Bone tissue engineering attempts at substituting critical size bone defects with scaffolds that can be primed with osteogenic cells, usually mesenchymal stem cells (MSC) from the bone marrow. Although overlooked, peripheral blood is a valuable source of MSC and circulating osteoprogenitors (COP), bearing a significant regenerative potential, and peripheral blood is easier to access than bone marrow. We thus studied osteodifferentiation of peripheral blood mononuclear cells (pbMNC) under different culture conditions, and how they compared to primary human osteoblasts. pbMNC were isolated from healthy adult volunteers by Ficoll density gradient centrifugation, and they were then cultured using media supplemented with 100nM Dexamethasone, 10mM sodium β-glycero phosphate and ascorbic acid (either 40mM or 0.05mM). For comparison, primary osteoblasts were isolated from the femoral heads of patients undergoing hip arthroplasty. After 4 weeks of culture, osteogenic activation was quantified with spectrometric measurement of alkalic phosphatase (ALP) and lactate dehydrogenase (LDH) levels. The extent of osteoid mineralization was measured with Alizarin red staining. We studied the effects of 1) varying cell concentration at seeding, 2) surface coating of culture wells with collagen and 3) high compared to low ascorbic acid (40mM and 0.05mM) media. Higher numbers of pbMNC (0.5–5.9 versus 0.062–0.25 million cells per well) at seeding resulted in a lower ALP/LDH-ratio (mean ± standard deviation), 0.39 ± 0.33 arbitrary units (AU) versus 1.36 ± 1.06 AU, but led to higher amount of osteoid production, 0.10 ± 0.06 versus 0.065 ± 0.02 AU, p < 0.05. Culture of pbMNC on collagen did not confer any difference in ALP/LDH-ratios, with 0.43 ± 0.3 AU for collagen-coated and 0.43 ± 0.41 AU for uncoated wells (p = 0.95), and we also observed no relevant difference in osteoid production (0.07 ± 0.01 AU for collagen-coated versus 0.1 ± 0.08 AU for uncoated wells, p = 0.28). Cultures of pbMNC on collagen in media supplemented with a higher concentration of ascorbic acid showed a 130% higher ALP/LDH-ratio when compared to cultures exposed to a lower ascorbic acid concentration (p < 0.05). Cultures with a low initial concentration of pbMNC (0.5 − 1 million cells) had no significantly different ALP/LDH-ratio when compared to primary human osteoblasts, but the cultures of pbMNC resulted in a 90% increase in osteoid mineralization when compared to primary human osteoblasts (p < 0.05). These findings indicate that progenitor cells derived from peripheral blood have a significant osteogenic potential, rendering them interesting candidates for seeding of scaffolds intended to fill critical sized bone defects. pbMNC produced almost double the amount of osteoid as primary osteoblasts. The isolation of pbMSC and COP is non-invasive and easy, and they might be seeded directly onto scaffolds without prior ex-vivo expansion, a question that we intend to pursue further

Patients who are Jehovah's witnesses do not accept blood transfusions. Thus, total hip arthroplasty can be challenging in this group of patients due to the potential for blood loss. Multiple strategies have been developed in order to prevent blood loss. A 76-year-old female, Jehovah's witness medicated with a platelet antiaggregant, presented to the emergency department after a fall from standing height. Clinically, she had pain mobilizing the right lower limb and radiological examination revealed an acetabular fracture with femoral head protrusion and ipsilateral isquiopubic fracture. Skeletal traction was applied to the femur during three weeks and no weight bearing was maintained during the following weeks. Posteriorly, there was an evolution to hip osteoarthritis with necrosis of the femoral head. The patient was submitted to surgery six months after the initial trauma, for a total hip arthroplasty. The surgery was performed with hypotensive anaesthesia, careful surgical technique and meticulous haemostasis and there was no need for blood transfusion. Posteriorly, there was a positive clinical evolution with progressive improvement on function and deambulation. Total hip arthroplasty may be safely carried out with good clinical outcomes in Jehovah's witnesses, without the need for blood transfusion, if proper perioperative precautions are taken, as has already been shown in previous studies

