Platelet-rich plasma (PRP) has been demonstrated to benefit a variety of disciplines. But there exists heterogeneity in results obtained due to lack of standardization of the preparation protocols employed in them. We aim to identify and standardize a preparation protocol for PRP with maximum recovery of platelets to obtain reproducible results across studies. Blood samples were collected from 20 healthy volunteers. The double spin protocol of PRP preparation was analyzed for variables such as centrifugal acceleration, time, and volume of blood processed and final product utilized. The final PRP prepared was investigated for platelet recovery, concentration, integrity, and viability. We noted maximum platelet recovery (86-99%) with a mean concentration factor of 6-times baseline, with double centrifugation protocol at 100xg and 1600xg for 20 minutes each. We also noted that 10 ml of blood in a 15 ml tube was the ideal volume of blood to be processed to maximize platelet recovery. We demonstrated that the lower 1/3rd is the ideal volume to be utilized for clinical application. We did not note a loss of integrity or viability of the platelets in the final product from the above-said protocol. Preparation of PRP by the double spin protocol of 10 ml of blood at 100xg and 1600xg for 20 minutes each in a 15ml tube and using the lower 1/3rd of the final product demonstrated consistent high platelet recovery (86-99%) and concentration (6x) without disturbing the platelet integrity or viability

Decellularization techniques have advanced to reduce the risk of immune rejection in transplantation. Validation of these protocols typically relies on Crapo's criteria. 1. , which include the absence of visible nuclei and low DNA content. In our study, five decellularization protocols were compared to determine the optimal approach for human fascia lata (HFL) samples. However, our findings raised questions as to why recipients can still develop immunity despite meeting validation criteria. HFL samples were decellularized using four protocols with SDS-Triton X100-DNase (D1 to D4-HFL) and one protocol using solvent-detergent-based baths (D5-HFL). The decellularized samples (D-HFL) were compared to native samples (N-HFL) using histology, and DNA content was measured. The human leukocyte antigen (HLA) content within the matrix was assessed using western blot analysis. Both D-HFL and N-HFL samples, along with negative control patches, were implanted in the backs of 28 Wistar rats. Anti-human IgG serum levels were evaluated after one month. H&E and Hoechst staining revealed the absence of residual cells in all decellularization protocols. DNA content was consistently below the critical threshold (p<0.05). All implanted D-HFL samples resulted in significantly lower anti-human IgG levels compared to N-HFL (p<0.01). However, 2.5 out of 4 rats developed immunity after being implanted with D1 to D4-HFL, with varying levels of anti-human IgG. Only rats implanted with D5-HFL showed undetectable levels of IgG and were considered non-immunized. Western blot analysis indicated that only D5-HFL had a residual HLA content below 1%. The literature on decellularization has primarily relied on Crapo's criteria, which do not consider the role of HLA mismatch in acute immune rejection. Our results suggest that a residual HLA content below 1% should also be considered to prevent immunization, even if other validation criteria are met. Further research is needed to evaluate the impact of residual HLA levels on human allotransplantation outcomes

To be able to assess the biomechanical and functional effects of ankle injury and disease it is necessary to characterise healthy ankle kinematics. Due to the anatomical complexity of the ankle, it is difficult to accurately measure the Tibiotalar and Subtalar joint angles using traditional marker-based motion capture techniques. Biplane Video X-ray (BVX) is an imaging technique that allows direct measurement of individual bones using high-speed, dynamic X-rays. The objective is to develop an in-vivo protocol for the hindfoot looking at the tibiotalar and subtalar joint during different activities of living. A bespoke raised walkway was manufactured to position the foot and ankle inside the field of view of the BVX system. Three healthy volunteers performed three gait and step-down trials while capturing Biplane Video X-Ray (125Hz, 1.25ms, 80kVp and 160 mA) and underwent MR imaging (Magnetom 3T Prisma, Siemens) which were manually segmented into 3D bone models (Simpleware Scan IP, Synopsis). Bone position and orientation for the Talus, Calcaneus and Tibia were calculated by manual matching of 3D Bone models to X-Rays (DSX Suite, C-Motion, Inc.). Kinematics were calculated using MATLAB (MathWorks, Inc. USA). Pilot results showed that for the subtalar joint there was greater range of motion (ROM) for Inversion and Dorsiflexion angles during stance phase of gait and reduced ROM for Internal Rotation compared with step down. For the tibiotalar joint, Gait had greater inversion and internal rotation ROM and reduced dorsiflexion ROM when compared with step down. The developed protocol successfully calculated the in-vivo kinematics of the tibiotalar and subtalar joints for different dynamic activities of daily living. These pilot results show the different kinematic profiles between two different activities of daily living. Future work will investigate translation kinematics of the two joints to fully characterise healthy kinematics

