Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes. Cite this article: Bone Joint Res 2024;13(9):462–473

Introduction: Metal-on-metal (MoM) articulations for THA are used successfully from CoCr-alloys. Low or high carbon hydride metals contain less or more than 0.2% carbon in the alloy. The systems show encouraging clinical results and lower rates of aseptic loosening in midterm results. Hypersensitivity reactions to high carbide MoM articulations were reported. The immune response is characterized by a perivascular T-/B-lymphocyte infiltration of the capsular tissue around the hip replacement. The present study examines if lymphocytic reactions are present in low carbide MoM THA and if distinct cytokines are released to joint fluids. Retrieval tissues from 28 patients were used. Joint fluids were aspirated at the time of surgery. Materials and Methods: Tissues were collected from 25 patients undergoing 26 aseptic revisions of MoM THA (CoCrMo, Sikomet. ®. , Plus Orthopaedics). The patients had following symptoms: Hip and femoral pain; recurrent dislocation and clicking noises. 8 patients had osteolysis, 12 patients showed a metallosis. The peripros-thetic tissues were examined by standard histology and immunohistochemistry. Joint fluids were frozen at the time of surgery. The control groups were patients with osteoarthritis of the hip (n=10), revisions from Al2O3-UHMWPE articulations (n=6), revisions of MoM with hypersensitivity reaction (n=18), and MoM without hypersensitivity reactions (n=8). The fluids were analyzed for various Interleukins, Il-1 receptor antagonist, G-CSF, GM-CSF, IFN gamma, MIP-1ß, and TNF-α. Results: 18 out of 26 cases showed diffuse and follicular lymphocyte infiltrations in the revision tissues. Perivas-cular T- and B-lymphocytes and few macrophages were also seen. In low and excessive metallosis no lymphocytes were observed. The tissue response in low carbide MoM is similar to high carbide MoM. Analysis of the cytokine profile in the joint fluids showed markers of osteoclast activation (Il-6 and −10) in all MoM articulations. TNF-α increase was increased in all loosening groups but was further increased in MoM. Il-5, IFN gamma, MIP-1ß, and GM-CSF were increased in all fluids from loosening cases but were further increased in MoM with lymphocyte activation. Discussion: Activation of lymphocytes in failed MoM THA’s is not necessarily related to high carbide MoM. Hypersensitivity also occurred in low carbide MoM THA. The cytokine profiles in the joint fluids showed distinct characteristics. Il-6 and Il-10, markers of osteo-clast activation, were elevated in all cases of bone loss and osteolysis. The increase in TNF-α may account for a regulation of the OPG/RANKL system TNF-a which can induce osteolysis. The elevated levels of Il-5, IFN gamma, MIP-1ß, and GM-CSF in MoM failures with hypersensitivity represent markers of chemotaxis and lymphocyte activation may account for index markers of hypersensitivity reaction

Introduction: Symptomatic abnormal soft-tissue masses relating to the hip joint, such as those described as pseudotumours, are being increasingly reported following metal-on-metal hip resurfacing arthroplasty (MoMHRA). These were found to be locally destructive, requiring revision surgery in a high proportion (75%) of patients. Lymphocyte infiltrations seen in pseudotumours were similar to aseptic lymphocyte vascular associated lesion (ALVAL), which is thought to represent a T-lymphocyte-mediated delayed type hypersensitivity. Therefore, a delayed hypersensitivity reaction to nickel (Ni), chromium (Cr) or cobalt (Co) has been suggested to play a role in pseudotumour aetiology. In patients with bilateral MoMHRA who presented with symptoms on one side, subsequent scans have demonstrated pseudotumours both on the symptomatic and asymptomatic side. Thus, there are concerns that there may be an appreciable number of asymptomatic pseudotumours that surgeons are unaware of and these may eventually become symptomatic. Aim: The aims of this study were:. to determine the prevalence of asymptomatic pseudotumours after MoMHRA; and. to measure Co and Cr ion levels as well as lymphocyte proliferation responses to Ni, Co and Cr (the principal elements in the CoCr alloy used in MoMHRA) in MoMHRA patients with and without asymptomatic pseudotumours. Methods: A total of 201 MoMHRA implanted hips in 158 patients (97 male, 61 female) with a mean age of 56 years (range 33–73 years) were evaluated. The mean follow-up was 61 months (range 13–88 months). Resurfacing devices implanted included 128 Birmingham Hip Resurfacing, 66 Conserve Plus and seven ReCap. The control groups included additional 20 patients, 10 male and 10 female (a mean age 68 years, range 57–80 years) with metal-on-polyethylene total hip arthroplasty and a further 22 age-matched patients (a mean age 55 years) without any metal implants. Ultrasound was used as the initial imaging modality and MRI was used to assess the extent of the identified masses. Patients with a soft-tissue mass had ultrasound-guided aspiration or core biopsy performed. Venous blood samples were collected in all patients for serum cobalt and chromium ion levels analysis using Inductively-Coupled Plasma Mass Spectrometer and lymphocyte transformation tests (LTT). The Oxford Hip Score (OHS) was used to measure the functional outcomes of patients. Acetabular component abduction angle was measured from standardised anteroposterior pelvis radiographs. Results: Prevalence – Pseudotumours were found in 7 patients (6 female and 1 male). The overall prevalence of asymptomatic pseudotumours was 4%, with a relatively very high (30%) prevalence in females with bilateral implants. Histological examinations showed extensive necrosis of connective tissue, in which there were scattered aggregates of metal particles and a diffuse lymphocyte infiltrate. Metal Ion Levels – The presence of pseudotumour was associated with significantly higher median serum cobalt levels (9.2mg/L vs. 1.9mg/L, p< 0.001), chromium levels (12.0mg/L vs. 2.1mg/L, p< 0.001), hip aspirate cobalt levels (1182 mg/L vs. 86.2mg/L, p=0.003), and aspirate chromium levels (883mg/L vs. 114.8mg/ L, p=0.006), as well as with inferior functional scores (OHS 41 vs. 47 p< 0.001). There was no significant difference in acetabular cup inclination angle (p=0.51). Lymphocyte Reactivity: A higher incidence and level of enhanced lymphocyte reactivity to Ni (p=0.001), but not to Co or Cr (the principal elements in the CoCr alloy used in metal-on-metal hip resurfacing implants), was found in patients with MoMHRA compared to the patients without MoM implants. However, lymphocyte reactivity to Co, Cr and Ni did not significantly differ in patients with pseudotumours compared to those patients without pseudotumours. Conclusion: The prevalence of asymptomatic pseudotumours in females was high, especially in females with bilateral MoMHRA implants (30%). The patients with ‘asymptomatic’ pseudotumours were in fact mildly symptomatic. Lymphocyte reactivity to Co, Cr and Ni did not differ in patients with pseudotumour compared to those patients without pseudotumours, suggesting that systemic hypersensitivity type IV reactions, mediated by lymphocyte reactivity to these metals, is not the dominant mechanism in pathogenesis of the soft tissue pseudotumours. Furthermore, pseudotumours were not detected in those patients who had normal levels of cobalt and chromium ions. This suggests that pseudotumours do not occur if MoM articulations are well functioning. Therefore, pseudotumours are likely to be a biological consequence of the large amount of metal debris generated in vivo due to excessive wear

We investigated the lymphocyte-mediated immune response to polymethylmethacrylate bone cement in 26 patients who had revision surgery for aseptic loosening of cemented total hip arthroplasties, at a mean time of seven years after the first replacement. We studied eight patients with cemented total hip arthroplasties which were not loose as controls. Patch tests to polymethylmethacrylate bone cement were positive in 13 patients with loosening, and these patients had higher lymphoblast transformation values against polymethylmethacrylate bone cement patients with a negative skin reaction (p < 0.01) or those in the control group (p < 0.001). Specific monoclonal antibodies were used to assess the percentage of certain cells of the immune system according to their cluster of differentiation (CD). There was a higher number of total T and B lymphocytes (CD2 and CD22) and interleukin-2 receptor-positive lymphocytes (activated cells, CD25) in patients with loose prostheses. More CD25 lymphocytes were found in patients with positive patch tests. The activation of the lymphocyte-mediated immune response was not related to the presence or absence of aggressive granulomatous lesions at the cement-bone interface

Recently, a series of locally destructive soft tissue pseudotumour has been reported in patients following metal-on-metal hip resurfacing arthroplasty (MoMHRA), requiring revision surgery in a high percentage of patients. Based on the histological evidence of lymphocytic infiltration, a delayed hypersensitivity reaction to nickel (Ni), chromium (Cr) or cobalt (Co) has been suggested to play a role in its aetiology. The aim of this study was to investigate the incidence and level of hypersensitivity reaction to metals in patients with pseudotumour. Materials and Methods: 25 patients were investigated in this Ethics approved study:. Group 1: MoMHRA patients with pseudotumours, detected on the ultrasound and confirmed with MRI (n=6, 5 F:1 M, mean age 53 years);. Group 2: MoMHRA patients without pseudotumours (n=13, 7 F:6 M, mean age 55 years); and. Group 3: age-matched control subjects without metal implants (n=6, 4 F:2 M, mean age 54 years). Lymphocyte transformation tests (LTT) were used to measure lymphocyte proliferation responses to metals. Peripheral blood mononuclear cells were isolated from heparinized blood samples using standard Ficoll–Hypaque® (Pharmacia). The PBMC were cultured at a cell density of 106 cells/mL. Culture was set up in the presence of either:. medium alone;. nickel chloride (Sigma; 10-4M-10-6M);. cobalt chloride (10-4M-10-6M); and. chromium chloride (10-4M-10-6M). After 5 days of culture, cells were pulsed with [3H]-thymidine and proliferation was assessed by scintillation counting. The stimulation index (SI) was calculated by the ratio of mean counts per minute of stimulated to unstimulated cultures. A SI value of greater than 2.0 was interpreted as a positive result. Results: A clinical history of metal allergy was reported in 2/6 in Group 1, 2/13 in Group 2, and none in Group 3. In pseudotumour group, the incidence of reactivity to Ni, Co and Cr was 60%, 17% and 0%, respectively. Within Group 2, the reactivity to Ni, Co and Cr was 69%, 8% and 15%, respectively. One control subject had reactivity to Ni. Inter-group comparisons of mean SI values (Kruskal-Wallis non-parametric analysis of variance) showed no significant differences (p> 0.05). Discussion: The incidence of enhanced lymphocyte response to metals in patients with MoMHRA was more common than the control group. However, in comparison with non-pseudotumour patients, there was no significant difference in the incidence or the level of lymphocyte reactivity in patients with pseudotumour. We conclude that patients with MoMHRA have an enhanced lymphocyte response to metal ions, reflecting exposure and immune reactivity. However, patients with pseudotumours have a similar proliferative response to those without pseudotumours, which suggests that type IV hypersensitivity may not be the cause of the pseudotumours