Introduction and Objective. In multiple trauma patients, as well as in the healing of isolated fractures (Fx) with heavy bleeding (trauma haemorrhage, TH), complications occur very often. This is particularly evident in elderly patients over 65 years of age. Since these accompanying circumstances strongly influence the clinical course of treatment, the influence of age on bone regeneration after femoral fracture and severe blood loss was investigated in this study. Materials and Methods. 12 young (17–26 weeks) and 12 old (64–72 weeks) male C57BL / 6J mice per group were examined. The fracture group Fx underwent an osteotomy after applying an external fixator. The THFx group also received blood pressure-controlled trauma hemorrhage (35 mmHg for 90 minutes) and reperfusion with Ringer's solution for 30 minutes. The Sham group received only the catheter and one external fixator. μCT scans of the femora were performed in vivo after 2 weeks and ex vivo after 3 weeks. Histological and biomechanical examinations were also carried out. The statistical significance was set at p ≤ 0.05. The non-normally distributed data were analyzed using the Mann-Whitney-U or Kruskal-Wallis test. Results. The histology showed less mineralized bone in the fracture gap in old animals of the Fx (25.41% [1.68%]) and THFx groups (25.50% [4.07%]) compared with the young ones (34.20% [6.36%], p = 0.003; 34.31% [5.12%], p=0.009). Moreover, a severe blood loss lead to more cartilage in both young (6.91% [5.08%]) and old animals (4.17% [1.42%]) compared to animals with only a fracture (2, 45% [1.04%], p=0.004; 2.95% [1.12%], p=0.032). In old animals (11.37 / nm. 2. [17.17 / nm. 2. ]) in contrast the young mice with an isolated fracture (33.6/nm. 2. [8.83/nm. 2. ]) fewer osteoclasts were present (p=0.009). Therefore, the severe blood loss further reduced the number of osteoclasts only in young animals (16.83/nm. 2. [6.07/nm. 2. ]) (p=0.004). In the in vivo μCT, after 2 weeks, a lower volume of bone, cortex and callus was found in old THFx animals (3.14 mm. 3. [0.64 mm. 3. ]); 1.01 mm. 3. [0.04 mm. 3. ]; 2.07 mm. 3. [0.57 mm. 3. ]) compared with the Fx animals (4.29 mm. 3. [0.74 mm. 3. ], p=0.008; 1.18 mm. 3. [0, 25 mm. 3. ], p=0.004; 3.02 mm. 3. [0.77 mm. 3. ], p=0.008) After 3 weeks, the ex vivo μCT scans also showed a reduced callus percentage in old THFx animals (61.18% [13.9 9%]), as well as a low number of trabeculae (1.81 mm. -1. [0.23 mm. -1. ]) compared to animals without blood loss (68.72% [15.71%], p = 0.030; 2.06mm. -1. [0.37mm. -1. ], p=0.041). In the biomechanical test, a reduced elasticity limit of the old THFx mice (7.75 N [3.33 N]) in contrast to the old Fx (10.24 N [3.32 N]) animals was shown (p=0.022). Conclusions. A severe blood loss has a higher negative effect on the healing, morphometry, and biomechanical properties of previously fractured femora in old compared to young individuals

Chronic Achilles tendinopathy is characterised by sub-acute inflammation with pro-inflammatory type 1 macrophages (M1), tissue degeneration and consequent partial or total tendon injury. Control of the inflammatory response and M1-to-M2 macrophage polarisation can favour tendon healing both directly and indirectly, by allowing for the regenerative process driven by local mesenchymal stem cells. Ten patients (3 females and 7 males aged between 32 and 71 years old) with partial Achilles tendon injury were treated with injections of autologous peripheral blood mononuclear cells (PB-MNCs). The cell concentrate was obtained from 100-120 cc of each patient's blood with a selective point-of-care filtration system. PB-MNCs remained trapped in the filter and were injected immediately after sampling. Around 60% of the PB-MNC concentrate was injected directly into the injured area, while the remaining 40% was injected in smaller amounts into the surrounding parts of the Achilles tendon affected by tendinosis. All patients were evaluated both clinically with the help of the American Orthopaedic Foot & Ankle Society (AOFAS) scale, and radiologically (MRI examination) at baseline and 2 months after the PB-MNC injection. A clinical reassessment with the AOFAS scale was also performed 6 months after the intervention. The rehabilitation protocol implied full weight-bearing walking immediately after the procedure, light physical activity 3-4 days after the injection, and physiotherapist-assisted stretching exercises and eccentric training. In all patients, functional and radiological signs of tendon healing processes were detected as early as 2 months after a single treatment and the AOFAS scale rose from the initial mean value of 37.5 (baseline) to 85.4 (6 months). Our preliminary results indicate that regenerative therapies with PB-MNCs can prove useful for partial Achilles tendon injuries as a valid alternative to surgical options, especially when other conservative approaches have failed. Advantages of this therapy include rapid execution, no need for an operating theatre, easy reproducibility, quick recovery and good tolerability regardless of the patient's age (the procedure is not to be performed in subjects who are below 18 years old). Further studies on the topic are recommended to confirm these observations