Implant manufacturers develop new products to improve existing fracture fixation methods or to approach new fracture challenges. New implants are commonly tested and approved with respect to their corresponding predecessor products, because the knowledge about the internal forces and moments acting on implants in the human body is unclear. The aim of this study was to evaluate and validate implant internal forces and moments of a complex physiological loading case and translate this to a standard medical device approval test. A finite elements model for a transverse femur shaft fracture (AO/OTA type 32-B2) treated with a locked plate system (AxSOS 3 Ti Waisted Compression Plate Broad, Stryker, Kalamazoo, USA) was developed and experimentally validated. The fractured construct was physiologically loaded by resulting forces on the hip joint from previously measured in-vivo loading experiments (Bergmann et. al). The forces were reduced to a level where the material response in the construct remained linear elastic. Resulting forces, moments and stresses in the implant of the fractured model were analysed and compared to the manufacturers’ approval data. The FE-model accurately predicted the behaviour of the whole construct and the micro motion of the working length of the osteosynthesis. The resulting moment reaction in the working length was 24 Nm at a load of 400 N on the hip. The maximum principle strains on the locking plate were predicted well and did not exceed 1 %. In this study we presented a protocol by the example of locked plated femur shaft fracture to calculate and validate implant internal loading using finite element analysis of a complex loading. This might be a first step to move the basis of development of new implants from experience from previous products to calculation of mechanical behaviour of the implants and therefore, promote further optimization of the implants’ design

The purpose of this study is to enhance massive bone allografts osseointegration used to reconstruct large bone defects. These allografts show >50% complication rate requiring surgical revision in 20% cases. A new protocol for total bone decellularisation exploiting the vasculature can offer a reduction of postoperative complication by annihilating immune response and improving cellular colonization/ osseointegration. The nutrient artery of 18 porcine bones - humerus/femur/radius/ulna - was cannulated. The decellularization process involved immersion and sequential perfusion with specific solvents over a course of one week. Perfusion was realized by a peristaltic pump (mean flow rate: 6ml/min). The benefit of arterial perfusion was compared to a control group kept in immersion baths without perfusion. Bone samples were processed for histology (HE, Masson's trichrome and DAPI for cell detection), immunohistochemistry (IHC : Collagen IV/elastin for intraosseous vascular system evaluation, Swine Leukocyte Antigen – SLA for immunogenicity in addition to cellular clearance) and DNA quantification. Sterility and solvent residues in the graft were also evaluated with thioglycolate test and pH test respectively. Compared to native bones, no cells could be detected and residual DNA was <50ng/mg dry weight. Intramedullary spaces were completely cleaned. IHC showed the preservation of intracortical vasculature with channels bounded by Collagen IV and elastin within Haversian systems. IHC also showed a significant decrease in SLA signaling. All grafts were sterile at the last decellularization step and showed no solvent residue. The control group kept in immersion baths, paired with 6 perfused radii/ulnae, showed that the perfusion is mandatory to ensure complete decellularisation. Our results prove the effectiveness of a new concept of total bone decellularisation by perfusion. These promising results could lead to a new technique of Vascularized Composite Allograft transposable to pre-clinical and clinical models

