Objectives

Rotator cuff tears are among the most common and debilitating upper extremity injuries. Chronic cuff tears result in atrophy and an infiltration of fat into the muscle, a condition commonly referred to as ‘fatty degeneration’. While stem cell therapies hold promise for the treatment of cuff tears, a suitable immunodeficient animal model that could be used to study human or other xenograft-based therapies for the treatment of rotator cuff injuries had not previously been identified.

Introduction. Modern processing techniques in bone banking are thought to decrease the presence of allogenic material in bone. This project was performed to observe any changes in peripheral blood lymphocyte subsets in response to allografted bone used in revision hip replacement. Methods. 87 patients were entered into this prospective study and grouped according to whether impaction allograft was used or not. Samples were collected pre-operatively and at set time intervals up to one year post-operatively. Using flow cytometry, analysis of venous blood allowed counts of the following cells: Helper T-lymphocytes, cytotoxic T-lymphocytes, memory T-lymphocytes, naïve T-lymphocytes, Natural Killer cells and B-lymphocytes. Results. All patients had a successful outcome at one year. 50 patients with radiologically defined host-graft union were compared with 37 patients who did not receive an allograft. Pre-operatively, a significant difference (p=0.03) was found between the groups of patients with respect to Natural Killer cells but other subsets showed no significant difference. Post-operatively, the significant difference between Natural Killer cells resolved. T-helper lymphocytes, cytotoxic lymphocytes, memory T-lymphocytes and naïve T-lymphocytes in both groups showed decreases in values immediately post-surgery, recovering to normal values within 6 weeks post-surgery. The allograft group showed significant increases from baseline in cytotoxic T-lymphocytes at 6 months (p<0.01) and memory T-lymphocytes one year post-operatively (p=0.04). B-lymphocyte numbers did not alter significantly from baseline. Discussion. Cytotoxic T-lymphocytes recognise HLA-class I molecules which are present on all nucleated cells and have been implicated in having a role in osteoclast regulation. Memory T-lymphocytes are produced after a naïve T-lymphocyte is exposed to an antigen. The observed increases of these subsets were not observed in the non-grafting group suggesting the allografted bone had elicited an immunological response. At 12 months all grafts appeared radiologically stable and the immunological response may have been beneficial to the outcome

Introduction: Freezing and storage of fresh frozen femoral heads destined for use in revision hip arthroplasty is thought to result in graft cell death. Washing of the graft following the morsellisation process also removes a large proportion of the marrow content of the allograft. However, the immunological load of the impaction allografting process remains unknown. The aim of this study was to investigate the immune response by observing any changes in peripheral blood lymphocyte subsets in response to allografted bone used in revision hip replacement. Methods: 87 patients were entered into this prospective study and grouped according to whether impaction allograft was used or not. Venous blood samples were collected pre-operatively and at set time intervals up to one year post-operatively. Using flow cytometry, analysis of venous blood allowed counts of the following cells: Helper T-lymphocytes, cytotoxic T-lymphocytes, memory T-lymphocytes, naïve T-lymphocytes, Natural Killer cells and B-lymphocytes. Results: All patients had a successful outcome at one year. 50 patients with radiologically defined host-graft union were compared with 37 patients who did not receive allograft. Pre-operatively, a significant difference (p=0.03) was found between the groups of patients with respect to Natural Killer cells but other subsets showed no significant difference. Post-operatively the significant difference between Natural Killer cells resolved. T-helper lymphocytes, cytotoxic lymphocytes, memory T-lymphocytes and naïve T-lymphocytes in both groups showed decreases in values immediately post surgery, recovering to normal values within 6 weeks post-surgery. The allograft group showed significant increases from baseline in cytotoxic T-lymphocytes at 6 months (p< 0.01) and memory T-lymphocytes one year postoperatively (p=0.04). B-lymphocyte numbers did not alter significantly from baseline. Conclusion: Cytotoxic T-lymphocytes recognise HLA-class I molecules which are present on all nucleated cells and have been implicated in having a role in osteoclast regulation. Memory T-lymphocytes are produced after a naïve T-lymphocyte is exposed to an antigen. The observed increases of these subsets were not observed in the non-grafting group suggesting the allografted bone had elicited an immunological response. At 12 months all grafts appeared radiologically stable and the immunological response may have been beneficial to the outcome