Blood management protocols attempt to reduce blood loss by strategies including autologous blood donation, red cell salvage, normovolaemic haemodilution and haemostatic agents such as tranexamic acid (TXA). TXA usage in particular has become increasingly commonplace with numerous studies demonstrating a significant reduction in peri-operative blood loss and proportion of patients requiring transfusion, without increasing the risk of venous thromboembolism. Tourniquet usage has now become ubiquitous in TKA operations with reported benefits of improved visualization, shorter operative time and decreased intra-operative bleeding. However, its use is not without considerable complications including wounding dehiscence, increased venous thromboembolism, superficial wound infection and skin blistering. It is therefore imperative that we review tourniquet usage in light of ever evolving blood management strategies. The aim of this study was to evaluate the effect of stopping tourniquet usage in primary TKRs, performed by an experienced surgeon, in light of new blood reduction measures, such as a TXA. A retrospective analysis identified a total of 31 patients who underwent primary TKR without the use of a tourniquet from January 2018 to March 2019. This was compared to an earlier group of patients from the same surgeon undergoing TKR with the use of a tourniquet; dating from July 2016 to November 2017. All surgeries were performed within the same hospital (CXH). Peri-operative factors and outcome measures were collected for analysis. There was no significant difference in post-operative haemoglobin drop (Tourniquet, 23.1 g/L; No Tourniquet, 24.4 g/: p=0.604) and fall in haematocrit (Tourniquet, 0.082; No Tourniquet, 0.087: p=0.604). Allogenic blood transfusion rates were the same in both groups at 12.9% (2 patients) and blood loss was not found to be significantly different (Tourniquet, 1067ml; No tourniquet, 1058mls). No significant difference was found in operative time (Tourniquet, 103 minutes; No Tourniquet, 111.7 minutes: p=0.152) or length of stay (Tourniquet, 5.5 days; No Tourniquet, 5.2 days: p=0.516). Tranexamic acid usage was not found to be significant (p=1.000). ROM of motion and analgesia requirement was significantly better in the no tourniquet group on one post-operative day out of five analysed (p=0.025, p=00.011). No post-operative thromboembolic events were reported in either group. There was no significant difference in readmission rates (p=0.492) or complications (p=0.238). The increase in minor complications and potential increased VTE risk with tourniquet usage must be balanced against an improved visual field and reduced blood loss in TKR patients. Our study found no difference in post-operative blood loss and transfusion rates between tourniquet and no tourniquet groups. With ever evolving and improving blood loss management strategies, including the use of TXA, the application of tourniquet may not be needed. Further prospective RCTs are needed to assess the impact of tourniquet usage in light of this

7–20 % of the patients with a total knee arthroplasty (TKA) are dissatisfied without an indication for revision. Therapeutic options for this patient population with mostly a lack of quadriceps strength are limited. The purpose of this study is to evaluate the effect of six weeks low load resistance training with blood flow restriction (BFR) on the clinical outcome in these unhappy TKA patients. Thirty-one unhappy TKA patients (of the scheduled fifty patients) without mechanical failure were included in this prospective study since 2022. The patients participate in a supervised resistance training combined with BFR, two times a week during nine weeks. Patients were evaluated by the Knee Osteoarthritis Outcome Score (KOOS), Knee Society Score: satisfaction (KSSs) and the Pain Catastrophizing Scale (PCS). Functionality was tested using the Six Minute Walk Test (6MWT) and the 30-Second Chair Stand Test (30CST). Follow-up took place at six weeks, three months and six months after the start. Six weeks training with BFR provided statistically significant improvements in all the KOOS subscales compared to the baseline, especially for symptoms (55.1 (±15.4) versus 48.0 (±16.5); p<0.001), activities in daily living (50.3 (±21.1) versus 43.7 (±17.2); p<0.00) and quality of life (24.6 (±18.5) versus 17.3 (±13.0); p<0.001). The PCS reduced from 27.4 (±11.0) to 23.2 (±11.4) at six weeks (p<0.01), whereas the KSSs increased from 11.8 (±6.5) to 14.9 (±7.6) (p=0.021). Both the 6MWT and the 30CST improved statistically significant from respectively 319.7 (±15.0) to 341.6m (±106.5) (p<0.01) and 8.6 (±3.9) to 9.3 times (±4.5) (p<0.01). Blood flow restriction appears to enhance the quality of life and functional performance of unhappy TKA patients. Based on these preliminary results, BFR seems to be a promising and valuable alternative for these TKA patients with limited therapeutic options

Abstract. Objectives. to evaluate the efficacy and safety of topically applied tranexamic acid (TXA) in thoracolumbar spinal tuberculosis surgery, posterior approach. Methods. Thoracolumbar spine tuberculosis patients who requiring debridement, pedicle screw fixation and fusion surgery were divided into two groups. In the TXA group (n=50), the wound surface was soaked with TXA (1 g in 100 mL saline solution) for 3 minutes after exposure, after decompression, and before wound closure, and in the control group (n=116) using only saline. Intraoperative blood loss, drain volume 48 hours after surgery, amount of blood transfusion, transfusion rate, the haemoglobin, haematocrit after the surgery, the difference between them before and after the surgery, incision infection and the incidence of deep vein thrombosis between the two groups. Results. EBL for the control group was 783.33±332.71 mL and for intervention group 410.57±189.72 mL (p<0.001). The operative time for control group was 3.24±0.38 hours and for intervention group 2.99±0.79 hours (p<0.695). Hemovac drainage on days1 and 2 for control group was 167.10±53.83mL and 99.33±37.5 mL, respectively, and for intervention group 107.03±44.37mL and 53.38±21.99mL, respectively (p<0.001). The length of stay was significantly shorter in the intervention group (4.8±1.1 days) compared to control group (7.0±2.3 days). There was bo different in incision side infection and DVT. Conclusions. Topical TXA is a viable, cost-effective method of decreasing perioperative blood loss in major spine surgery with fewer overall complications than other methods. Further studies are required to find the ideal dosage and timing