Introduction. Metal on metal articulations produce chromium (Cr) and cobalt (Co) debris, particularly when the articulations are worn in. High levels in the peripheral blood are indicative of excess wear and may cause adverse effects. The present RCT investigates metal ion levels and the relationship of Co, Cr ions and lymphocyte counts during the running-in period. Materials and Methods. Following randomization to RHA (ASRTM, DePuy) or THA we obtained whole blood (wb), and serum (s) samples at baseline, 8 w, 6 m and 1 y. We measured the Co and Cr concentrations, the total lymphocyte count as well as the CD3+, CD4+, CD8+, CD19+ and CD16+/CD56+ sub populations. Cup inclination and anteversion angles came from conventional radiographs. Activity was measured as steps by pedometer and UCLA activity. Data are presented as median (range). Results. We had 19 patients in each group. Age 57 (46–64) y (RHA) and 55 (44–64) y (THA). RHA only: head size: 51 (47 to 57)mm, cup inclination: 45.0 (40- 56)° and anteversion: 23 (7–38)°. The ion concentrations stayed below 0.2 ppb for THAs. For RHAs the Co and Cr concentrations generally rose markedly in the initial 8 weeks, followed by a slower ascent up to 1 year to wb Co: 1.0 (0.6–5.2), s Co: 1.3 (0.7–6.5), wb Cr: 1.3 (0.4–8.0) and s Cr: 1.7 (0.6–15) ppb. A high ion level was correlated to a small head size (p<0.03) and a cup inclination around 45° (p<0.04). We could not correlate the ion concentrations to the anteversion or the activity levels. The absolute lymphocyte counts were not always identical in the groups (eg. lower CD8+ for RHA) but there was no group difference when we analysed the change from baseline. We did not demonstrate any correlation between metal ion levels and lymphocyte subpopulations. Discussion. The findings of this study does not support the theory that metal ions suppress the lymphocytes or the CD8+ in particular (Hart et al., 2009). Our conclusions are limited by low numbers, but we suspect the theory could be biased by missing baseline values. Our study did not demonstrate the same 6–9 months ion peak found in other running-in studies (Back et al., 2005, Heisel et al., 2008), but rather a slow continuous rise with lower median ion concentrations. It could indicate lower wear or perhaps a longer running in period in our slightly older population. Contrary to our expectations we found that cups placed in optimal inclination displayed a higher ion level. Most of the smallest cups were found in this group, and the majority of the steep cups had very large heads. For this implant head size may be more important than cup position

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

We compared the peripheral blood and periprosthetic tissues of 53 patients at revision arthroplasty with those of 30 patients at primary arthroplasty to determine whether there is a systemic difference in lymphocytes in patients with worn hip implants. The absolute number and relative proportion of lymphocytes bearing CD2, CD3, CD4, CD8, CD16, CD19, HLA-DR, kappa and lambda antigens were compared with the levels of IL-1β, IL-6 and PGE. 2. in the pseudosynovial membrane as well as with a semiquantitative estimate of metal and polyethylene particles, necrosis and chronic inflammation and the total concentration of metals within the periprosthetic tissues. There was a significant increase in the relative proportion of CD2-positive T-cells and CD16-positive natural killer cells in the peripheral blood at revision arthroplasty compared with primary arthroplasty and an increased proportion of CD8-positive T-cells and a decreased ratio of CD4 to CD8 (helper inducer/suppressor cytotoxic cells). Three control patients, who went on to have revision surgery, had values at primary arthroplasty which were similar to those of patients at the time of revision surgery. These differences did not correlate with the local concentration of metal, plastic or cement or inflammatory response or the type of prosthesis. An inverse correlation was noted between the necrosis in the periprosthetic tissue and both the local production of IL-6 and the absolute numbers of T-cells in peripheral blood. We conclude that there may be several cell-mediated systemic immune responses to aseptic loosening, at least one of which may be directly related to events in the periprosthetic tissues. We cannot exclude the possibility that the changes in the proportion of CD8-positive cells reflected a predisposition, rather than a reaction, to loosening of the implant

A major concern in metal on metal bearings has been the elevated serum concentrations of cobalt and chromium. Recent papers have suggested that metal hypersensivity in a few cases could cause periprostetic lymphocyte accumulation leading prosthetic loosening. To measure the lymphocyte activation and proliferation in vitro by re-exposure of the cells to cobalt, chromium, nickel and titanium. To correlate the lymphocyte assay data to the serum concentration of metals and plasma cytokines. A prospective clinical study with the ASR (DePuy) and ReCap (Biomet) resurfacing hip implants. Blood samples were collected one and two years postoperatively, lymphocytes were isolated by density gradient centrifugation, cultured in a medium containing the patient’s serum and exposed to metal salts. Cells were analyzed by flow cytometry, evaluating number, viability, size and CD69 activation. A negative control and a positive control (phytohaemagglutinine) were included in the assay, and the responses to the metals were calculated in proportion to controls. 11 patients were assessed at one and two years follow up, 16 patients were assessed only at two years. Serum chromium and cobalt were measured preoperatively, six months, one year and two years postoperatively by graphite furnace absorptiometry. Plasma cytokines were measured by multiplexed immunoassay. In the assay the negative and positive controls gave the expected responses. When exposed to metals no response was found in the lymphocytes in any patients. There were no difference in response between one and two years. The results seems to indicate that the metal hypersensitivity is a rare condition in metal on metal arthroplasty. The results indicate that the method can be used to monitor hypersensitivity to implant metals

Introduction. The utilization of lymphocyte transformation testing (LTT) has increased for diagnosing metal sensitivity associated with TKA, but its validity for the diagnosis of TKA failure due to an immune reaction has not been established. Methods. We performed a retrospective study of 27 well-fixed, aseptic primary TKAs with persistent pain and/or stiffness, revised by a single, experienced surgeon for suspected metal allergy to nickel based on a positive LTT. Periprosthetic tissue samples obtained at the time of revision surgery were scored using the aseptic lymphocytic vasculitis-associated lesion (ALVAL) scoring system. Results. Eight patients were categorized as mildly reactive, 8 reactive, and 11 highly reactive to nickel by LTT. The predominant findings on routine histology were fibrosis and varying degrees of lymphocytic infiltration in 17/27 (63%) of the cases. The average ALVAL score of the cohort was 3.1 ± 1.9, out of a maximum score of 10. Average Knee Society Scores (KSS) improved post-revision, as did range of motion (all p<0.01). Neither LTT stimulation index as a continuous variable nor as a categorical variable (mild, moderate, high) was correlated with ALVAL score, pre-operative function, or change in function at last follow-up (0.015 < r < 0.30, 0.13 < p < 0.95) as measured by KSS. In addition, the ALVAL score did not correlate significantly with either pre-operative or post-operative KSS or range of motion (0.061 < r < 0.365, 0.09 < p < 0.88). Conclusions. Based on this analysis including histopathology, LTT results alone are insufficient for the diagnosis of TKA failure due to an immune reaction. A positive LTT may not indicate that an immune reaction is the cause of pain and stiffness post-TKA. The role of LTT in assessing immune failure of TKA needs further investigation. Diagnostic criteria for immune failure of a TKA need to be established

Total joint arthroplasty has proven to be efficient to relieve pain and regain mobility. In fact, most patients undergoing a total knee arthroplasty (TKA) are satisfied with their surgery (80 to 90%), yet 4 to 7% still complain of unexplainable pain and stiffness. Several authors have proposed that reactivity to the implant could explain this phenomenon. Still, no strong evidence supports this theory as of today. We aimed to determine the prevalence of metal and cement hypersensitivity in a cohort of patients with unexplained pain and stiffness after TKA. We retrieved data for a group of patients presenting unexplained pain and stiffness. We excluded all other potential known causes of pain. All patients were tested with a Lymphocyte Transformation Test from whole blood taps. We analysed data of hypersensitivity to metals (alloy particles of titanium and cobalt, aluminum, cobalt, nickel, zirconium, vanadium, molybdenum, cobalt, chromium and iron) and PMMA cement (bone cement monomer and particles). Fifty-three patients underwent a LTT for unexplained pain and stiffness after total knee arthroplasty between May 2012 and May 2015. The cohort consisted of 26 men and 27 women with a mean age of 66.3(±8.0) years. Six patients had no hypersensitivity (11.3%), leaving 88.7% of the cohort with hypersensitivity to metal and/or cement. Almost half the cohort of patients tested for PMMA was hypersensitive to cement (44.0%). The most common metal hypersensitivity was nickel (69.8%). Twelve patients presented sensitivity to only one metal (22.6%), whereas 35 patients were hypersensitive to more than one metal (66.0%). Eleven patients had revision surgery with a hypoallergenic prosthesis. Patients reported a significant diminution of pain as well as better knee function compared to preoperative status as early as 6 weeks postop, although some reported residual stiffness. The results of this study suggest that metal and/or cement hypersensitivity could play a role in cases of total knee arthroplasty with unexplained pain and stiffness. Randomised controlled clinical trials on the subject will be initiated by our team to further investigate this phenomenon

The long-term biological effects of wear debris are unknown. We have investigated whether there is any evidence of cumulative mutagenic damage in peripheral blood lymphocytes of patients undergoing revision arthroplasty of predominantly metal-on-plastic total hip replacements compared with those at primary arthroplasty. There was a threefold increase in aneuploidy and a twofold increase in chromosomal translocations which could not be explained by the confounding variables of smoking, gender, age and diagnostic radiographs. In the patients with TiVaAl prostheses there was a fivefold increase in aneuploidy but no increase in chromosomal translocations. By contrast, in patients with cobalt-chrome prostheses there was a 2.5-fold increase in aneuploidy and a 3.5-fold increase in chromosomal translocations. In six patients with stainless-steel prostheses there was no increase in either aneuploidy or chromosomal translocations. Our results suggest that future epidemiological studies of the putative long-term risks of joint replacement should take into account the type of alloy used in the prosthesis

