Rheumatism 52:3110–24). In OA, chondrocytes increasingly produce the enzymes and stably transmit the abnormal gene expression to daughter cells. This aberrant expression has been shown to be associated with an epigenetic “un-silencing” via demethylation of specific CpG sites within the promoter regions. Why and how this demethylation takes place is not known. The pro-inflammatory cytokine IL-1beta is of potential importance in OA, where temporary synovitis could provide the cytokine. Moreover, it is well established that IL-1beta upregulates MMPs in chondrocyte monolayer cultures. We investigated whether the IL-1 mediated induction of MMPs was associated with DNA demethylation. Control chondrocytes were isolated from non-OA articular cartilage, obtained with ethical permission from patients with a femoral neck fracture, and expanded in monolayer culture. The cells from each patient were divided into pre-culture control, no-treatment control culture and IL-1 treated culture. When confluent, simultaneous RNA and DNA extraction was carried out. mRNA expression was analysed by RT-PCR and the methylation status of specific CpG sites within the promoters of MMP-3, -13, and IL-1â was determined in the same samples, using methylation-sensitive restriction enzymes and PCR. The pre-culture controls expressed type II collagen and low levels of MMP-3, but not MMP-13 nor IL-1beta. All IL-1 treated samples expressed high levels of MMP-3, -13, and, surprisingly, IL-1beta itself. As predicted, the large increases in MMP-3 and IL-1beta were associated with some loss of methylation at specific CpG sites in the promoter of these mediators with the strongest correlation between IL-1beta expression and promoter demethylation. IL-1beta thus induced its own expression, which was associated with loss of DNA methylation at one specific CpG site in the IL-1 promoter. If these in vitro results have relevance for the in vivo situation, then these findings suggest the following mechanisms for OA progression: An initial inflammatory episode in the synovium could induce IL-1beta in surface chondrocytes. Since this induction is associated with loss of DNA methylation, IL-1beta is now part of the expression repertoire of these chondrocytes and this abnormal expression is stably transmitted to daughter cells. IL-1 then could diffuse deeper into the cartilage to induce its own expression in adjacent chondrocytes, thus providing a continuous supply of IL-1beta even after synovial inflammation had abated. This may explain the unremitting progression of OA

Extracorporeal shock-wave (ESW) treatment hasbeen shown to be effective in promoting the healing of fractures. We aimed to determine whether ESW could enhance the growth of bone-marrow osteoprogenitor cells. We applied ESW to the left femur of rats 10 mm above the knee at 0.16 mJ/mm. 2. in a range of between 250 and 2000 impulses. Bone-marrow cells were harvested after ESW for one day and subjected to assessment of colony-forming unit (CFU) granulocytes, monocytes, erythocytes, megakaryocytes (CFU-Mix), CFU-stromal cells (CFU-S) and CFU-osteoprogenitors (CFU-O). We found that the mean value for the CFU-O colonies after treatment with 500 impulses of ESW was 168.2 CFU-O/well (. sem. 11.3) compared with 88.2 CFU-O/well (. sem. 7.2) in the control group. By contrast, ESW treatment did not affect haematopoiesis as shown by the CFU-Mix (p = 0.557). Treatment with 250 and 500 impulses promoted CFU-O, but not CFU-Mix formations whereas treatment with more than 750 impulses had an inhibiting effect. Treatment with 500 impulses also enhanced the activity of bone alkaline phosphatase in the subculture of CFU-O (p< 0.01), indicating a selective promotion of growth of osteoprogenitor cells. Similarly, formation of bone nodules in the long-term culture of bone-marrow osteoprogenitor cells was also significantly enhanced by ESW treatment with 500 impulses. The mean production of TGF-β1 was 610 pg/ml (. sem. 84.6) in culture supernatants from ESW-treated rats compared with 283 pg/ml (. sem. 36.8) in the control group. Our findings suggest that optimal treatment with ESW could enhance rat bone-marrow stromal growth and differentiation towards osteoprogenitors presumably by induction of TGF-β1