This study aims to investigate that a single dose of tranexamic acid (TXA) will reduce blood loss and transfusion rates in elderly patients, undergoing intertrochanteric (IT) or femoral neck fractures surgery. Consecutive elderly patients receiving hip fracture surgery for stable or unstable IT fracture, treated with short intramedullary nail (IMN) insertion as well as cemented hemiarthroplasty for acute femoral neck (subcapital) hip fracture, were screened for inclusion in this single-centre randomized trial. Patients were randomly allocated to a study group by sealed envelope. One TXA dose of 15 mg/kg i.v. diluted in 100 ml N/S or one placebo dose i.v. in 100 ml N/S were administered 5 mins before the skin cut. Haemoglobin (Hb) concentration was measured at admission time and prior to surgery. Post-operatively it was measured on a daily basis until day 4, giving a total of four Hb measurements (days 1 to 4). The transfusion trigger point was determined in accordance with the French guidelines for erythrocyte blood transfusion. The transfusion trigger was 10 g/dl for patients at risk, while in all other cases, it was 9 g/dl. Information regarding the transfusions number was assessed directly by the hospital blood bank database. Blood loss was calculated by the Hb dilution method. Nadler's formula was used to calculate patients' blood volume. For calculation of total blood loss (TBL) expressed to total Hb loss and total Volume loss, the number of transfusions (55 grams of Hb per transfusion), the Hb concentration on preoperatively (Hgbi) and the Hb concentration on the last measure (Hgbe) were used. (Hb balance method). The primary efficacy outcome was the number of transfusions of allogeneic RBC from surgery up to day 4. The secondary ones were the total blood loss from surgery to day 4 as it was calculated by Hb-balance method. After randomization, 35 patients with femoral neck fracture and 30 patients with IT fracture received TXA prior to surgery. Respectively, 30 patients with femoral neck fracture and 55 with IT fracture didn't receive TXA. The groups did not differ significantly in their basic demographics (age, gender, BMI, injury mechanism, ASA score, co-morbidities). Results showed that patients undergoing hemiarthroplasty after receiving TXA, were transfused with less allogeneic RBC and had less total blood loss than patients that didn't receive TXA, but without statistical significance. While patients treated with IMN in the TXA group received a significantly lower number of RBC units than the control group (1.28 ± 1.049 vs 2.075 ± 1.685), (P = 0.0396), had a significantly lower loss of Hb (98.59 ± 55.24 vs 161.6 ± 141.7), (P = 0.0195) and a lower total blood volume loss (951.3 ± 598.9 ml vs 1513 ± 1247 ml), (P = 0.023). This trial confirmed TXA administration efficacy in reducing blood loss and transfusion rate in elderly patients undergoing hip fracture surgery. A TXA single dose may be a safer option, taking into account these patients' physiological status and co-morbidities

Pre-operative anaemia can present in up to 30% of elective arthroplasty patients. The presence of anaemia increases the risk of requiring blood transfusion post-operatively as well as acts as an independent risk factor for poor outcome such as prosthetic joint infection. Recent international consensus on this topic has recommended a specific care pathway for screening patients with pre-operative anaemia using a simple bedside Heaemacue finger-prick test to detect in a simple and cost-effective manner, and then allow treatment of preoperative anaemia. This pathway was therefore incorporated in our trust. This was a retrospective study done at a single tertiary-referral arthroplasty centre. Our data collection included the Heamacue test results and formal haemoglobin levels if they were performed as well as compliance and costs of each of the tests for patients listed for an elective shoulder, hip and knee arthroplasty between September and December 2018. Medical records and demographics were also collected for these patients for subgroup analysis. Our exclusion criteria comprised patients listed for revision arthroplasty surgery. 87 patients were included in this study. Our compliance rate was 15%. The mean difference between a Haemacue test and a formal FBC result was only 17.6g/L suggesting that it has a reasonably high accuracy. With regards to costs, we found that a Haemacue test costs £2, compared to £7.50 for a full blood count and Haematinics combined. This gave an overall cost saving of £5.50 per patient. Extrapolation of this date locally for 2017 at our hospital, where 1575 primary joint arthroplasties were done, a cost saving of £8,662.5 could have been achieved. Within the UK using data extrapolated from the National Joint Registry a total of £1,102,205.5 (1,221,894 Euros) could have been saved. The use of a single, Haemacue test to screen for pre-operative anaemia in elective arthroplasty patients is more cost effective compared to a formal full count and haematinics tests. However, we found that compliance with the care pathway is variable due to system limitations. This may be addressed through implementing changes to our electronic system in which patients are booked for surgery. We also noted a significant cost reduction if this pathway were to be used Nation-wide. Thus, we encourage other centres to consider the use of the Haemacue test pre-operatively in elective arthroplasty instead of formal full blood counts at the time of decision to treat with arthroplasty; this allows sufficient time for correction of pre-operative anaemia thus improving patient outcomes from arthroplasty