Abstract. Skeletal kinematics are traditionally measured by motion analysis methods such as optical motion capture (OMC). While easy to carry out and clinically relevant for certain applications, it is not suitable for analysing the ankle joint due to its anatomical complexity. A greater understanding of the function of healthy ankle joints could lead to an improvement in the success of ankle-replacement surgeries. Biplane video X-ray (BVX) is a technique that allows direct measurement of individual bones using highspeed, dynamic X-Rays. Objective. To develop a protocol to quantify in-vivo foot and ankle kinematics using a bespoke High-speed Dynamic Biplane X-ray system combined with OMC. Methods. Two healthy volunteers performed five level walks and step-down trials while simultaneous capturing BVX and synchronised OMC. participants undertook MR imaging (Magnetom 3T Prisma, Siemens) which was manually segmented into 3D bone models (Simpleware Scan IP, Synopsis). Bone position and orientation for the Talus, Tibia and Calcaneus were calculated by manual matching of 3D Bone models to X-Rays (DSX Suite, C-Motion, Inc.). OMC markers were tracked (QTM, Qualisys) and processed using Visual 3D (C-motion, Inc.). Results. Initial results for level walking showed that OMC overestimated the rotational range of motion (ROM) in all three planes for the tibiotalar joint compared with BVX (Sagittal: OMC 30°/BVX 20°, Frontal: OMC 16°/BVX 15° and Transverse: OMC 20°/BVX 17°). For the subtalar joint, OMC (22°) over-estimated sagittal ROM compared with BVX (14°) and underestimated the ROM in the other planes (Frontal: OMC 8°/BVX 15° and Transverse: OMC 18°/BVX 20°). Conclusions. The results highlight the discrepancy between OMC and BVX methods. However, the BVX results are consistent with previous literature. The protocol developed here will form the foundation of future patient-based studies to investigate in-vivo ankle kinematics. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Objectives. This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocols. Methods. Two manual preparation procedures (single-spin (SS) at 900 g for five minutes; double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects. Both preparations were tested for platelet activation by one of three activation protocols: no activation, activation with calcium (Ca) only, or calcium with a low dose (50 IU per 1 ml PRP) of thrombin. Each preparation was divided into four aliquots and incubated for one hour, 24 hours, 72 hours, and seven days. The cytokine-release kinetics were evaluated by assessing PDGF, TGF, VEGF, FGF, IL-1, and MMP-9 concentrations with bead-based sandwich immunoassay. Results. The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols. Ca-only activation had a significant effect on the DS PRPs (where the VEGF, FGF, and IL-1 concentrations were sustained) while Ca/thrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short). The IL-1 content showed a significant increase with Ca-only or Ca/thrombin activation while these activations did not increase the MMP-9 concentration. Conclusion. The SS and DS methods differed in their effect on cytokine release, and this effect varied among the cytokines analysed. In addition, low dose of thrombin/calcium activation increased the overall cytokine release of the PRP preparations over seven days, relative to that with a calcium-only supplement or non-activation. Cite this article: Professor J. H. Oh. Cytokine-release kinetics of platelet-rich plasma according to various activation protocols. Bone Joint Res 2016;5:37–45. doi: 10.1302/2046-3758.52.2000540

Restoration of anatomy is paramount in total hip arthroplasty (THA) to optimise function and stability. Leg-length discrepancy of ≥10mm is poorly tolerated and can be the subject of litigation. We routinely use a multimodal protocol to optimise soft tissue balancing which involves pre-operative templating, leg-length measurement supine and in the lateral position after positioning, and the use of an intra-operative leg-length measurement device to ensure optimisation of leg-length. We have analysed the results of our protocol in restoring leg-length in primary THA. Radiological leg-length was measured in a consecutive series of 50 patients who had THA for unilateral arthritis by an independent observer pre- and post-operatively using validated methods utilising radiological software. The measurements pre- and post-operative were compared. Patients with bilateral hip arthritis and poor imaging were excluded. Leg-length was successfully restored to within 5.0mm of the target leg-length in 84.0% of patients (mean +0.7mm (95% CI +0.2 to +1.1)). The other 14.0% of patients were restored to within 5.1–8.0mm (mean +2.2mm (95% CI −2.7 to +7.1)) and 2.0% of patients were restored to within 8.1–10.0mm. Leg length was accurately restored across the subset of patients within a narrow range of either side of the mean target leg length. Intra-operative measurement of leg length can be difficult but is vital in ensuring appropriate restoration of leg-length. We recommend a similar multimodal protocol to ensure restoration of leg-length within narrow limits to maximise function and patient satisfaction