Hip fractures are a significant cause of mortality and morbidity in the elderly. Malnutrition is a major element of this but no consensus exists as to the detection or management of this condition. Reported incidence in elderly hip fracture patients varies widely between 9.0% and 88.6%. The aim of this study was to evaluate the nutritional status of 415 patients with operatively managed hip fractures and determine the prognostic relevance of admission serum albumin and total lymphocyte count (TLC) assays. Protein-energy malnutrition (PEM) was defined as serum albumin < 3.5g/dl and a TLC < 1,500 cells/mm3. Delay to operation, duration of in-patient stay, re-admission (< 3 months) and in-patient, 3- and 12-month mortality were assessed as outcome variables. Survival data was available for 377 patients at 12 months. Of 377 patients, 53% (n=200) had both a serum albumin and TLC levels taken at admission, while 47% (n=177) had not. The incidence of PEM was 51%. Inhospital mortality for PEM patients was 9.8%, compared with 0% for patients with normal values of both laboratory parameters. Older patients were more likely to have lower albumin (p=0.017) and TLC (p=0.023). Nursing home patients were also more likely to have lower albumin (p=0.033). Multivariate analysis revealed a significant difference in 12-month mortality, with patients who had both a low albumin and a low TLC 4.6 times (95% CI: 1.0–21.3) more likely to die within 12 months postoperatively than patients who had normal values of both laboratory parameters. This was significant after adjusting for age, gender and domicile (p=0.049). Serum albumin and TLC in combination are accurate predictors of 12-month mortality in hip fracture patients. These results highlight the relevance of assessing the nutritional status of patients with hip fractures at the time of admission and emphasises the relationship between nutrition and outcome

Background: Protein energy malnutrition (PEM) is an accepted predictor of poor outcome in hip fracture patients. There is no universally accepted definition of PEM. Admission screening for PEM is not routinely performed for hip fracture patients. The reported incidence in elderly hip fracture patients varies widely between 9.0% and 88.6%. Aims: To determine the prognostic relevance of admission serum albumin and total lymphocyte count (TLC), as clinical markers of PEM and predictors of outcome for hip fracture patients. Methods: Retrospective review of 415 patients with operatively managed hip fracture. Protein-energy malnutrition was defined as albumin < 3.5g/dl and TLC < 1,500cells/mm3. Delay to operation, duration of in-patient stay, readmission (< 3 months) and in-patient, 3- and 12-month mortality were assessed as outcome variables. Results: Survival data was available for 377 patients at 12 months. Of 377 patients, 53% (n=200) had both a serum albumin and TLC levels taken at admission (study), while 47% (n=177) had not (control). Incidence of PEM was 51%. Older patients were more likely to have lower albumin (p=0.03) and TLC (p=0.012). Nursing home patients were also more likely to have lower albumin (p=0.049). In-hospital mortality for PEM patients was 9.8%, compared with 0% for patients with normal values of both laboratory parameters. Patients with PEM had a higher 12-month mortality compared to patients who had normal values of both laboratory parameters (Odds Ratio=4.52; p=0.049). Conclusion: Serum albumin and TLC in combination are accurate predictors of 12-month mortality in hip fracture patients. These results underscore the clinical relevance of assessing the nutritional status of patients with hip fractures at the time of admission and emphasises the relationship between nutrition and outcome in these patients

Background: Protein energy malnutrition (PEM) is an accepted predictor of poor outcome in hip fracture patients. There is no universally accepted definition of PEM. Admission screening for PEM is not routinely performed for hip fracture patients. The reported incidence in elderly hip fracture patients varies widely between 9.0% and 88.6%. Aims: To determine the prognostic relevance of admission serum albumin and total lymphocyte count (TLC), as clinical markers of PEM and predictors of outcome for hip fracture patients. Methods: Retrospective review of 415 patients with operatively managed hip fracture. Protein-energy malnutrition was defined as albumin < 3.5g/dl and TLC < 1,500 cells/ mm3. Delay to operation, duration of in-patient stay, readmission (< 3 months) and in-patient, 3- and 12-month mortality were assessed as outcome variables. Results: Survival data was available for 377 patients at 12 months. Of 377 patients, 53% (n=200) had both a serum albumin and TLC levels taken at admission (study), while 47% (n=177) had not (control). Incidence of PEM was 51%. Older patients were more likely to have lower albumin (p=0.03) and TLC (p=0.012). Nursing home patients were also more likely to have lower albumin (p=0.049). In-hospital mortality for PEM patients was 9.8%, compared with 0% for patients with normal values of both laboratory parameters. Patients with PEM had a higher 12-month mortality compared to patients who had normal values of both laboratory parameters (Odds Ratio=4.52; p=0.049). Conclusion: Serum albumin and TLC in combination are accurate predictors of 12-month mortality in hip fracture patients. These results underscore the clinical relevance of assessing the nutritional status of patients with hip fractures at the time of admission and emphasises the relationship between nutrition and outcome in these patients

Achilles tendinopathy is classically defined as a tendinosis devoid of an inflammatory cell population. However, recent literature suggests inflammation as a mediator in the pathogenesis. These finding were mainly based on semi-quantative immunohistochemistry. We therefore used flow cytometry to obatain a more accurate identification and quantification of the different cell types involved. Thirty-two samples were obtained from twelve patients with chronic tendinopathic lesions undergoing Achilles tendon surgery. Samples obtained from three patients with hemiplegia requiring surgical release due to spastic Achilles tendons served as control. We used two panels to identify the myeloid and lymphoid population targeting the following markers: CD45, CD3, CD8, CD4, CD19, CD11b, CD56, CD14, CD16, Vα7.2, 6b11, CD161, TCRγδ. To assess the presence of fibroblasts CD90 was targeted. The mean count of CD45+ hematopoietic cells in the tendinopathic samples was significantly higher than in the control samples, respectively 13.27% and 3.24% of the total cell count (P<0.001). The mean fraction of CD3+ cells present in the complete cell population was significantly higher in pathological samples than in control samples, respectively 1.70% and 0.37% (P<0.05). Presence of CD19+ B cells was not reported. The mean fraction of γδ T cells was significantly higher in tendinopathic samples compared to blood samples of the same patient and consisted of 12.9% and 5.8% γδ T cells respectively (P<0.05). These findings support an inflammatory cell infiltration in midportion Achilles tendinopathy that show similarities to enthesitis in SpA. This implies a potential target to investigate in novel treatment modalities.

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.

Aims

Metal particles detached from metal-on-metal hip prostheses (MoM-THA) have been shown to cause inflammation and destruction of tissues. To further explore this, we investigated the histopathology (aseptic lymphocyte-dominated vasculitis-associated lesions (ALVAL) score) and metal concentrations of the periprosthetic tissues obtained from patients who underwent revision knee arthroplasty. We also aimed to investigate whether accumulated metal debris was associated with ALVAL-type reactions in the synovium.

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.

Aims. The modified Glasgow Prognostic Score (mGPS) uses preoperative CRP and albumin to calculate a score from 0 to 2 (2 being associated with poor outcomes). mGPS is validated in multiple carcinomas. To date, its use in soft-tissue sarcoma (STS) is limited, with only small cohorts reporting that increased mGPS scores correlates with decreased survival in STS patients. Methods. This retrospective multicentre cohort study identified 493 STS patients using clinical databases from six collaborating hospitals in three countries. Centres performed a retrospective data collection for patient demographics, preoperative blood results (CRP and albumin levels and neutrophil, leucocyte, and platelets counts), and oncological outcomes (disease-free survival, local, or metastatic recurrence) with a minimum of two years' follow-up. Results. We found that increased mGPS, tumour size, grade, neutrophil/lymphocyte ratio, and disease recurrence were associated with reduced survival. Importantly, mGPS was the best at stratifying prognosis and could be used in conjunction with tumour grade to sub-stratify patient survival. Conclusion. This study demonstrated that prognosis of localized STS strongly correlates with mGPS, as an increasing score is associated with a poorer outcome. We note that 203 patients (41%) with an STS have evidence of systemic inflammation. We recommend the mGPS and other biochemical blood indicators be introduced into the routine diagnostic assessment in STS patients to stratify patient prognosis. Its use will support clinical decision-making, especially when morbid treatment options such as amputation are being considered. Cite this article: Bone Joint J 2022;104-B(1):168–176

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). Methods. Studies were identified by searching electronic databases: Cochrane Central Register of Controlled Trials, Ovid MEDLINE, and Embase, from their inception to November 2020, for studies evaluating TKA patients with metal hypersensitivity/allergy. All studies reporting on diagnosing or managing metal hypersensitivity in TKA were included. Data were extracted and summarized based on study design, study population, interventions and outcomes. A practical guide is then formulated based on the available evidence. Results. We included 38 heterogeneous studies (two randomized controlled trials, six comparative studies, 19 case series, and 11 case reports). The evidence indicates that metal hypersensitivity is a rare complication with some histopathological features leading to pain and dissatisfaction with no reliable screening tests preoperatively. Hypoallergenic implants are viable alternatives for patients with self-reported/confirmed metal hypersensitivity if declared preoperatively; however, concerns remain over their long-term outcomes with ceramic implants outperforming titanium nitride-coated implants and informed consent is paramount. For patients presenting with painful TKA, metal hypersensitivity is a diagnosis of exclusion where patch skin testing, lymphocyte transformation test, and synovial biopsies are useful adjuncts before revision surgery is undertaken to hypoallergenic implants with shared decision-making and informed consent. Conclusion. Using the limited available evidence in the literature, we provide a practical approach to metal hypersensitivity in TKA patients. Future national/registry-based studies are needed to identify the scale of metal hypersensitivity, agreed diagnostic criteria, and management strategies. Cite this article: Bone Jt Open 2021;2(10):785–795

Objectives. Cortical and cancellous bone healing processes appear to be histologically different. They also respond differently to anti-inflammatory agents. We investigated whether the leucocyte composition on days 3 and 5 after cortical and cancellous injuries to bone was different, and compared changes over time using day 3 as the baseline. Methods. Ten-week-old male C56/Bl6J mice were randomized to either cancellous injury in the proximal tibia or cortical injury in the femoral diaphysis. Regenerating tissues were analyzed with flow cytometry at days 3 and 5, using panels with 15 antibodies for common macrophage and lymphocyte markers. The cellular response from day 3 to 5 was compared in order to identify differences in how cancellous and cortical bone healing develop. Results. Between day 3 and 5, the granulocytes increased in the cancellous model, whereas the lymphocytes (T cells, B cells, NK cells) and monocytes (CD11b+, F4/80+, CD206+, CD14+) increased in the cortical model. Conclusion. These results suggest an acute type of inflammation in cancellous bone healing, and a more chronic inflammation in cortical healing. This might explain, in part, why cancellous healing is faster and more resistant to anti-inflammatory drugs than are diaphyseal fractures. Cite this article: L. Tätting, O. Sandberg, M. Bernhardsson, J. Ernerudh, P. Aspenberg. Different composition of leucocytes in cortical and cancellous bone healing in a mouse model. Bone Joint Res 2018;7:620–628. DOI: 10.1302/2046-3758.712.BJR-2017-0366.R2