Summary. Our meta-analysis showed that pooled mean blood loss during spinal tumour surgeries was 2180 ml. Standardised methods of calculating and reporting intra-operative blood loss are needed as it would be beneficial in the pre-operative planning of blood replenishment during surgery. Introduction. The vertebral column is the commonest site of bony metastasis, accounting for 18,000 new cases in North America yearly. Patients with spinal metastasis are often elderly, have compromised cardiovascular status, poor physiological reserve and altered immune status, all of which render them more susceptible to the complications of intra-operative blood loss and associated transfusion. Currently no consensus exists regarding the expected volume of blood lost during metastatic spine tumour surgery with various papers quoting anywhere between 1L to 6L. Knowledge of the expected blood loss prior to surgery however is important as it facilitates pre-operative planning, intra- and post-operative management of fluid balance and blood transfusion. We conducted a meta-analysis of published literature on spine tumour surgery to answer the question: “What is the expected blood loss in major spinal tumour surgery for metastatic spinal disease?”. Methods. A comprehensive online search of the English literature using Medline, Embase, and the Cochrane Central Register of Controlled Trials was performed. We included articles published from 31 January 1992 until 31 January 2012. This initial online search yielded 98 relevant articles. Two senior investigators independently reviewed all abstracts. The full text of articles that were deemed eligible for further consideration obtained and reviewed. Eighty five articles were excluded at this stage, largely due to lack of clear blood loss data, leaving 13 eligible articles. A hand search of the reference lists of relevant articles yielded 5 more articles. A total of 18 articles were included in the final meta-analysis of blood loss data. Disagreements regarding eligibility of articles for analysis were resolved by consensus. Selected articles for final analysis were independently graded according to the Centre for Evidence-Based Medicine (CEBM) Levels of Evidence. We evaluated the possibility of publication bias by obtaining a funnel plot (created by plotting the sample size against the effect estimate). The Egger's regression asymmetry test was used to assess the existence of publication bias. Results. Eighteen selected articles had a total of 785 patients who had undergone major spine tumour surgery for metastatic spinal disorders. The pooled estimate of the blood loss occurring during spinal tumour surgeries was calculated to be 2180ml (95%CI: 1805–2554ml). Apart from two studies which reported significant mean blood loss of more than 5500 ml, the resulting funnel plot suggested absence of publication bias. This was confirmed by Egger's test which did not show any small-study effects (p=0.119). However, there was strong evidence of heterogeneity between studies with I2=90% (p<0.001). Conclusions. The expected blood loss of a patient undergoing major surgery for spinal tumour constitutes more than a third of the circulating blood volume in a typical cancer patient with significantly impaired physiological reserve. Moreover, cases of catastrophic blood loss exceeding 5L exist in almost every series evaluated in this paper, with some reaching as much as 17–18L. Blood loss is a significant problem during spine tumour surgery and concerted effort is needed to address it

Background. Post-traumatic immunosuppression (PTI) after surgery increases vulnerability to nosocomial infections, sepsis, and death. Knee arthroplasty offers a sterile clinical model to characterise PTI and explore its underlying mechanisms. Methods. This prospective non-randomised cohort study of primary total knee arthroplasty was approved by the Local Ethics Committee. Exclusion criteria included revision-arthroplasty, pre-existing infections, blood-transfusions, malignancy, and auto-immune disease. 48 recruited patients fell into two groups, the first received unwashed anti-coagulated autologous salvaged blood transfusions after surgery (ASBT cohort, n=25). The second received no salvaged blood transfusions (NSBT cohort, n=18). Venous blood was sampled pre-operatively and within 3–7 days post-operatively. Salvaged blood was sampled at one and six hours post-operatively. Biomarkers of immune status included: interleukins (IL) or cytokines (x15), chemokines (x3), Damage-Associated-Molecular-Patterns (DAMPS) (x5), anti-microbial proteins (x3), CD24, and Sialic-acid-binding-Immunoglobulin-type-Lectin-10 (Siglec-10). Results were expressed as fold-change over pre-operative values. Only significant changes are described. Results. Certain biomarkers associated with sterile trauma were common to all 43 patients, including supra-normal: IL-6, IL-1-Receptor-Antagonist, IL-8, Heat-Shock-Protein-70 (HSP70), Calprotectin, CD24 and Siglec-10. But, whereas in NSBT patients post-operative pro-inflammatory biomarkers were sub-normal consistent with PTI, they were supra-normal in ASBT patients implying its reversal. These PTI-biomarkers included: IL-1β, IL-2, IL-17A, Interferon-gamma (IFN-γ), Tumour-Necrosis-Factor-alpha (TNF-α), and Annexin-A2. Reversal of PTI by salvaged blood was further endorsed in ASBT by sub-normal levels of the anti-inflammatory biomarkers: IL-4, IL-5, IL-10, and IL-13. Salvaged blood analyses revealed sustained supra-normal levels of DAMPs, CD24 and Siglec-10; and increasingly elevated levels of cytokines and chemokines during the six hour collection period. Interestingly, plasma CD24, Siglec-10, HSP70 and Calprotectin levels were significantly correlated, implying physical association within the circulation. Conclusions. Several anti-inflammatory processes triggered by traumatised tissue induce systemic PTI, thereby increasing vulnerability to infections. Reversal of PTI by re-infusion of anti-coagulated salvaged blood suggests a novel source of immuno-stimulants