Introduction. It is well-known that wear debris generated by metal-on-metal hip replacements leads to aseptic loosening. This process starts in the local tissue where an inflammatory reaction is induced, followed by an periprosthetic osteolysis. MOM bearings generate particles as well as ions. The influence of both in human bodies is still the subject of debate. For instance hypersensitivity and high blood metal ion levels are under discussion for systemic reactions or pseudotumors around the hip replacement as a local reaction. The exact biopathologic mechanism is still unknown. The aim of this study was to investigate the impact of local injected metal ions and metal particles. Material and Methods. We used an established murine inflammation model with Balb/c mice and generated three groups. Group PBS (control group, n=10) got an injection of 50µl 0.1 vol% PBS-suspension, Group MI (Metal-ion, n=10) got an injection of 50µl metal ion suspension at a concentration of 200µg/l and Group MP (Metal-particles, n=10) got an injection of 50µl 0.1 vol% metal particle suspension each in the left knee. After incubation for 7 days the mice were euthanized and the extraction of the left knee ensued. Followed by immunhistochemical treatment with markers of inflammation that implied TNFα, IL-6, IL-1β, CD 45, CD 68, CD 3, we counted the positive cells in the synovial layer in the left knees by light microscopy, subdivided into visual fields 200× magnified. The statistical analysis was done with Kruskal-Wallis test and a post hoc Bonferroni correction. Results. The Group with metal particles showed significantly elevated inflammatory markers (TNFα, IL-6, IL-1β, CD 68, CD 45) compared to all other groups. Interestingly, CD 3 as a marker for T-lymphocytes showed no increased levels in all groups. The metal ion group showed significant elevated CD 45 expressions compared to the control group. Conclusion. The results clearly demonstrate that especially metal wear particles lead to an intensive inflammatory reaction. The tissue formations in the metal particle group show an osseous destructive behavior in previously demonstrated results, comparable to pseudotumors. But, in this study, the expression of the immunohistological markers CD 3, CD 45 and CD 68 indicate that the tissue consists of leucocytes and macrophages, whereas lymphocytes could not be detected. This might be due to an acute inflammatory reaction, whereas the adaptive immune response by T-lymphocytes seems not (yet) to be activated. Overall it must be stated that solid metal wear particles are responsible for local inflammatory reactions, whereas it is still unknown whether wear particles corrode in vivo and release a potentially high level of locally toxic metal ions

The T-lymphocyte secreted pro-inflammatory cytokine, interleukin-17F (IL-17F), was found to be a key mediator in the cellular response of the immune system in the early phase of fracture repair but its intracellular signaling processes are currently not known in osteoblasts. The objective of this study was to identify the signaling proteins and crucial gene targets involved in osteoblast activation via IL-17F. It was hypothesised that IL-17F stimulated osteoblast maturation through a novel GSK3beta / beta-catenin independent pathway. Mouse pre-osteoblast cell line (MC3T3-E1) was used for IL-17F or Wnt3a treatment. Desired proteins were detected using western blot analysis (antibodies: Phospho-GSK-3beta (Tyr 216), Phospho-GSK-3beta (Ser9), Runx2/cbfa1, TRAF6, Act1, p-ERK2, p-JNK and p-MAPK, C/EBP-beta and & delta). Gene-specific siRNAs of mouse IL-17Ra, IL-17Rc and a non-targeting siRNA (control) were utilised. MC3T3-E1 were transfected with IL-17Ra, IL-17Rc or Negative Control and treated with IL-17F. Chromatin Immunoprecipitation (ChIP-qPCR) was used to evaluate the mouse Runx2 P1 promoter region. IL-17F increased expression of Col1, BSP, Runx2/cbfa1 and osteocalcin in MC3T3-E1 cells. Western blot analysis confirmed expression of known Wnt signaling proteins TRAF6, Act1, p-ERK2, p-JNK and p-MAPK in both IL-17F and Wnt3a treated cultures, including up-regulation of Runx2/cbfa1, a key transcription factor associated with osteoblast differentiation. IL-17F up-regulation of Runx2/cbfa1 appears independent of the Wnt/beta-catenin pathway as phosphorylated GSK-3beta at the Ser9 site was not detected with IL-17F treatment. Despite this, IL-17F treatment still increased expression of Runx2/cbfa1 downstream, lending evidence for a GSK3beta/beta-catenin independent manner of IL-17F stimulated osteogenesis. While IL-17F and Wnt3a both induced expression of C/EBP-delta, only IL-17F treatment induced expression of C/EBP-beta, an upstream transcription factor of Runx2/cbfa1. Further, siRNA knock down of the IL-17 receptors directly decreased Act1, C/EBP-beta and Runx2/cfba1 expression. By ChIP analysis, IL-17F was shown to upregulate C/EBP-beta expression and stimulated its binding to the P1 Promoter of the Runx2/cbfa1 gene. The C/EBP-beta transcription factor was shown to be a key regulator of early osteogenesis. C/EBP-beta up-regulates Runx2/cbfa1 expression by directly binding to the Runx2/cbfa1 P1 promoter in osteoblasts. C/EBP-beta was activated in the osteoblast by IL-17F but not by Wnt3a adding further support to a novel GSK3beta/beta-catenin independent pathway. Our data shows that IL-17F, a cytokine secreted by T-lymphocytes, stimulates osteoblast maturation through a novel GSK3beta/beta-catenin independent pathway and reveals a crucial interaction between C/EBP-beta and the Runx2/cbfa1 P1 promoter not previously been shown in osteogenesis signaling further