Introduction and Objective. Osteochondral allograft (OCA) transplants have been used clinically for more than 40 years as a surgical option for joint restoration, particularly for young and active patients. While immediate graft rejection responses have not been documented, it is believed that the host's immunological responses may directly impact OCA viability, incorporation, integrity, and survival, and therefore, it is of the utmost importance to further optimize OCA transplantation outcomes. The influences of sub-rejection immune responses on OCA transplantation failures have not been fully elucidated therefore aimed to further characterize cellular features of OCA failures using immunohistochemistry (IHC) in our continued hopes for the successful optimization of this valuable surgical procedure. Materials and Methods. With IRB approval, osteochondral tissues that were resected from the knee, hip, and ankle of patients undergoing standard-of-care revision surgeries (N=23) to treat OCA failures and tissues from unused portions of OCAs (N=7) that would otherwise be discarded were recovered. Subjective histologic assessments were performed on hematoxylin and eosin-stained and toluidine blue-stained sections by a pathologist who was blinded to patient demographics, outcomes data, and tissue source. IHC for CD3, CD8, and CD20 were performed to further characterize the and allow for subjective assessment of relevant immune responses. Results. Eleven (48%) of the failed OCAs had aggregates of CD3+, CD8+, and CD20+ lymphocytes around small blood vessels in the bone marrow spaces and adipose/collagenous tissue of the allograft, while the non-implanted healthy control OCA tissues did not show any evidence of inflammation. The remaining failed OCAs (52%) did not show a similar pattern of T- and B-cell infiltrates around blood vessels. Other histologic abnormalities associated with failed OCAs included avascular necrosis, subchondral micro and macro fractures, subchondral collapse, bacterial infection, and/or articular cartilage erosion or delamination. Conclusions. The results from the present study support this possibility in that mixed aggregates of CD3+, CD8+, and CD20+ lymphocytes were observed around small blood vessels in approximately half of the failed OCAs. This potentially cytotoxic immune response may have contributed to the lack of functional survival of the OCA noted in these cases, and warrants further investigation as a possible failure mechanism that may be mitigated using post-transplantation management strategies

We carried out a cross-sectional study with analysis of the demographic, clinical and laboratory characteristics of patients with metal-on-metal hip resurfacing, ceramic-on-ceramic and metal-on-polyethylene hip replacements. Our aim was to evaluate the relationship between metal-on-metal replacements, the levels of cobalt and chromium ions in whole blood and the absolute numbers of circulating lymphocytes. We recruited 164 patients (101 men and 63 women) with hip replacements, 106 with metal-on-metal hips and 58 with non-metal-on-metal hips, aged < 65 years, with a pre-operative diagnosis of osteoarthritis and no pre-existing immunological disorders. Laboratory-defined T-cell lymphopenia was present in13 patients (15%) (CD8. +. lymphopenia) and 11 patients (13%) (CD3. +. lymphopenia) with unilateral metal-on-metal hips. There were significant differences in the absolute CD8. +. lymphocyte subset counts for the metal-on-metal groups compared with each control group (p-values ranging between 0.024 and 0.046). Statistical modelling with analysis of covariance using age, gender, type of hip replacement, smoking and circulating metal ion levels, showed that circulating levels of metal ions, especially cobalt, explained the variation in absolute lymphocyte counts for almost all lymphocyte subsets

Introduction and Objective. The use of microfragmented adipose tissue (mFAT) for the treatment of musculoskeletal disorders, especially osteoarthritis, is gaining popularity following the positive results reported in recent case series and clinical trials. The purpose of this study is to characterize mFAT in terms of structure, cell content and secretome (i.e. protein and microvescicles released as paracrine mediators), and to compare it with unprocessed lipoaspirate tissue, in order to understand the possible mechanisms of action and the benefit derived from tissue processing. Materials and Methods. Unprocessed lipoaspirate (LA) and mFAT were obtained from 7 donors. Each tissue sample was divided in four aliquots: A) fixed in formalin for histological evaluation; B) enzymatically digested to harvest cells with the exclusion of adipocytes; C) cultured for 24 hours in serum-free DMEM to harvest secretome; D) freshly frozen for proteomic evaluation. Hematoxylin and eosin staning, as well as immunohistochemistry for CD31, CD90, CD146 were performed on aliquot A. Cell count, viability, senescence and immunophenotype were assessed on aliquot B. Culture medium from aliquot C was collected and used for proteomic analysis and micro-RNA extraction and quantitation from extracellular vesicles. Aliquot D was lysed, protein were extracted and analyzed using a high-throughput proteomic approach. Results. Histological investigations showed a lower red blood cell content in mFAT with respect to LA, while the presence of blood vessels (CD31+), stromal cells (CD90) and pericytes (CD146) was similar in all samples. These results were confirmed by flow cytometry, with reduction of erythrocytes (CD235a+) by 76% and reduction of lymphocytes (CD45+) by 79% in mFAT compared to LA. Otherwise, the proportions of stromal cells, pericytes and endothelial cells in LA and mFAT remained comparable. The percentage of senescent cells resulted similar before and after tissue processing, with very low values (< 5%). The analysis of the miRNAs contained in the extracellular vesicles in culture media identified 376 miRNAs in LA secretome and 381 in mFAT secretome. A high correlation in the expression of these miRNAs within subjects (LA and mFAT of each donor) was observed (R2> 0.8), indicating that processing in mFAT does not significantly alter the portfolio of miRNAs associated with extracellular vesicles. Proteomic analysis of secretome revealed that 217 proteins significantly differ between LA and mFAT. In particular, protein associated with acute phase were less represented in mFAT secretome, while intracellular proteins were more frequent. Proteomic analysis of tissues demonstrated a reduction of protein related to extracellular matrix and of proteins closely related to peripheral blood contamination in mFAT with respect to LA. Conclusions. Taken together, these results suggest that processing of LA into mFAT allow for removal of blood elements, in terms of red blood cells, lymphocytes, acute phase and complement system proteins, and for the reduction of extracellular matrix components. Otherwise, tissue structure, cell populations, cell viability and senescence are not influenced by tissue processing. Then, microfragmentation process represents a safe and efficient method for the application of adipose tissue properties to musculoskeletal disorders, allowing for the maintenance of all the effector elements for tissue regeneration while removing possible detrimental agents such as inflammatory mediators

Soft tissue sarcomas (STS) are rare, aggressive malignancies derived from connective tissues such as muscle and fat. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common STS in adults. UPS is an aggressive, highly metastatic sarcoma, and is resistant to chemotherapy. New therapies for UPS are desperately needed. STS have an immune desert tumour immune microenvironment (TIME), characterized by a paucity of tumour infiltrating lymphocytes and subsequent resistance to immunotherapies such as immune checkpoint inhibitors. Strategies capable of creating an immune-rich, inflamed TIME may improve immunotherapy efficacies for sarcoma. Activation of the STING (stimulator of interferon genes) receptor can induce potent innate and adaptive immune responses within immunogenic solid tumours. However, this approach has never been attempted in immune-inert sarcomas. Purpose: To determine the therapeutic anti-tumour effects of STING activation in UPS tumours. We have developed an inducible, immune-competent mouse model of UPS. We evaluated intra-tumoural injection of the murine STING receptor agonist, DMXAA, into UPS-bearing immune-competent mice. DMXAA was injected into palpable UPS tumours of the hindlimb. Tumour volume and bioluminescence imaging was recorded bi-weekly. DMXAA treated UPS tumours were also evaluated for necrosis and immune infiltration at defined time points. UPS tumours developed necrosis and lymphocytic infiltration 72 hours after DMXAA treatment. A single intra-tumoural dose of DMXAA into UPS tumours resulted in durable cure in 50% of mice. All survivors rejected a re-challenge of the UPS tumours in both the contralateral hindlimb and lung, suggesting adaptive immunity. The therapeutic effects of DMXAA were mitigated in lymphocyte deficient Rag2 knockout mice. STING therapy is a promising immunotherapeutic opportunity for immune-inert sarcomas. Our data warrants further preclinical investigations in other sarcoma models and in combination with other immune-based therapies

The interleukin-6/gp130-associated Janus Kinases/STAT3 axis is known to play an important role in mediating inflammatory signals, resulting in production of matrix metalloproteinase-3 (MMP-3). The hip joints with rapidly destructive coxopathy (RDC) demonstrate rapid chondrolysis, probably by increased production of MMP-3 observed in the early stage of RDC. In the recent study, no apparent activation of STAT3 has been shown in the synovial tissues obtained from the osteoarthritic joint at operation. However, no data are currently available on STAT3 activation in the synovial tissues in the early stage of RDC. This study aimed to elucidate STAT3 activation in the synovial tissues in the early stage of RDC. Synovial tissues within 7 months from the disease onset were obtained from four RDC patients with femoral head destruction and high serum levels of MMP-3. RDC synovial tissues showed the synovial lining hyperplasia with an increase of CD68-positive macrophages and CD3-positive T lymphocytes. STAT3 phosphorylation was found in the synovial tissues by immunohistochemistry using anti-phospho-STAT3 antibody. The majority of phospho-STAT3-positive cells were the synovial lining cells and exhibited negative expression of macrophage or T cell marker. Treatment with tofacitinib, a Janus Kinase inhibitor, resulted in a decrease in phospho-STAT3-positive cells, especially with high intensity, indicating effective suppression of STAT3 activation in RDC synovial tissues. Inhibitory effect of tofacitinib could act through the Janus Kinase/STAT3 axis in the synovial tissues in the early stage of RDC. Therefore, STAT3 may be a potential therapeutic target for prevention of joint structural damage in RDC. Acknowledgements: This study was supported by Katakami Foundation for Clinical Research