Objectives. The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods. Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically. Results. The total number of PBMNCs was decreased after QQ-culture, however, the number of CD34+ and CD206+ cells were found to have increased as assessed by flow cytometry analysis. In addition, gene expression of angiogenic factors was upregulated in QQMNCs. In the animal model, the rate of bone union was higher in the QQMNC group than in the other groups. Radiographic scores and bone volume were significantly associated with the enhancement of angiogenesis in the QQMNC group. Conclusion. We have demonstrated that QQMNCs have superior potential to accelerate fracture healing compared with PBMNCs. The QQMNCs could be a promising option for fracture nonunion. Cite this article: K. Mifuji, M. Ishikawa, N. Kamei, R. Tanaka, K. Arita, H. Mizuno, T. Asahara, N. Adachi, M. Ochi. Angiogenic conditioning of peripheral blood mononuclear cells promotes fracture healing. Bone Joint Res 2017;6: 489–498. DOI: 10.1302/2046-3758.68.BJR-2016-0338.R1

Musculoskeletal disorders is one of most important health problems human population is facing includes. Approximately 310 thousand of hip protheses have been used in 45 years and older patients in total according to the recent studies have been done. [1, 2]. Many factors, including poor osseointegration or relaxation of the implant due to stress, limit the life of the load-bearing implants [3]. To overcome these difficulties and to protect metal implants inside the body, the surfaces of the implants were coated with silver ion doped hydroxyapatite/bioglass. In this study, silver doped hydroxyapatite ceramic powder and 6P57 bioglass were synthesized. Two different coating suspensions, 100% bioglass and 70% Ag-HAp / 30% bioglass, were prepared in methyl alcohol with a solid content of 1% by weight. Two layers were coated on the external fixator nails by using electrospray method with the bioglass and Ag-Hap/Bioglass suspensions respectively. The coated implants were cut with an equal surface area and kept in human blood plasma for different time. The scanning electron microscopy (SEM, Zeiss Supra 50VP and Zeiss Evo 50EP) and stereo microscope (Zeiss Axiocam Stemi 2000-C) were used to characterize microstructure and thickness of coated surface. Energy dispersive X-ray Spectroscopy was used characterized of chemical composition of coating. Changing of pH value of plasma was measured by pH meter (Hanna HI83414). In addition, the ICP method was used to determine the elements contained in the plasma fluid after dissolution. As a result of this study, physical and chemical changes occurring on the coating surface in different time periods are presented in detail

Background. There are multiple documented advantages of undertaking total knee arthroplasty (TKA) without tourniquet, however, increased rates of blood loss and transfusion are often cited as contraindications to this approach. The aim of this study was to examine the effect of intra-operative TA administration on blood loss and transfusion rates in TKA without pneumatic tourniquet, using Rivaroxaban as thrombo-embolic prophylaxis. Method. 120 patients split into two continuous data sets, (A+B), underwent TKA without application of above knee tourniquet, receiving a post operative dose of oral Rivaroxaban within 8 hours. Group B patients received an intra-operative dose of 1 gram of Tranexamic Acid intravenously before the first cut, whilst those in group A did not. Haemoglobin and haematocrit levels were recorded peri-operatively. A revised Gross formula was used to calculate blood loss. Four patients were excluded from the study for incomplete data. Results. 58 patients (M34F24) in Group A, average age 6, had a mean haemoglobin drop of 33gram/litre, haematocrit drop of 0.097litre/litre (9.7%), with an average calculated blood loss of 1393ml. 58 (M34, F24) patients in group B, average age 67, had a haemoglobin drop of 25.2gram/litre, haemotocrit drop of 0.076litre/litre (7.6%) with an average calculated blood loss of1079ml. Thus Group A patients were seen to sustain significantly more blood loss without TA administration, with a 29.1% larger calculated blood loss, a 25.5% larger drop in haemoglobin and a 27.6% larger fall in haematocrit. Transfusion rate was 5.2% (3 patients) per group. Conclusion. TA was shown to be effective in reducing blood loss in TKA without tourniquet using Rivaroxaban. Transfusion rates of 5.2% across both groups is close to 1/10th of the transfusion rate reported for some major studies of TKA using Rivaroxaban with tourniquet application, and 1/8th of the transfusion rate in studies of TKA with administration of TA and use of tourniquet. Level of Evidence. Level-III. The authors report there are no relevant disclosures to make. Ethical approval was granted for the study

Tissue reconstruction, based on stem cell activity has become an important part of orthopaedic practice. It is now possible to develop cell lines which are able to produce the fundamental cells which can be used in musculoskeletal regeneration, especially in fracture healing, cartilage regeneration, and muscle repair. However, for the newly implanted cells to be effective, it is vital to have an adequate and developing blood supply to that area. Human and animal studies have demonstrated the marked contribution of bone marrow derived precursor cells in the normal bone healing process. Studies of the application of bone marrow graft have shown that there is greater bone growth when more precursor cells are grafted and these cells are thought to be a mixed population of stems cells and their associated progeny. CD34+ cells have shown remarkable ability to differentiate into many cells types which include chondrocytes and osteocytes. They have also been shown to home on to sites of bone injury and mature into bone cells which take part in the repair process. Colleagues in our laboratories have described a plastic adherent sub-population of CD34+ cells which have been able to reconstitute and sustain hematopoeisis over 5 weeks, similar to long-term marrow culture. This sub-population of cells are called omnicytes. Using this sub-population, we have conducted in vitro and animal experiments using a fracture healing model to assess the role of stem cells in accelerating the fracture healing process. However, it is clear that in order for these cells to be effective, the blood supply needs to be viable. In this paper, the importance of the blood supply and the role of blood flow will be discussed particularly in relation to fracture healing and intervertebral disc regeneration. In fracture healing, the increase of blood flow occurs within the first 6 weeks after the fracture has occurred and CD34+ cells applied to the fracture site via the nutrient artery could accelerate the process of fracture union. In the same way, intervertebral disc patients with chronic low back pain for more than 3 months could be treated with enhanced CD34+ cells in order to allow disc cartilaginous type cells to regenerate. This will be a review of the role of the blood supply, the development of CD34+ cells (namely omnicytes), and the clinical application of these cells to patients with long bone fractures and low back pain