Aims

Hip resurfacing remains a potentially valuable surgical procedure for appropriately-selected patients with optimised implant choices. However, concern regarding high early failure rates continues to undermine confidence in use. A large contributor to failure is adverse local tissue reactions around metal-on-metal (MoM) bearing surfaces. Such phenomena have been well-explored around MoM total hip arthroplasties, but comparable data in equivalent hip resurfacing procedures is lacking. In order to define genetic predisposition, we performed a case-control study investigating the role of human leucocyte antigen (HLA) genotype in the development of pseudotumours around MoM hip resurfacings.

Summary Statement. Cross-talk between cells from immune and bone system might play a role in molecular regulation of subchondral bone sclerosis in osteoarthritis. Macrophages, B-lymphocytes and tartrate-resistant acid phosphatase activity are specifically increased in sclerotic subchondral bone of patients with knee osteoarthritis. Background. Recent investigations have provided substantial evidence that distinct molecular and morphological changes in subchondral bone tissue, most notably sclerosis, play an active and important role in the pathogenesis of OA. The cellular and molecular regulation of this pathological process remains poorly understood. Here, we investigated whether osteoimmunology, the reciprocal signaling between cells from the immune and bone system, is involved in OA subchondral bone sclerosis. Patients & Methods. Tibial plateaus and informed consent were obtained from patients undergoing total knee arthroplasty due to end-stage OA. Subchondral bone mineralization distribution was analyzed using computed tomography osteoabsoptiometry (CT-OAM) and standardised cryosections of low (non-sclerotic) and high (sclerotic) bone mineralization were prepared (n=18 each). Cartilage degeneration was graded in Safranin-O-stained sections using the Mankin scoring system. The presence of T-lymphocytes, B-cells and macrophages was assessed using immunohistochemical staining of their respective surface markers CD3, CD20 and CD68. Osteoclast activity was visualised by staining of the enzyme marker tartrate-resistant acid phosphatase (TRAP). Cellular characterization of ex vivo subchondral bone outgrowth cultures was performed using alkaline phosphatase (ALP), TRAP staining. Correlation between histological parameters was assessed using Spearman's rank correlation. Statistical differences were calculated using Wilcoxon signed rank test or paired t-test, where appropriate. Results. CT-OAM revealed a heterogeneous distribution of subchondral bone mineralization in OA tibial plateaus, displaying focal areas of sclerosis that overlapped macroscopically with areas of cartilage damage. These data were confirmed at the histological level by a strong correlation between Mankin score and grade of sclerosis (r=0.7, p<0.001). Immunohistochemistry showed that CD20. +. , but not CD3. +. , lymphocytes and CD68. +. mononuclear (macrophage) and multinucleated (osteoclast) cells were present in subchondral marrow spaces. Notably, the number of CD20. +. lymphocytes and CD68. +. cells was significantly (p<0.05) increased in sclerotic subchondral bone. Enhanced osteoclast activity was confirmed by a significantly increased (p<0.05) number of multinucleated and mononuclear TRAP. +. cells in sclerotic bone. Finally, the number of CD68. +. cells was strongly correlated (p<0.001) with Mankin score (r=0.7), grade of sclerosis (r=0.8), CD20. +. lymphocytes (r=0.8), and TRAP-positive cells (r=0.9). Outgrowth cultures of subchondral bone showed cells of different morphologies including fibroblast-shaped osteoblasts and macrophage-like cells. Expression of ALP was detected in the prior, while TRAP expression was evident in the latter. Corresponding with histological analyses, the number of TRAP. +. cells was increased in ex vivo outgrowth cultures of sclerotic compared to non-sclerotic subchondral bone. Conclusions. Together, our data suggest that osteoimmunological mechanisms, specifically the interaction of CD68. +. macrophages with bone-resident cells, play a - previously unknown - role in regulating subchondral bone sclerosis in progressive OA. Targeting osteoimmunology might hold potential as a disease-modifying treatment for OA