Soft tissue sarcomas (STS) have not demonstrated favourable clinical responses to emerging immunotherapies such as checkpoint inhibitors. Studies in carcinomas and melanoma have demonstrated that tumours lacking T-cell infiltrates are associated with poor responses to immunotherapies. It is postulated that STS lack tumour asscoiated lymphocytes which renders these tumours insensitive to checkpoint inhibitors. Our objective was to develop a novel syngeneic mouse model of STS and characterize the immune phenotype of these tumours. Additionally, we sought to evaluate the therapeutic responses of these sarcomas to checkpoint inhibitors and a Type I interferon agonist. K-ras mutagenesis and p53 deletion was induced using a Lenti-Cre-recombinase injection into the hindlimb of 3 week old C57BL/6 mice. Tumours were harvested and characterized using standard histopathology techniques and whole trascriptome sequencing (RNAseq). Full body necrospy and histopathology was performed to identify metastases. Flow cytometry and immunohistochemistry was used to evaluate tumour immune phenotypes. Tumours were implanted into syngeneic C57BL/6 mice and the therapeutic responses to anti-CTLA4, anti-PD1 and DMXAA (Type I interferon agonist) were performed. Tumour responses were evaluated using bioluminescent imaging and caliper measurements. Soft tissue sarcomas developed in mice within 2–3 months of Lenti-Cre injection with 90% penetrance. Histologic analyses of tumours was consistent with a high-grade myogenic sarcoma characterized by smooth muscle actin, Desmin and Myogenin D positive immunostaining. Using crossplatform normalization protocols, geneexpression signatures of the mouse tumours most closely correlated with human undifferentiated pleomorphic sarcoma (UPS). Collectively, gene expression signatures of this murine sarcoma correlated with all muscle-derived human sarcomas (ERMS, ARMS, Synovial sarcoma, UPS). No lung or other visceral metastases were observed in all mice who developed spontaneous tumours. Immune phenotyping demonstrated a paucity of tumour-infiltrating lymphocytes (TILs, (TAMs). 50% of identified TILs in these murine sarcomas expressed PD-1, yet tumours were not responsive to anti-PD1 therapy or anti-CTLA4 therapy. A single intra tumoural (i.t.) injection of the Type I interferon agonist, DMXAA resulted in 80–90% tumour necrosis 72 hrs post-injection, decreased tumour viability up to 2 weeks post-injection and a marked infiltration of CD8+ T-cells and anitgen presenting dendritic cells and macrophages. Additional longitudinal experiments demonstrate a sustained and progressive anti-tumour effect in 83% (5/6) mice up to 6weeks following a single i.t. injection of DMXAA. All control treated mice (6/6) reached humane endpoint within 14 days. At 3 months post-DMXAA treatment, 4/6 mice were free of disease. We re-injected UPS tumours into these mice and tumours did not grow, suggesting abscopal effects after DMXAA treatment of primary tumours. We have characterized a new orthotopic and syngeneic mouse model of a myogenic soft tissue sarcoma. Like most human STS sub-types, these tumours have an immune inert tumour microenvironment and are not sensitive to checkpoint inhibitors. This model, syngeneic to C56BL/6 mice will enable future opportunities to investigate how various branches of the immune system can be targetted or manipulated to unearth new immunotherapeutic strategies for sarcoma. Using this model we have demonstrated that a single, intra-tumoural injection of a Type I interferon agonist can result in anti-tumour effects, recruit cytotoxic lymphocytes and antigen presenting cells with into the the tumour microenvironment. Abscopal tumour rejection after DMXAA treatement suggest adaptive T-cell responses against UPS are active in this model. Future work is needed to determine if upregulation of Type I inferferon pathways can be used as a therapeutic strategy for sarcoma or as a sensitization strategy for checkpoint inhibitors

Malnutrition is considered a risk factor for postoperative complications in total hip and knee arthroplasty, though prospective studies investigating this assumption are lacking. The aim of this study was to prospectively analyse the 90-day postoperative complications, postoperative length of stay (LOS) and readmission rates of patients undergoing primary total hip and total kneearthroplasty using albumin, total lymphocyte count (TLC) and transferrin as serum markers of potential malnutrition. 603 primary hip and 823 primary knee arthroplasties over a 3-year period from a single centre wereprospectively analysed. BMI, demographic and comorbidity data were recorded. Complications werecategorised as surgical site infection, venous thromboembolism (deep vein thrombosis andpulmonary embolus), implant related (such as dislocation), and non-implant related (such aspneumonia). Outcomes were compared between groups, with malnutrition defined as serumalbumin <3.5g/dL, transferrin <200 mg/dL, or TLC <1,500 cells/mm³. Potential malnutrition was present in 9.3% of the study population. This group experienced a longeraverage LOS at 6.5 days compared to the normal albumin group at 5.0 days (p=0.003). Surgical siteinfection rate was higher in the malnourished group (12.5 vs 7.8%, p=0.02). There was no differencebetween the two groups in implant related complications (0.8 vs 1.0%, p=0.95) medicalcomplications (7.8 vs 13.3%, p=0.17), rate of venous thromboembolism (2.3 vs 2.7%) or 90-dayreadmission rate (14.1 vs 17.0%, p=0.56). TLC and transferrin were not predictive of any of theprimary outcomes measured (p<0.05). Pacific Island (p<0.001), Indian (p=0.02) and Asian (p=0.02) patients had lower albumin than NZ European. This study demonstrates an association between low albumin levels and increased postoperativeLOS and surgical site infection in total joint arthroplasty, providing rationale for consideration ofpreoperative nutritional screening and optimisation

Tendinopathy can commonly occur around the foot and ankle resulting in isolated rupture, debilitating pain and degenerative foot deformity. The pathophysiology and key cells involved are not fully understood. This is partly because the dense collagen matrix that surrounds relatively few resident cells limits the ability of previous techniques to identify and target those cells of interest. In this study, we apply novel single cell RNA sequencing (CITE-Seq) techniques to healthy and tendinopathic foot/ankle tendons. For the first time we have identified multiple sub-populations of cells in human tendons. These findings challenge the view that there is a single principal tendon cell type and open new avenues for further study. Healthy tendon samples were obtained from patients undergoing tendon transfer procedures; including tibialis posterior and FHL. Diseased tendon samples were obtained during debridement of intractable Achilles and peroneal tendinopathy, and during fusion of degenerative joints. Single cell RNA sequencing with surface proteomic analysis identified 10 sub-populations of human tendon derived cells. These included groups expressing genes associated with fibro-adipogenic progenitors (FAPs) as well as ITGA7+VCAM1- recently described in mouse muscle but, as yet, not human tendon. In addition we have identified previously unrecognised sub-classes of collagen type 1 associated tendon cells. Each sub-class expresses a different set of extra-cellular matrix genes suggesting they each play a unique role in maintaining the structural integrity of normal tendon. Diseased tendon harboured a greater proportion of macrophages and cytotoxic lymphocytes than healthy tendon. This inflammatory response is potentially driven by resident tendon fibroblasts which show increased expression of pro-inflammatory cytokines. Finally, identification of a previously unknown sub-population of cells found predominantly in tendinopathic tissue offers new insight into the underlying pathophysiology. Further work aims to identify novel proteins targets for possible therapeutic pathways

Aims. As the population ages and the surgical complexity of lumbar spinal surgery increases, the preoperative stratification of risk becomes increasingly important. Understanding the risks is an important factor in decision-making and optimizing the preoperative condition of the patient. Our aim was to determine whether the modified five-item frailty index (mFI-5) and nutritional parameters could be used to predict postoperative complications in patients undergoing simple or complex lumbar spinal fusion. Methods. We retrospectively reviewed 584 patients who had undergone lumbar spinal fusion for degenerative lumbar spinal disease. The 'simple' group (SG) consisted of patients who had undergone one- or two-level posterior lumbar fusion. The 'complex' group (CG) consisted of patients who had undergone fusion over three or more levels, or combined anterior and posterior surgery. On admission, the mFI-5 was calculated and nutritional parameters collected. Results. Complications occurred in 9.3% (37/396) of patients in the SG, and 10.1% (19/167) of patients in the CG. In the SG, the important predictors of complications were age (odds ratio (OR) 1.036; p = 0.002); mFI-5 (OR 1.026 to 2.411, as score increased to 1 ≥ 2 respectively. ;. p = 0.023); albumin (OR 11.348; p < 0.001); vitamin D (OR 2.185; p = 0.032); and total lymphocyte count (OR 1.433; p = 0.011) . In the CG, the predictors of complications were albumin (OR 9.532; p = 0.002) and vitamin D (OR 3.815; p = 0.022). Conclusion. The mFI-5 and nutritional status were effective predictors of postoperative complications in the SG, but only nutritional status was successful in predicting postoperative complications in the CG. The complexity of the surgery, as well as the preoperative frailty and nutritional status of patients, should be considered when determining if it is safe to proceed with lumbar spinal fusion. Cite this article: Bone Joint J 2020;102-B(12):1717–1722

Abstract. Introduction. It is increasingly evident that synovium may play a larger role in the aetiology of osteoarthritis. We compared gene expression in whole tissue synovial biopsies from end-stage knee osteoarthritis and knee trauma patients with that of their paired explant cultures to determine how accurately cultured cells represent holistic synovial function. Methodology. Synovial tissue biopsies were taken from 16 arthroplasty patients and 8 tibial plateau fracture patients with no osteoarthritis. Pairs of whole tissue fragments were either immediately immersed in RNAlater Stabilisation Solution at 4o C before transfer to -80o C storage until RNA extraction; or weighed, minced and cultured at 500mg tissues/5ml media in a humidified incubator at 37oC, 5% CO2. After sub-culturing total RNA was extracted using RNAeasy Plus Mini Kit with gDNA removal. Following RT-PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed. Results. PCA analysis illustrates the clear separation of expression array data from cultured cells compared with their parental whole tissues and no segregation between cells derived from osteoarthritic or trauma tissues. A differentially expressed gene heat map demonstrated the hierarchical independence of cultured cells from their paired sample parental tissues. The biological pathways enriched by these gene expression differences emphasise the activities of macrophages and lymphocytes lost from culture. Conclusion. Adherent synovial cells grown from different knee pathologies lose the expression patterns characteristic of their originating pathology. Interpretation of data needs caution as the cells are not representative of whole synovium