Objectives. Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs. Methods. Human ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS). Results. HPL and HPL+Hplasma had a significantly greater growth-promoting effect than FBS, while Hplasma exhibited a similar growth-promoting effect to that of FBS. ADMSCs cultured in HPL and/or Hplasma generated more colony-forming unit fibroblasts (CFU-F) than those cultured in FBS. After long-term culture, ADMSCs cultured in HPL and/or Hplasma showed reduced cellular senescence, retained typical cell phenotypes, and retained differentiation capacities into osteogenic and adipogenic lineages. Conclusion. HPL and Hplasma prepared from blood products after their recommended transfusion date can be used as an alternative and effective source for large-scale ex vivo expansion of ADMSCs. Cite this article: J. Phetfong, T. Tawonsawatruk, K. Seenprachawong, A. Srisarin, C. Isarankura-Na-Ayudhya, A. Supokawej. Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture. Bone Joint Res 2017;6:414–422. DOI: 10.1302/2046-3758.67.BJR-2016-0342.R1

Introduction. Severe ‘discogenic’ back pain may be related to the ingrowth of nerves and blood vessels, although this is controversial. We hypothesise that ingrowth is greater in painful discs, and is facilitated in the region of annulus fissures. Methods. We compared tissue removed at surgery from 22 patients with discogenic back pain and/or sciatica, and from 16 young patients with scoliosis who served as controls. Wax-embedded specimens were sectioned at 7μm. Nerves and blood vessels were identified using histological stains, and antibodies to PGP 9.5 and CD31 respectively. Results. Blood vessels were identified in 77% of ‘painful’ discs compared to 44% of scoliotic discs (p=0.013), and they were more common in the anterior anulus compared to the posterior (p=0.026). Maximum penetration of blood vessels from the peripheral anulus was 4.7 mm (in ‘painful’ discs) and 2.0 mm (in control discs), and penetration increased with histological grade of disc degeneration in the ‘painful’ discs (p=0.002). In 16/17 ‘painful’ discs, blood vessels were within 1 mm of an anulus fissure, or the disc periphery. Nerves were found in 36% of ‘painful’ discs (all with blood vessels) and 25% of control discs. Nerve ingrowth was always less than or equal to blood vessel ingrowth, with a maximum observed penetration of 1.5 mm from the annulus periphery. Discussion. In degenerated and painful discs, the ingrowth of nerves appears to follow that of blood vessels, and is facilitated in the region of annulus fissures. No nerves were seen >2mm from the annulus periphery, suggesting that previous reports of nerves in the disc nucleus may refer to vertical growth from a vertebral endplate rather than radial growth through the annulus. Results support the view that discogenic back pain is associated with pain-sensitisation events in the disc periphery. Acknowledgements. Research funded by BackCare. M Stefanakis would like to thank the Greek Institute of Scholarships (I.K.Y) for financial support

Background. Hitherto, no study has compared blood loss (BL) after different thromoprophylactic regimes (TR). The objective of this study was to quantify and compare BL in total hip arthroplasty (THA) under three different TRs. Methods. Between September 2013 and July 2014, sixty primary, unilateral, same-implant THAs entered a randomised, double-blind clinical trial. The patients were randomised to receive manufacturers' recommended doses of enoxaparin, dabigatran or rivaroxaban. Complete blood counts were obtained preoperatively and on the third day postoperatively. BL was calculated according to the Nadler formula. We also evaluated the occurence of wound healing disturbances (WHDs). All data were analysed using R statistical software. Results. The mean BL and standard deviations were 844 ± 222 ml for enoxaparin, 854 ± 205 ml for dabigatran and 806 ± 227 ml for rivaroxaban. The BL did not significantly differ between groups (Kruskall-Wallis, p=0.92). More WHDs occured in the rivaroxaban group (5/20), compared to enoxaparin (2/20) and dabigatran (3/20). Conclusions. None of the chemical TR is superior to others in terms of reducing the BL. There seems to be more WHDs with the use of oral agents - this finding needs further studies. Level of evidence. 1b (Centre for Evidence Based Medicine, Oxford). Approval. This study was approved by The Medical Centre of Postgraduate Education Ethical Committee. Disclosure. The authors disclose no competing interests