Epigenetic DNA de-methylation at specific CpG promoter sites is associated with abnormal synthesis of matrix-degrading enzymes in human osteoarthritis (Arthritis Rheum 52:3110–24), but the mechanisms that trigger or cause loss of DNA methylation are not known. Since inflammatory cytokines are known to induce abnormal gene expression in cultured chondrocytes, we wanted to know whether this induction also involved loss of DNA methylation. If so, the abnormal gene expression would be permanent and transmitted to daughter cells rather than a simple up-regulation. To test this hypothesis, we selected IL-1b as the abnormally expressed gene. Healthy chondrocytes, harvested from human femoral head cartilage following a fracture, were divided into five groups: non-culture; control culture; culture with the de-methylating agent 5-aza-deoxycyti-dine (5-aza-dC); culture with the inflammatory cytokine IL-1b; or with TNF-a/oncostatin M. Total RNA and genomic DNA were extracted at confluency, relative mRNA expression of IL-1b was quantified by Syb-rGreen-based real-time PCR, and a method for quantifying the percent of cells with DNA methylation at a specific CpG site was developed (Epigenetics 2: 86–95). The methylation status of 16 CpG sites in the promoter of IL-1b was determined by the bisulfite modification method. The two CpG sites important for the epigenetic regulation of IL-1b were at -247bp and -290bp, the latter was selected to quantify DNA methylation. 5-aza-dC halved DNA methylation, which resulted in 4–8 fold increases in IL-1b expression; showing that DNA de-methylation per se increases gene expression. However, far greater effects were seen with the inflammatory cytokines. IL-1b increased its own expression 50–100 fold, whereas TNF-a/OSM increased IL-1b expression 500–1000 fold. DNA methylation varied inversely, IL-1b reducing methylation to ~15% and TNF-a/OSM abolishing DNA methylation almost completely. This is the first demonstration that inflammatory cytokines have the capacity to cause loss of DNA methylation. We also confirmed previous work that IL-1b induces its own expression in healthy chondrocytes, thus setting up a dangerous positive feed-back mechanism. If true in vivo, both the auto-induction and the heritable expression of IL-1b by a growing number of chondrocytes could explain the unrelenting progression of osteoarthritis

Experiments have been performed on rabbits and sheep which demonstrate that pure carbon, in a flexible and filamentous form of great strength, can be used successfully to induce the formation of new tendons. A concept fundamentally different from that underlying the use of other artificial tendon replacements is involved, in which rapidly developing tendon-like tissue is induced to form around the implant. This gradually takes over the function of the implant. The early results in rabbits and the late results in sheep suggest that filamentous carbon may have a place in the replacement of the calcaneal tendon and the collateral ligaments of the knee.

Introduction: The highest goal after meniscus damage is the preservation of the meniscus, which is often not possible due to the bad healing of meniscus lesions in the avascular zone. Therefore, the goal of our investigations was the analysis of expression of different angiogenic factors, growth hormones and cytokines in human meniscus cells (fibrochondrocytes). The mutual influence of the fibrochondrocytes by endothelial cell cocultures was analyzed, in order to examine the molecular bases of the healing of meniscus tears in vascularized zones more exactly. For this purpose, commercially available HUVEC [human umbilical vein endothelial cells] were used as well established and stable endothelial cell model.

Material and Methods: Meniscal fibrochondrocytes were expanded in DMEM medium enriched with antibiotics and 10 % FCS. Cocultures of mensical cells and HUVEC were incubated in transwells over four and twelve days, separated by a semipermeable membrane. The expression of Angiopoietin-1, Angiopoietin-2, End-ostatin, VEGF, SMAD-4, Thrombospondin-1, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, IL-1β, iNOS, MMP-1, MMP-3, MMP-13, collagen-I, -II, -III, -VI, X, and -XVIII were examined by RT-PCR and immunhistochemistry in fibrochondrocytes in the comparison to cultures without endothelial coculture. A proliferation assay was used to investigate the mitotic activity in the coculture compared to the control culture after 4 and 12 days.

Results: In presence of HUVEC, meniscal fibrochon-drocytes expressed the following factors at rates comparable to cells w/o HUVECS: Angiopoietin-1, Angiopoietin-2, VEGF, SMAD-4, Aggrecan, Biglycan, Fibronectin, Vimentin, Connexin-43, iNOS, MMP-1, MMP-3, MMP-13, Thrombostatin-1, collagen-I, -II, -III, -VI, X, and -XVIII. In contrast, expression of end-ostatin (5.1-fold ± 1.2, p< 0.01) and IL-1β (10.3-fold ± 2.3, p< 0.003) were expressed significantly higher in the coculture when compared to the individual cell cultures. The proliferation rate of HUVEC was significantly decreased in coculture when compared to controls: 22 % after 7 days and 35 % after 14 days (p< 0.001).

Discussion/ Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint. We could show that coculture of meniscus cells with endothelial cells revealed an increased expression of the anti-angiogenetic factor endostatin and the pro-inflammatory IL-1β. This suggests that meniscus cells are trying to inhibit proliferation of endothelial cells in their neigbourhood, which implicates huge problems in the research field of neoangiogenisis and tissue engineering in meniscus tissue for new healing methods after meniscus trauma.