Introduction and Objective. Hip osteoarthritis (OA) is the leading cause for total hip arthroplasty (THA). Although, being considered as the surgery of the century up to 23% of the patients report long-term pain and deficits in physical function and muscle strength may persist after THA. Progressive resistance training (PRT) appear to improve several outcomes moderately in patients with hip OA. Current treatment selection is based on low-level evidence as no randomised controlled trials have compared THA to non-surgical treatment. The primary objective of this trial is to determine the effectiveness of THA followed by standard care compared to 12 weeks of supervised PRT followed by 12 weeks of optional unsupervised PRT, on changes in hip pain and function, in patients with severe hip OA after 6 months. Materials and Methods. This is a protocol for a multicentre, parallel-group, assessor blinded, randomised controlled superiority trial. Patients aged ≥50 years with clinical and radiographic hip OA found eligible for THA by an orthopaedic surgeon will be randomised to THA or PRT (allocation 1:1). The primary outcome will be change in patient-reported hip pain and function, measured using the Oxford Hip Score. Key secondary outcomes will be change in the Hip disability and Osteoarthritis Outcome Score subscales, University of California Los Angeles Activity Score, 40-meter fast-paced walk test, 30-second chair stand test, and number of serious adverse events. Results. The trial has been approved by The Regional Committees on Health Research Ethics for Southern Denmark (Project-ID: S-20180158) in February 2019 and registration was performed at . ClinicalTrials.gov. (NCT04070027) in August 2019. Recruitment was initiated on the 2. nd. of September 2019 and the final deadline will be on the 30. th. of June 2021, or when a sample size of 120 patients has been accomplished. Conclusions. The results of the current trial are expected to enable evidence-based recommendations, which may be used to facilitate the shared-decision making process in the discussion of treatment strategy for the individual patient with severe hip OA. All results will be presented in peer-reviewed scientific journals and international conferences

This protocol describes a pragmatic multicentre randomised controlled trial (RCT) to assess the clinical and cost effectiveness of arthroscopic and open surgery in the management of rotator cuff tears. This trial began in 2007 and was modified in 2010, with the removal of a non-operative arm due to high rates of early crossover to surgery. . Cite this article: Bone Joint Res 2014;3:155–60

We reviewed renal function of 22 patients who had undergone total knee replacements using the enhanced recovery protocol (Caledonian technique) between August 2012 and November 2012 at a district general hospital in the west of Scotland. Pre-operative and post operative data were compared to determine if there was any change. We observed that 4 out of 22 (18%) of patients had a significant rise in creatinine, and 6 out of 22 (27%) had an abnormal eGFR. These findings were significant and were classed as (Acute Kidney Injury) AKI type 1, which should be treated actively. Subsequently, we collected data in the same way for 22 patients who underwent total knee replacements without using the enhanced recovery protocol. In this group, only one (5%) had a significant rise in creatinine and 2 (9%) had an abnormal eGFR. Significant difference is noted in the two groups. We conclude that the enhanced recovery protocol has some adverse effect on a patient's renal function. Our hypothesis is that this is due to restriction of fluids after surgery but a larger study is needed to find the cause and ways to avoid this