Introduction. Standard surgical exposure reduces blood flow to the patella during total knee arthroplasty (TKA). Reduction of patellar blood flow has resulted in patellofemoral complications including osteonecrosis and patellar fracture, necessitating revision surgery. Eversion of the patella is typically used to gain access to the knee joint in most TKA surgical approaches. More recently, the development of minimally invasive surgery (MIS) techniques has avoided patellar eversion by subluxing the patella. The present study is the first to measure patellar blood flow during MIS TKA with the knee in both extension and 90 degrees of flexion followed by lateral retraction and then eversion of the patella. Methods. Patellar blood flow was measured using laser Doppler flowmetry in 40 patients during MIS TKA. Patients included 32 women and 8 men who had a mean age of 73 years (range, 52 to 88 years) and a mean weight of 59 kg (39 to 85 kg). The pre-operative diagnoses were osteoarthritis in 36 patients and rheumatoid arthritis in four patients. All patients underwent MIS TKA using the mini-midvastus approach. After initial blood flow was assessed with the leg in full extension, further measurements were performed after lateral retraction and after eversion of the patella. Then, blood flow was assessed with the knee in 90 degrees of flexion followed by lateral retraction and then eversion of the patella. Results. For measurements made during knee extension, a significant reduction in flow was observed during eversion of the patella compared with the neutral patellar position (P < 0.001). However, no significant difference was found between lateral retraction of the patella and the neutral patellar position. A significant reduction in flow was noted when the leg was flexed from full extension to 90 degrees (P < 0.001). For measurements made with the knee in 90 degrees of flexion, a significant reduction in flow was observed during eversion of the patella (P = 0.002) and a significant increase was noted during lateral retraction of the patella compared with the neutral position (P < 0.001). Conclusion. Patella eversion may result in traction both on medial vessels and those within the quadriceps mechanism, whereas, lateral retraction reduces the force exerted on the arteries within the quadriceps after the medial blood supply was interrupted by medial arthrotomy. In addition, the position of the leg had a great impact on patellar blood flow. MIS TKA without patellar eversion may be useful for preventing a reduction in patellar blood flow

Summary Statement. Bone stress fracture triggers a rapid increase in blood flow in association with mast cell production of inducible nitric oxide synthase (iNOS). NOS inhibition blocks the increase in blood flow and reduces woven bone formation needed for stress fracture healing. Introduction. Vascular-bone interactions are critical in skeletal development and fracture healing. We recently showed that angiogenesis is required for stress fracture healing. However, the changes in vascularity that occur in the first 72 hours after stress fracture can not be explained by angiogenesis. Here, we evaulated early changes in blood flow and vasodilation after either damaging (stress fracture) or non-damaging mechanical loading in rats. Methods. The right forelimbs of adult rats were subjected to cyclic axial compression in vivo. We used two established protocols: damaging loading that creates a stress fracture and leads to woven bone formation (WBF loading), or non-damaging loading that stimulates lamellar bone formation (LBF loading). PET imaging was used to evaluate blood flow and fluoride kinetics based on uptake of . 15. O water and . 18. F fluoride radioisotopes, respectively, at the site of bone formation. To quantify vasodilation, the area of the anterior interosseous artery was measured. Inducible nitric oxide synthase (iNOS) expression was evaluated by immunostaining. Finally, NO production was impaired by administration of L-NAME (N. ω. -nitro-L-arginine methyl ester), a NOS inhibitor. Results. PET Imaging: Damaging WBF loading induced early and persistent increases in blood flow. Blood flow rate was increased ∼30% at 4 hours through 14 days in WBF loaded limbs. Fluoride uptake peaked 7 days after WBF loading, then declined from 7 to 14 days, consistent with the dynamics of woven bone formation described previously. Non-damaging LBF loading did not affect blood flow or fluoride kinetics. Histology: WBF loaded limbs had significantly increased arterial area (+50%) compared to non-loaded limbs at days 1 and 3, with return to normal by day 7. LBF loading did not affect arterial area. Since mast cells are a possible effector of vasodilation, mast cell infiltration and iNOS expression were quantified following loading. iNOS+ mast cells in WBF-loaded limbs were significantly increased on days 1 and 3, with return to normal by day 7. LBF loading was not associated with increases in iNOS+ mast cells. NOS Inhibition: L-NAME blocked the expression of iNOS in mast cells following WBF loading. Additionally, L-NAME treatment abolished the increase in blood flow rate at days 1 and 3, and diminished fluoride uptake at day 3. Finally, L-NAME treatment decreased woven bone formation, with significant decreases in woven bone volume (−27%) and BMD (−26%), compared to vehicle controls. Discussion/Conclusion. Damaging loading produces a stress fracture and leads to woven bone formation (WBF). Prior to bone formation, there is a rapid increase in blood flow rate in association with vasodilation and infiltration of iNOS+ mast cells in the expanded periosteum. Inhibition of NOS blocks the increase in blood flow rate, and ultimately impairs woven bone formation. In contrast, non-damaging (LBF) loading does not affect blood flow rate, vasodilation, or iNOS expression in mast cells. Thus, the vascular response after stress fracture involves an early increase in blood flow by vasodilation, followed by angiogenesis to maintain increased blood flow. Disruption of either response affects subsequent bone formation during stress fracture healing