Wear and corrosion debris generated from total hip replacements (THR) can cause adverse local tissue reactions (ALTR) or osteolysis, often leading to premature implant failure. The tissue response can be best characterized by histopathological analysis, which accurately determines the presence of cell types, but is limited in the characterization of biochemical changes (e.g. protein conformation alteration). Fourier transform infrared micro-spectroscopy imaging (FTIRI) enables rapid analysis of the chemical structure of biological tissue with a high spatial resolution, and minimal additional sample preparation. The data provides the most information through multivariate method carried out by hierarchical clustering analysis (HCA). It is the goal of this study to demonstrate the beneficial use of this multivariate approach in providing pathologist with biochemical information from cellular and subcellular organization within joint capsule tissue retrieved from THR patients. Joint capsule tissue from 2 retrieved THRs was studied. Case 1: a metal-on-polyethylene THR, and Case 2: a dual modular metal-on-metal THR. Prior to FTIRI analysis, tissue samples were formalin-fixed paraffin-embedded and 5μm thick microtome sectioned samples were prepared and mounted on BaF. 2. discs and deparaffinized. FTIRI data were collected using high-definition transmission mode (pixel size: ∼1.1 μm. 2. ). Hyperspectral images were exported to CytoSpec V2.0.06 for processing and reconstruction into pseudo-color maps based on cluster assignments. Case 1 exhibited a strong presence of lymphocytes and macrophages (Fig. 1a). Since the process of taking second derivatives reduces the half width of the spectral peaks, it increases the sensitivity toward detecting shoulders or second peaks that may not be apparent in the raw spectra (Fig. 1b). Thus, areas occupied by lymphocytes and macrophages can be easily distinguished providing a fast tissue screening method. Here, HCA was able to distinguish macrophages and lymphocytes based on the infrared response, even in areas where both occurred intermixed. (Fig. 1c) The tissue in direct proximity to cells had a slightly altered collagenous structure. Case 1 also exhibited multiple glassy, green particles which can typically observed around THRs that underwent taper corrosion (Fig. 2a). HCA image was able to visualize and distinguish large CrPO. 4. particles, embedded within fibrin exudate rich areas, collagenous tissue without inflammatory cells, and a nearby area with a strong macrophage presence and some finer CrPO. 4. particles (Fig. 2d). Moreover, this method can not only locate macrophages, but distinguish particle-laden macrophages depending the type of particles within the cells. In Case 2 (Fig. 3a), clustering results (Fig. 3 b&c) are consistent with the fact that different particle types are associated with MoM bearing surface wear (Co rich particles), corrosion of the CoCrMo taper junctions (Cr-oxides and –phosphate), fretting of Ti-alloy dual modular tapers (Ti-oxides, Ti alloy particles), and even suture debris, which all occurred in this case. Although details of debris types are not available, specifications are possible by coupling other techniques. The results demonstrate that multivariate FTIRI based spectral histopathology is a powerful tool to characterize the chemical structure and foreign body response within periprosthetic tissue, thus providing insights into the biological impact of different types of implant debris. For any figures or tables, please contact the authors directly

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common and aggressive adult soft tissue sarcomas (STS). Once metastatic, UPS is rapidly fatal. Most STS, including UPS, are resistant to conventional immunotherapies as these tumours have low numbers of spontaneous tumour infiltrating lymphocytes (TILs) and are densely populated with immune suppressive macrophages. Intra-tumoural activation of the STimulator of INterferon Genes (STING) pathway is a novel immunotherapeutic strategy to recruit anti-tumour TILs into the tumour microenvironment. In a murine model of UPS, we have demonstrated that intra-tumoural injection of a murine-specific STING agonist, DMXAA, results in profound immune mediated tumour clearance. Recently, molecules capable of activating both human and mouse STING pathways have been developed. In pursuit of clinically relevant therapeutic opportunities, the purpose of this study is to evaluate the anti-tumour potential of two agonists of the human and murine STING receptors: ADU-S100 and MSA-2 as monotherapies and in combination with the immune checkpoint inhibitor, anti-PD1 in a murine model of UPS. Immune competent mice were engrafted with murine UPS cells in the hindlimb muscle. Once palpable, mice in the monotherapy group were treated with a single intra-tumoural dose of 1) ADU-S100 or 2) MSA-2 or 3) DMXAA. In additional experimental groups, mice were treated with the different STING agonists and monoclonal anti-PD1. Tumour volume measurements and tumour bioluminescence were measured over time. To quantify dynamic changes in immune populations and in the tumour immune microenvironment, STING treated UPS tumours were evaluated using flow cytometry and mRNA quantification at various timepoints after therapy. DMXAA monotherapy produced complete tumour eradication in 50% of mice, whereas both ADU-S100 or MSA-2 monotherapy only extended survival but did not result in complete tumour clearance. Flow cytometry and transcriptional profiling of tumours at multiple timepoints post-treatment showed similar inflammatory changes and increased TILs numbers across all STING agonists. The addition of anti-PD1 treatment to STING therapy significantly extended survival times with both ADU-S100 and MSA-2, and resulted in 14% complete tumour clearance with ADU-S100. No complete survivors were observed with MSA-2-anti-PD1 combinations therapy. STING activation is a promising immunotherapeutic strategy for UPS. Recently developed human STING agonists are not as effective as DMXAA despite similar immunologic responses to treatment. STING and anti-PD-1 treatment were therapeutically synergistic for both human STING agonists. These results justify further research around STING activation as a therapeutic modality for STS. DMXAA may possess additional off-target therapeutic properties beyond STING activation which warrants further investigation. Elucidating these differences may be critical to further optimize STING therapy for human STS

Aims. This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. Methods. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results. Macrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor. Conclusion. The results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss. Cite this article: Bone Joint Res 2020;9(11):827–839

Cobalt chrome alloy is commonly used in joint replacement surgery. However, it is recognised that some patients develop lymphocyte mediated delayed type hypersensitivity (DTH) responses to this material, which may result in extensive bone and soft tissue destruction. Phase 1. United Kingdom: From an existing database, we identified extreme phenotype patient groups following metal on metal (MoM) hip resurfacing or THR: ALVAL with low wearing prostheses; ALVAL with high wear; no ALVAL with high wear; and asymptomatic patients with implants in situ for longer than ten years. Class I and II HLA genotype frequency distributions were compared between these patients’ groups, and in silico peptide binding studies were carried out using validated methodology. Phase 2. United Kingdom: We expanded the study to include more patients, including those with intermediary phenotypes to test whether an algorithm could be developed incorporating “risk genotypes”, patient age, sex and metal exposure. This model was trained in phase 3. Phase 3. United Kingdom, Australia, United States. Patients from other centres were invited to give DNA samples. The data set was split in two. 70% was used to develop machine learning models to predict failure secondary to DTH. The predictions were tested using the remaining blinded 30% of data, using time-dependent AUROCs, and integrated calibration index performance statistics. A total of 606 DNA samples, from 397 males and 209 female patients, were typed. This included 176 from patients with failed prostheses, and 430 from asymptomatic patients at a mean of >10 years follow up. C-index and ROC(t) scores suggested a high degree of discrimination, whilst the IBS indicated good calibration and further backed up the indication of high discriminatory ability. At ten years, the weighted mean survival probability error was < 4%. At present, there are no tests in widespread clinical use which use a patient's genetic profile to guide implant selection or inform post-operative management. The algorithm described herein may address this issue and we suggest that the application may not be restricted to the field of MoM hip arthroplasty

Aims. The aim of this study was to evaluate the diagnostic value of preoperative serum CRP, white blood cell count (WBC), percentage of neutrophils (%N), and neutrophil to lymphocyte ratio (NLR) when using the fracture-related infection (FRI) consensus definition. Methods. A cohort of 106 patients having surgery for suspected septic nonunion after failed fracture fixation were studied. Blood samples were collected preoperatively, and the concentration of serum CRP, WBC, and differential cell count were analyzed. The areas under the curve (AUCs) of diagnostic tests were compared using the z-test. Regression trees were constructed and internally cross-validated to derive a simple diagnostic decision tree. Results. Using the FRI consensus definition, 46 patients (43%) were identified as infected. Sensitivity, specificity, and AUC of CRP were 67% (95% confidence interval (CI) 52% to 80%), 61% (95% CI 47% to 74%), and 0.64 (95% CI 0.54 to 0.74); of WBC count were 17% (95% CI 9% to 31%), 95% (95% CI 86% to 99%), and 0.57 (95% CI 0.50 to 0.62); of %N 13% (95% CI 6% to 26%), 87% (95% CI 76% to 93%), and 0.50 (95% CI 0.43 to 0.56); and of NLR 28% (95% CI 17% to 43%), 80% (95% CI 68% to 88%), and 0.54 (95% CI 0.46 to 0.63), respectively. A better performance of serum CRP was shown in comparison to the leucocyte count (p = 0.006), %N (p < 0.001), and NLR (p = 0.001). A statistically lower serum CRP level was shown in patients with an infection caused by a low virulence microorganism in comparison to high virulence bacteria (p = 0.008). We found that a simple decision tree approach using only low serum neutrophils (< 3.615 × 10. 9. /l) and low CRP (< 2.45 mg/l) may allow better identification of aseptic cases. Conclusion. The evaluated serum inflammatory markers showed limited diagnostic value in the preoperative diagnosis of FRI when using the uniform FRI Consensus Definition. Therefore, they should remain as suggestive criteria in diagnosing FRI. Although CRP showed a higher performance in comparison to the other serum markers, it is insufficiently accurate to diagnose a septic nonunion, especially when caused by low virulence microorganisms. Cite this article: Bone Joint J 2020;102-B(7):904–911

Introduction. In 2010, a widely used metal-on-metal hip implant was voluntarily recalled from the market by the manufacturer. Our lab performed detailed retrieval analysis of 548 of the explanted devices and 165 periprosthetic tissue specimens. We reported a high degree of variability in the multiple measurements that we performed on the components, including volumetric wear. Other studies using semi-quantitative tissue grading methods to describe the histology of the periprosthetic tissues from metal-on-metal hips have looked for relationships between component wear and histological features. Grammatopoulos et al found higher ALVAL scores in 45 hip resurfacing patients with pseudotumors compared to 11 without pseudotumors and a moderately positive correlation between wear and the histological rankings for tissue necrosis and lymphocytic response. We examined correlations between the component wear measurements from retrieval analysis and the tissue features in this large group of one metal-on-metal hip design. Materials and Methods. Tissues were processed routinely into paraffin sections, stained with H&E and characterized with light microscopic semi-quantitative scores for ALVAL features and for the number of macrophages and lymphocytes, extent of metal debris and the estimated percentage of necrosis per slide. Spearman correlation analysis was conducted to evaluate any correlations between retrieval wear measurements and histological findings. Results. The median combined cup and ball wear volume was 17.5mm3 in these 165 cases. The median tissue ALVAL score was 5.5 out of 10. Component total wear volume was not predictive of the ALVAL score (P = 0.09, R = −0.007) or the number of lymphocytes (P= 0.170, R= −0.077) but there was a positive correlation between wear volume and the ranking for macrophages in the tissues (P< 0.001, R=0.215). No other correlations were found for component wear. The median time to revision was 4.84 years. There was a negative correlation between tissue inflammation ranking (P=0.002, R=−0.183) and for lymphocyte ranking and time to revision (P=0.003, R=−0.173). There was a positive correlation between time to revision and the macrophage ranking (P=0.005, R=0.166). Conclusion. From this large-scale retrieval analysis of one design of metal-on-metal total hip, a high degree of variability was observed over the multiple analyses performed, including volumetric wear and tissue features. This likely reflects our previous observation that no single particular failure mechanism was predominant in this large cohort. Funded by DePuy Synthes Companies of Johnson & Johnson