Abstract. Objective. To compare the periprosthetic fracture mechanics between a collared and collarless fully coated cementless femoral stem in a composite femur. Methods. Two groups of six composite femurs (‘Osteoporotic femur’, SawBones, WA USA) were implanted with either a collared (collared group) or collarless (collarless group) cementless femoral stem which was otherwise identical by a single experienced surgeon. Periprosthetic fractures of the femur were simulated using a previously published technique. High speed video recording was used to identify fracture mechanism. Fracture torque and angular displacement were measured and rotational work and system stiffness were estimated for each trial. Results were compared between collared and collarless group and the comparison was evaluated against previously published work using fresh frozen femurs and the same protocol. Results. In composite femur testing median fracture torque (IQR) was greater with a collared versus collarless implant (48.41 [42.60 to 50.27] Nm versus 45.12 [39.13 to 48.09] Nm, p= 0.4). Median rotational displacement (IQR) was less with a collared versus collarless implant (0.29 [0.27 to 0.31] radians versus 0.33 [0.32 to 0.34] radians, p= 0.07). Estimated rotary work was similar between groups (5.76 [4.92 to 6.64] J versus 5.21 [4.25 to 6.04] J, p= 0.4). Torsional stiffness was greater with a collared versus collarless implant (158.36 [152.61, 163.54] Nm per radian versus 138.79 [122.53, 140.59] Nm per radian, p= 0.5). Collarless stems were seen to move independently of the femur and fracture patterns originated at the calcar. Conclusions. Testing with composite femurs using an established protocol produced similar results to previously published studies using human femurs, but the difference between collared and collarless stems was smaller. The internal homogenous foam material in composite femurs does not accurately represent the heterogeneous cancellous bone which supports a femoral stem in vivo and may lead to overestimation of implant stability. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Allogeneic blood transfusion is associated with many complications and significant cost. The RD&E has looked at the use of autologous drains after our study of 100 cases showed an improved post-operative haemoglobin and reduced length of stay. There is a need to identify those patients of increased need for an autologous drain, in order to decrease the frequency of allogeneic transfusion. In 2007 a protocol was drawn up using information from our study of 191 cases which showed an average haemoglobin drop post-operatively of 3.05g/dl and average intra-operative blood loss of 285 ml. This protocol gives the surgeon triggers for autologous drain use; preoperative haemoglobin of <13g/dl, intra-operative blood loss of >400ml, tourniquet use, patient weight <50kg and patients refusing donated blood. In 2007-08, 65% of a further 275 cases analysed met the triggers for use of an autologous system. The remaining patients received low vacuum drains. Of the 275 patients, only 2 (<1%) of those who did not fulfil the criteria for an autologous drain required allogeneic blood, compared with 43 patients (24%) of those deemed high risk of transfusion, and assigned autologous drains. The protocol was therefore deemed to be successful in identifying those patients who required additional support and expenditure to minimise allogeneic blood transfusion. Analysis of this data led to recommended changes to the protocol in order to maximise the efficiency of the autologous drain use. In 2010 a further patient cohort studied showed a reduction in allogeneic blood transfusion to <10% of those receiving autologous drains, and an increase to 5% of those with low vacuum drains. Due to the increased cost of autologous drains (£68) compared with the low vacuum systems (£32), and the cost of allogeneic units at £141, the expenditure per patient was calculated and shown to fall from £92 in 2007 to £78 in the 2010. In conclusion, this protocol allows the clinician to appropriately target the use of the more expensive autologous drains to those of increased risk of transfusion. This protocol helps to minimise unnecessary allogeneic blood transfusion risks, and this has been shown to be more cost effective

Component malrotation in total knee arthroplasty (TKA) is a reason for early failure and revision. Assessment of possible component malrotation using computed tomography (CT) might be useful when other differentials have been excluded. The aims of our study were to determine the proportion of symptomatic patients with component malrotation on CT, and review the subsequent management of such patients. A retrospective review of case notes was performed locally for all patients who had a CT scan for a painful TKA. Measurements of the femoral and tibial component rotations were done according to the standard Berger protocol, giving net degrees of either external rotation (ER) or internal rotation (IR). Any subsequent surgery was noted, and patients were followed up as per local practice. Between 2007 and April 2012, 69 knees in 68 patients had CT scans. There were 25 males and 43 females, and mean age at primary surgery was 65.03 years. The mean femoral component rotation for all knees was 0.1° ER (range 7.0° ER – 6.7° IR), and the mean tibial component rotation for all knees was 19.1° IR (6.6° ER – 37.0° IR). No statistically significant difference was found comparing the mean femoral and tibial component rotations between patients with and without further surgery. Further surgery was performed on 39 (56.5%) knees. Overall, there were ten cases (14.5%) of isolated femoral malrotation, 26 tibial malrotation (37.7%), and two cases (2.9%) had malrotation of both components. Out of these 38 cases, secondary surgery was performed in 22 knees (57.9%), of which a satisfactory outcome was achieved in fifteen cases (68.1%). It is impossible to establish component malrotation as the only cause of pain following TKA, however, our study does show that the Berger protocol has its uses when other causes have been excluded