Objectives: To evaluate the outcome of a cementless, low carbon MOM (metal on metal) THR with a 28mm ball-head (PPF STRATEC-SYNTHES, since 2002 BIOMET) and the concentration of metal ions within the intra-articular fluid. Methods: 173 unilateral MOM THR’s performed in 1995 were investigated. Average follow up time was 12 yrs (range 9–14yrs). During the study period there were, 11(6.4%) deaths, 2 (1.2%) lost to F.U. and 5 (2.9%) refused follow up x-rays because of lack of symptoms, and a self perceived perception that these were not necessary. 41 effusions in part of this group could be analysed for levels of Co, Cr and Ni. Results: 112 THR’s (64.7%) were completely symptom free and did not show any signs of osteolysis. 7 THR’s had revision surgery, unrelated to metallosis (1 cup loosening, 1 early infection and 5 late infections that could be attributed to a large amount of necrotic tissue within the joint space). 36 hips (20.8%) showed over time, progressive signs of a metallosis. Clinically none or moderate complaints in the groin were reported, and massive effusions of up to 90ml were present. The osteolysis began in the majority of these cases in the periosteal region of the acetabulum and the trochanter. No loosening of the implants was seen. A quarter of these implants had late dislocations. The CRP was up to three times above normal levels. Only 26 patients (15.0%) could be convinced to undergo further surgery (synovectomy, exchange of head and liner and bone grafting as required). Typical histology showed massive necrosis within the joint and a peri-vascular infiltrate of CD-3 pos. activated T-lymphocytes and L26 pos. B-lymphocytes. These findings have been recently published as ALVAL. In the 41 joint effusions, the mean level of Co was 595.6 μg/l (max 4802.2), Cr 481.1 μg/l (max 4602.9) and Ni 3.7 μg/l (max 14.4). The serum ion levels were up to four times the maximal permitted level (Co 3 μg/l, Cr 3 μg/l, Ni 3 μg/l). Conclusion: High levels of toxic metal ions in the joint space over time can lead to a severe allergic reaction and tissue necrosis. The current literature reports, that almost all MOM bearings show slightly elevated serum metal ion levels, and therefore a much higher concentration must be calculated within the joint space. In our experience, because of the serious consequences and unpredictable onset of metallosis, we no longer use MOM articulations