Aim. The demonstration of the in vivo bactericidal efficacy of a new bone cement with rifampicin contained in microcapsules and its intra-articular release profile. Method. Fifteen New Zealand White rabbits were employed to reproduce periprosthetic infection by intra-articular inoculation of 10. 5. CFU/mL of Staphylococcus aureus ATCC® 29213 using as a target implant a 3D printed stainless steel tibial insert. 7 days after inoculation, the first stage of the two stage exchange was carried out and at this time the animals were divided into two study groups: group C (7 rabbits) that received a spacer with gentamicin and group R (8 rabbits) that received a spacer with gentamicin and rifampicin microcapsules. Response to infection was monitored by clinical (weight and temperature), hematological (leukocyte, lymphocyte and platelet counts) and biochemical (erythrocyte sedimentation rate) analyses at the time of inoculation, at the first stage of exchange, 4 days after first stage and weekly until the fourth week when animals were euthanized. Microbiological counts were performed at the first stage of exchange and at the end of the study. Results. 14/15 animals (93.3%) developed a PJI 1 week after the inoculation. A statistically significant elevation of the leukocyte and platelet count and a decrease in the percentage of lymphocytes (p=0.0001) was found and positive microbiological cultures. Four weeks after the placement of the spacer, no bacterial growth was found in the soft tissue or bone samples of the group with rifampicin microcapsules (group R), being these differences statistically significant with p=0.01 and 0.03 respectively. The rifampicin intra-articular release kinetics showed concentrations above the staphylococcal MIC at all time points. Conclusions. The bone cement with microencapsulated rifampicin is effective in the in vivo treatment of prosthetic joint infection due to biofilm-forming S. aureus

Little is known about the relationship between head-neck corrosion and its effect on the periprosthetic tissues and distant organs of patients hosting well-functioning devices. The purpose of this study was to investigate in postmortem retrieved specimens the degree and type of taper damage, and the corresponding histologic responses in periprosthetic tissues and distant organs. Fifty postmortem THRs (34 primaries, 16 revisions) retrieved after 0.5 to 26 years were analyzed. Forty-three implants had a CoCrMo stem and seven had a Ti6Al4V stem. All heads were CoCrMo and articulated against polyethylene cups (19 XLPE, 31 UHMWPE). H&E sections of joint pseudocapsules, liver, spleen, kidneys and lymph nodes were graded 1–4 for the intensity of various inflammatory cell infiltrates and tissue characteristics. Corrosion damage of the taper surfaces was assessed using visual scoring and quantitated with an optical coordinate measuring machine. SEM analysis was used to determine the acting corrosion mode. Polyethylene wear was assessed optically. The majority of tapers had minimal to mild damage characterized by local plastic deformation of machining line peaks. Imprinting of the stem topography onto the head taper surface was observed in 18 cases. Column damage on the head taper surface occurred in three cases. All taper surfaces scored moderate or severe exhibited local damage features of fretting and/or pitting corrosion. Moderate or severe corrosion of the head and/or trunnion was present in nine hips. In one asymptomatic patient with bilateral hips, lymphocyte-dominated tissue reactions involving perivascular infiltrates of lymphocytes and plasmacytes were observed. In this patient, mild, focal lymphocytic infiltrates were also present in the liver and kidneys, and there was focal histiocytosis and necrosis of the para-aortic lymph nodes. These two implants, which had been in place for 58.6 and 60.1 months, had severe intergranular corrosion of the CoCrMo trunnion, and column damage and imprinting on the head taper. In the other 41 hips, macrophage responses in the joint pseudocapsule to metallic and/or polyethylene wear particles ranged widely from minimal to marked. Focal necrosis in the pseudocapsules of 12 arthroplasties was related to high concentrations of CoCrMo, TiAl4V, TiO, BaSO4 and polyethylene wear particles. High concentrations of these particles were also detected in para-aortic lymph nodes. Rare to mild macrophages were observed in liver and spleen. This is a comprehensive study of wear and corrosion within well-functioning postmortem retrieved THRs, and the resulting local and distant tissue reactions. One of eight patients with moderate or severe corrosion did have a subclinical inflammatory response dominated by lymphocytes after five years. To what extent such an inflammatory process might progress to become symptomatic is not known. Ionic and particulate products generated by corrosion disseminated systemically. The minor lymphocytic infiltrate in the liver and kidneys of one subject with bilateral severely corroded head-neck junctions might suggest possible metal toxicity. The diagnosis of adverse tissue reactions to corrosion of modular junctions can be challenging. Postmortem retrieval studies add to our understanding of the nature and progression of lymphocyte-dominated adverse local and potentially systemic tissue reactions to corrosion of modular junctions

Introduction and Objective. The early pro-inflammatory hematoma phase of bone healing is characterized by platelet activation followed by growth factor release. Bone marrow mesenchymal stromal cells (MSC) play a critical role in bone regeneration. However, the impact of the pro-inflammatory hematoma environment on the function of MSC is not fully understood. We here applied platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of MSC in comparison to a non-inflammatory control environment simulated by fibrin (FBR) hydrogels. Materials and Methods. MSC were isolated from acetabular bone marrow of patients undergoing hip arthroplasty. PRP was collected from pooled apheresis thrombocyte concentrates. The phenotype of MSC was analyzed after encapsulation in hydrogels or exposure with platelet-derived factors with regards to gene expression changes, cell viability, extracellular vesicle (EV) release and immunomodulatory effects utilizing cellular and molecular, flow cytometry, RT-PCR, western blot and immunofluorescence stainings. Results. Our results showed that encapsulation of MSC in PRP induced changes in cell metabolism increasing lactate production and reducing mitochondria membrane potential. This was followed by significantly decreased mTOR phosphorylation and differential gene regulation. While PRP-released factors could support EV-biogenesis and immunoregulation-related gene expression, FBR hydrogel reduced CD63+ and CD81+ EV release by MSC. In co-cultures with mitogen stimulated PBMC, pre-exposure of MSC with PRP reduced the proliferation rate and frequency of peripheral blood CD4. +. and favored the persistence of FOXP3. +. regulatory T lymphocytes (32±4.7% compared to 9±2.3% in control co-cultures where MSC were exposed to FBR). Conclusions. Our data indicate that exposure of MSC with a hematoma environment causes metabolic adaptation of MSC followed by increased immune regulatory functions, which in turn might contribute to resolution of inflammation required for successful bone healing

Cytological analysis of material aspirated from the effusion which occasionally develops around a polyglycolic acid (PGA) osteosynthesis implant showed a predominance of inflammatory monocytes and in particular lymphocytes. In order to discover whether PGA implants are immunologically inert, density gradient-isolated peripheral blood mononuclear cells were cultured in 0.2 ml of 10% delta FCS-RPMI 1640 culture medium supplemented with 10 mg PGA. Phytohaemagglutinin (PHA) lectin, a purified protein derivate of tuberculin (PPD) antigen and culture medium alone were used as positive and negative controls. We studied lymphocyte activation kinetics on days 0, 1, 3 and 5. Major histocompatibility complex locus II antigen (MHC locus II antigen) and interleukin-2 receptor (IL-2R) expression were analysed using the avidin-biotin-peroxidase complex (ABC) method and lymphocyte DNA synthesis by using 3H-thymidine incorporation and beta-scintillation counting. Especially on culture days 0 and 1, lymphocytes and monocytes were seen by light microscopy to be attached to PGA particles. However, our results show no PGA-induced lymphocyte DNA synthesis, but PGA-induced MHC locus II antigen and IL-2R activation marker expression was seen, greater than in negative controls, but less than that seen in PPD antigen driven lymphocyte response. This suggests that PGA is an immunologically inert implant material, but it does seem to induce inflammatory mononuclear cell migration and adhesion, leading to a slight non-specific lymphocyte activation. This activation is lower than that seen in mitogen and antigen-driven lymphocyte responses

We have studied the relationship between metal ion levels and lymphocyte counts in patients with metal-on-metal hip resurfacings. Peripheral blood samples were analysed for lymphocyte subtypes and whole blood cobalt and chromium ion levels in 68 patients (34 with metal-on-metal hip resurfacings and 34 with standard metal-on-polyethylene total hip replacements). All hip components were radiologically well-fixed and the patients were asymptomatic. Cobalt and chromium levels were significantly elevated in the patients with metal-on-metal hip resurfacings, compared with the patients with standard metal-on-polyethylene designs (p < 0.0001). There was a statistically significant decrease in the level of CD8. +. cells (T-cytotoxic/suppressor) (p = 0.005) in the metal-on-metal hip resurfacing group. A threshold level of blood cobalt and chromium ions was associated with reduced CD8. +. T-cell counts. We have no evidence that our patients suffered as a result of this reduced level of CD8. +. T-cells

Aim. The diagnosis of septic arthritis mostly relies on clinical examination, several blood parameters including white blood cell count, C-reactive protein, sedimentation, and the analysis of the joint aspiration. However, the diagnosis can be difficult when the symptoms are vague and the information obtained from laboratory might be insufficient for definitive diagnosis. This study aimed to evaluate several ratios obtained from routine blood tests for a possible use in the diagnosis of septic arthritis. Method. The adult patients who were operated in our clinic due to septic arthritis between 2014–2020 were identified and retrospectively evaluated. The patients with any blood disorders or missing file information were excluded. A total of 36 patients were found to be eligible for inclusion. The control group included 40 patients without any sign of infection who underwent total knee arthroplasty due to knee osteoarthritis. Preoperative blood tests of each patients were examined. In addition to CRP and sedimentation values, neutrophil-lymphocyte, monocyte-lymphocyte, platelet-lymphocyte, and platelet count-mean platelet volume were calculated and receiving operating characteristics (ROC) curve analysis was made to determine the sensitivity, specificity and area under curve (AUC) values of these parameters. Results. The distribution of affected joint in septic arthritis group was as follow; 22 knees, 6 hips, 4 shoulders, 2 elbows, 1 wrist and 1 ankle. The cultures of joint aspiration yielded positive result in 19 patients while the cultures were negative in 17 patients. All of the analyzed parameters were significantly different between the groups (p<0.001). ROC curve analysis results are given in detail, in Table 1 and Figure 1. The AUC value was 97.3 when only CRP and sedimentation values were used but increased to 98.6 when neutrophile/ lymphocyte ratio was added and increased to 100 when all analyzed parameters were included. Conclusions. The analyzed parameters were found to increase the overall sensitivity and specificity when used together with acute phase reactants. However, when evaluated separately, CRP and sedimentation were still found as the most valuable parameters in the diagnosis of septic arthritis. In the diagnosis of septic arthritis, 35 mm/hr cut-off value for sedimentation and 10 mg/L cut-off value for CRP were found more sensitive and specific compared to standard laboratory cut-off values of 20 mm/hr and 5 mg/L. For any tables or figures, please contact the authors directly