Background: Increased use of metal on metal (MOM) hip replacements has stimulated interest in immunological effects of chronic CoCr elevation. Unlike metal-on-polyethelylene, MOM implants are associated with a perivascular infiltrate of lymphocytes & plasma cells. This may be the mode of failure of MOM implants. A reduction in CD8+ T lymphocyte counts associated with MOM implants has previously been described. CoCr therefore seems to affect the adaptive immune response even though it is not a proteinous antigen. We therefore analyzed the effects of CoCr particles on T cells & B cells. We also analyzed it effects on dendritic cells, which are the key antigen presenting cells to T helper cells. Methods: CoCr nano particles were produced by repetitive short spark discharges between electrodes of prosthetic CoCr alloy. Electron micrography & BET both confirmed nanoparticle size. Dendritic cells (DCs) were harvested from mouse bone marrow & cultured in medium supplemented with GM-CSF for 6 days, generating DCs typically 80–90% CD11c+. These were incubated with CoCr in concentrations of 25, 10 & 2.5 μg/ml, for 24 hours, or lipopolysaccharide 1 μg/ml as a positive control. Following incubation, activation status of CD11c+ DCs was characterized by MHC Class II, CD40, CD80 & CD86 expression by FACS analysis. T-Lymphocytes were harvested from mouse lymph nodes & cultured in medium without phenol red. These were incubated at 5 ×105 cells/well with either CoCr, conA (positive control) or CoCr + conA & repeated using 2.5 ×105 cells/well. Other positive controls (CD3 & CD 28) were studied in repeating the experiment. At 48 hours Almar Blue was added & further incubation for 24 hrs. Light absorbance at 570nm & 600nm was then used to determine T cell proliferation. B-Lymphocytes were harvested from the lymph nodes of mice which were only able to mount a B-cell reaction to Hen egg Lysozyme (HEL). These were incubated with medium with CoCr, HEL (positive control) or CoCr+ HEL. The concentration of the CoCr was varied between 25, 10 & 2.5 μg/ml. FACS analysis for markers of B cell regulation was performed after 48 hours incubation. Results: CoCr did not significantly increase CD 40 expression on DCs, although such expression was increased significantly by lipopolysaccaride CoCr did not significantly up or down regulate B cells as compared to the effects of HEL. CoCr did inhibit proliferation of T-cells & this was more pronounced where the ratio of CoCr/cell density was higher. Conclusion: Both dendritic & B cells are unaffected by CoCr in vitro. However, CoCr inhibited T cell proliferation. This demonstrates the observed reduction in CD + T cells are probably due to a direct effect of CoCr, & not mediated through another cell type. The perivascular response to MOM implants on the other hand probably requires cell interaction in an in vivo environment

Aims

Metal allergy in knee arthroplasty patients is a controversial topic. We aimed to conduct a scoping review to clarify the management of metal allergy in primary and revision total knee arthroplasty (TKA).

A balanced inflammatory response is important for successful fracture healing. The response of osteoporotic fracture healing is deranged and an altered inflammatory response can be one underlying cause. The objectives of this review were to compare the inflammatory responses between normal and osteoporotic fractures and to examine the potential effects on different healing outcomes. A systematic literature search was conducted with relevant keywords in PubMed, Embase, and Web of Science independently. Original preclinical studies and clinical studies involving the investigation of inflammatory response in fracture healing in ovariectomized (OVX) animals or osteoporotic/elderly patients with available full text and written in English were included. In total, 14 articles were selected. Various inflammatory factors were reported; of those tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 are two commonly studied markers. Preclinical studies showed that OVX animals generally demonstrated higher systemic inflammatory response and poorer healing outcomes compared to normal controls (SHAM). However, it is inconclusive if the local inflammatory response is higher or lower in OVX animals. As for clinical studies, they mainly examine the temporal changes of the inflammatory stage or perform comparison between osteoporotic/fragility fracture patients and normal subjects without fracture. Our review of these studies emphasizes the lack of understanding that inflammation plays in the altered fracture healing response of osteoporotic/elderly patients. Taken together, it is clear that additional studies, preclinical and clinical, are required to dissect the regulatory role of inflammatory response in osteoporotic fracture healing.

Cite this article: Bone Joint Res 2020;9(7):368–385.

Aims

This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss.

Objectives

The diagnosis of periprosthetic joint infection (PJI) is difficult and requires a battery of tests and clinical findings. The purpose of this review is to summarize all current evidence for common and new serum biomarkers utilized in the diagnosis of PJI.

Objectives

T-cells are considered to play an important role in the inflammatory response causing arthroplasty failure. The study objectives were to investigate the composition and distribution of CD4+ T-cell phenotypes in the peripheral blood (PB) and synovial fluid (SF) of patients undergoing revision surgery for failed metal-on-metal (MoM) and metal-on-polyethylene (MoP) hip arthroplasties, and in patients awaiting total hip arthroplasty.