Introduction. The combined incidence of anatomic (aTSA) and reverse total shoulder arthroplasties (rTSA) in the US is 90,000 per annum and rising. There has been little attention given to potential long-term complications due to periprosthetic tissue reactions to implant debris. The shoulder has been felt to be relatively immune to these complications due to lower acting loads compared to other joint arthroplasties. In this study, retrieved aTSAs and rTSAs were examined to determine the extent of implant damage and to characterize the nature of the corresponding periprosthetic tissue responses. Methods. TSA components and periprosthetic tissues were retrieved from 23 (eleven aTSA, twelve rTSA). Damage to the implants was characterized using light microscopy. Head/stem taper junction damage was graded 1–4 as minimal, mild, moderate or marked. Damage on polyethylene (PE) and metal bearing surfaces was graded 1–3 (mild, moderate, marked). H&E stained sections of periprosthetic soft tissues were evaluated for the extent and type of cellular response. A semi-quantitative system was used to score (1=rare to 4=marked) the overall number of particle-laden macrophages, foreign body giant cells, lymphocytes, plasma cells, eosinophils, and neutrophils. Implant damage and histopathological patterns were compared between the two TSA groups using the Mann-Whitney and Spearman tests. Results. The PE bearing surfaces of aTSAs were dominated by three-body wear and plastic deformation, whereas the rTSA PE components exhibited mainly polishing and scratching. Metal surface damage occurred in a few cases of both groups. Only one aTSA case exhibited marked taper corrosion. In both groups the primary nature of the inflammatory response was a moderate to marked macrophage response to wear particles (78% of cases). The particle-laden macrophages tended to occur in broad sheets and contained metal, PE, bone cement and suture debris. The extent of macrophage and foreign body giant cell responses was greater in the aTSA group (p≤0.001). Metal particles were seen in 63% of aTSAs and 83% of rTSAs. In the aTSA group, bone cement was seen in all cases and suture was observed in 9 cases, and their presence was larger compared to the rTSA group (p≤0.022). There was no difference in the number of other cell types between the groups. A mild lymphocyte response and chromium-phosphate debris was present within the tissue of the aTSA case with marked corrosion, which may be indicative of an early stage adverse local tissue reaction (ALTR) analog to total hip replacements with taper corrosion. Conclusion. Both groups exhibited a strong macrophage response to a combination of different types of implant debris—PE, metal, bone cement and suture. The prevalence of a marked macrophage response was larger in the aTSA group which may be explained by the larger overall presence of cement and suture within this group. PE particles may differ in size between groups due to different acting wear mechanisms which may also affect the extent of the macrophage response. Although corrosion within modular junctions was overall rare, the presence of one case with marked corrosion shows that taper corrosion and subsequent ALTRs are possible in TSAs. For any figures or tables, please contact authors directly

Currently, there is considerable interest in the role that metal allergy may play in the clinical performance of orthopaedic devices. The extant literature suggests that metal allergy is a real clinical phenomenon, albeit the prevalence and clinical impact are not defined. Degradation products in the form of ionic or particulate debris can complex with local proteins and alter their conformation so that they may not be recognised as self-proteins. This can result in an adaptive immune response. The typical paradigm proposed for such an allergy is that of a delayed type hypersensitivity response (Type 4) whereby the antigenic stimulus interacts with antigen presenting cells and T lymphocytes to elicit a cell mediated immune response. There is some evidence that patients with metal-on-metal bearings and/or high serum metal levels elicit more response to metal antigen challenge measured as either patch test sensitivity or lymphocyte proliferation. Thus, while there is an idiosyncratic aspect of the allergic response, there is also a dose response component. The diagnosis of metal allergy remains a challenge as patch testing has not been shown to correlate well with clinical symptoms. In-vitro assays, such as lymphocyte transformation testing, have promise but await robust clinical validation before they can be considered reliable diagnostic testing modalities. Allergy to implanted metal orthopaedic devices is a rare clinical event, and is a diagnosis of exclusion. Revision surgery should be considered a last resort with the understanding that the outcomes are unpredictable. Given the limitations of current diagnostic modalities, widespread screening of patients for metal allergies prior to TKA is not recommended

Early failure associated with adverse reactions to metal debris is an emerging problem after hip resurfacing but the exact mechanism is unclear. We analysed our entire series of 660 metal-on-metal resurfacings (Articular Surface Replacement (ASR) and Birmingham Hip Resurfacing (BHR)) and large-bearing ASR total hip replacements, to establish associations with metal debris-related failures. Clinical and radiological outcomes, metal ion levels, explant studies and lymphocyte transformation tests were performed. A total of 17 patients (3.4%) were identified (all ASR bearings) with adverse reactions to metal debris, for which revision was required. This group had significantly smaller components, significantly higher acetabular component anteversion, and significantly higher whole concentrations of blood and joint chromium and cobalt ions than asymptomatic patients did (all p < 0.001). Post-revision lymphocyte transformation tests on this group showed no reactivity to chromium or cobalt ions. Explants from these revisions had greater surface wear than retrievals for uncomplicated fractures. The absence of adverse reactions to metal debris in patients with well-positioned implants usually implies high component wear. Surgeons must consider implant design, expected component size and acetabular component positioning in order to reduce early failures when performing large-bearing metal-on-metal hip resurfacing and replacement

The dynamic association between the immune system and the skeletal system has recently been appreciated. It has been suggested that cells involved in the inflammatory cascade might modulate the bone fracture repair process. Interestingly a number of studies have demonstrated that ability of the T lymphocyte to affect bone remodelling and health profoundly. For example the presence of T lymphocytes has been shown to increase bone resorption during experimental induced arthritis. We wanted to investigate the role of specific T lymphocytes in fracture repair and required an in vivo model to deplete CD4 and CD8 T lymphocytes selectively. Method The cell lines of Anti-L3T4 (CD4), Anti-Lyt-2 (CD8) and Anti-phytochrome were obtained from ECACC and produced by Edinburgh University group, Immunosolv. Anti-phytochrome (AFRC MAC 51) antibody was used as the control. To each group of 5 murine models 50ìL of CD4 or CD8 or control antibody was injected ip on days 0, 1, 2, 7, 8, and 9. The body weight and behaviour were measured. On day 20 the spleens were sampled and a single cell suspension was created for each murine model. PE CD8 antibody and FITC CD4 antibody were then added to each sample. Each spleen sample was then cell sorted using the FACS machine. Results Compared to the control group the murine model injected with CD4 antibody had only 1.14 % of CD4 T cells remaining (mean 2.462 % +/− 0.270). Similarly, the murine models injected with CD8 antibody had only 1.8% of CD8 T cells remaining (mean 1.723 % +/− 1.036). Conclusion Our results suggest that to investigate the role of T lymphocytes in fracture repair, specific T lymphocytes can be successfully depleted with the repeated use of antibodies

Aim. Although established serum inflammatory biomarkers, such as serum C-reactive protein (CRP) and serum white blood cell count (WBC), showed low accuracies in the literature, they are still commonly used in diagnosing periprosthetic joint infections (PJI). For a sufficient preoperative diagnosis novel more accurate serum parameters are needed. The aim of our study was to evaluate the performances of the established and novel routinely available serum parameters in diagnosing periprosthetic joint infections when using the proposed European Bone and Joint Infection Society (pEBJIS) criteria. Method. In this retrospective study, 177 patients with an indicated revision surgery after a total joint replacement were included from 2015 to 2019. The easily accessible and routinely available serum parameters CRP, WBC, the percentage of neutrophils (%N), the neutrophils to lymphocytes ratio (NLR), fibrinogen and the platelet count to mean platelet volume ratio (PC/mPV) were evaluated preoperatively. The performances were examined via receiver operating characteristic (ROC) curve analysis (AUC). The curves were compared using the z-test. Seventy-five cases (42%) showed a PJI based on the pEBJIS-criteria. Results. The sensitivities of serum CRP (cut-off: ≥10mg/L), WBC (≥10×10^9 cells/L), %N (≥69.3%), NLR(≥ 3.82), fibrinogen (≥ 457 mg/dL), and PC/mPV (≥ 29.4) were calculated with 68% (95% CI: 57–78), 36% (26 – 47), 66% (54 – 76), 63% (51 – 73), 69% (57 – 78), and 43% (32 – 54), respectively. Specificities were 87% (79 – 93), 89% (81 – 94), 67% (57 76), 73% (63 – 81), 89% (80 – 93), and 81% (72 – 88), respectively. Serum CRP and fibrinogen showed better performances than the other evaluated serum parameters (p<0.0001). The median serum CRP (17.6 mg/L) in patients with PJI caused by a low virulence microorganism was lower compared with infections caused by high virulence organisms (49.2 mg/L; p=0.044). Synovial fluid leucocyte count and histology showed better accuracies than serum CRP, serum WBC, %N, NLR, serum fibrinogen, and PC/mPV (p<0.0001). Conclusions. Although serum CRP and fibrinogen showed the best performances among the evaluated serum inflammatory markers, their results should be interpreted with caution in clinical practice. Serum parameters may remain normal in chronic infections or may be elevated in patients with other inflammatory conditions. In addition, they also correlated poorly with synovial fluid leukocyte count and histology. Therefore, serum parameters are still insufficient to confirm or exclude a periprosthetic joint infection. Hence, they can only be recommended as suggestive criteria in diagnosing PJI

Mesenchymal stem cells (MSC) are multipotent cells that possess regenerative functions that are of interest for in osteoarticular diseases such as osteoarthritis (OA). These functions are thought to be primarily mediated by mediators released within extracellular vesicles (EV). The aim of this study was to compare the immunomodulatory effects of two major types of EV, exosomes and microparticles, secreted by MSCs. EV subsets were isolated from murine primary MSCs by ultracentrifugation. Size and structure were evaluated by Dynamic Light Scattering and electron microscopy. Expression of membrane and endosomal markers was tested by flow cytometry. Proliferation of murine splenocytes was quantified after 72h of incubation with EVs after CFSE-labelling. Phenotypic analysis of T lymphocyte subpopulations was also performed by flow cytometry. In vivo, EVs were injected in the knee joint in the collagenase-induced osteoarthritis (CIOA) model and histological score was performed. In vitro functional analysis indicated that addition of microparticles or exosomes in proliferative assays inhibited the proliferation of total splenocytes in a dose-dependent manner. Analysis of T cell subpopulations revealed a decrease in CD8. +. IFNγ. +. lymphocytes and an increase in both CD4. +. IL10. +. Tr1 and CD4. +. CD25. +. FOXP3. +. Treg cells. This immunomodulatory function of EVs was also observed in vivo in the CIOA model. In summary, our data indicated that the immunosuppressive effect of MSCs is in part mediated by exosomes and microparticles that play in vivo a major role in MSC-mediated therapeutic effect by reducing osteoarthritic symptoms