Abstract

Surgical interventions for the treatment of chronic neck pain, which affects 330 million people globally [1], include fusion and cervical total disc replacement (CTDR). Most of the currently clinically available CTDRs designs include a metal-on-polymer (MoP) bearing. Numerous studies suggest that MoP CTDRs are associated with issues similar to those affecting other MoP joint replacement devices, including excessive wear and wear particle-related inflammation and osteolysis [2,3]. A device with a metal-on-metal (MoM) bearing has been investigated in the current study. Six MoM CTDRs made from high carbon cobalt-chromium (CoCr) were tested in a six-axis spine simulator, under standard ISO testing protocol (ISO-18192-1) for a duration of 4 million cycles (MC). Foetal bovine calf serum (25%v/v), used as a lubricant, was changed every 3.3×10. 5. cycles and saved for particle analysis. Components were taken down for measurements after each 10. 6. cycles; surface roughness, damage modes and gravimetric wear were assessed. The mean wear rate of the MoM CTDRs was 0.24mm. 3. /MC (SD=0.03), with the total volume of 0.98mm. 3. (SD=0.01) lost over the test duration. Throughout the test, the volumetric wear was linear; no significant bedding-in period was observed. The mean pre-test surface roughness decreased from 0.019μm (SD=0.005) to 0.012μm (SD=0.002) after 4MC of testing. Prior to testing, fine polishing marks on the bearing surfaces were observed using light microscopy. Following 4MC of testing, these polishing marks had been removed. Consistently across all components, surface discolouration and multidirectional, criss-crossing, circular wear tracks, caused by abrasive wear, were observed. The wear results showed low wear rates exhibited by MoM CTDRs (0.24mm. 3. /MC), when compared CTDR designs incorporating metal-on-polymer bearings (0.56mm. 3. /MC) [4] as well as MoM lumbar CTDRs [5,6] (0.76mm3/MC – 6.2mm. 3. /MC). These findings suggest that MoM CTDRs are more wear resistant than MoP CTDRs, however the particle characterisation and biological consequences of wear remain to be determined

Osteoarthritis is a joint condition affecting an estimated eight million people in the UK. The kinematics of walking and the impact experienced are thought to play an important role in the initiation and progression of the disease. Previous studies have looked the effect of osteoarthritis on the kinematics of walking in a laboratory environment. This work is part of the Newcastle Thousand Families Study which has followed a cohort of 1142 members since birth in 1947. Optoelectronic gait analysis methods are unsuitable for this environment, so inertial measurement units are being used. This study focuses on the validation of a protocol using inertial sensors to assess gait in the clinical environment. The sensors measure orientation in three dimensions. Our hypothesis was that an attachment position that minimises the movement of the sensor relative to the segment during gait was more important than the proximity of the sensor to anatomical landmarks. The effect of sampling rate, fatty tissue movement and material type were also tested Seven sensors (Xsens, Netherlands) were attached to participants on top of the foot, on the tibial plateau, on the lateral surface of the femur 10cm proximal to the lateral epicondyle, and over the sacrum. Attachment is by Velcro straps over the top of clothing for the waist, thigh and shank sensors, and with double-sided hypoallergenic tape on the foot. Four calibration movements are performed followed by a walking trial of ten paces down a corridor at a self-selected speed. Data is recorded wirelessly at a sampling rate of 50Hz. The calibration movements and trials are repeated twice and the time taken is 20 minutes. Measurement of the joint angles in the sagittal plane was used to assess the effect of changing the sensor position, simulating fatty tissue movement, and variation of material type underneath the sensor. The foot and thigh sensors were displaced in the distal direction by up to 10cm, the shank and waist sensors were displaced in the proximal direction by 5cm. Material types of different elasticity were tested. Fatty tissue movement beneath the straps was simulated using hydration gel packs. Each attachment scenario was repeated five times on a single subject. A “normal” attachment scenario was used to establish a baseline for repeatability of hip, knee and ankle angle measurement (mean±standard deviation of 49±1.28°, 61.5±1.28° and 33.5±0.69° respectively). Repeatability is comparable to that reported for an opto-electronic system (45±1.8°, 63±1.9° and 36±1.5°). Displacement of the foot, shank and waist sensors had no effect on the repeatability. Displacement of the thigh sensor decreased the repeatability for the knee and hip joint angles (52±3.22° and 62.5±2.91°). As the thigh sensor moved closer to the knee the movement artefact experienced increased. Altering sampling rate and simulated fatty tissue did not decrease repeatability. Of the materials tested, denim had the greatest affect, decreasing hip and knee angle repeatability (50.0±2.04° and 61.0±1.75°). A sensor attachment position that minimises sensor movement relative to the segment has been shown to produce the greatest repeatability, irrespective of their proximity to bony landmarks. This is particularly true for the femur sensor

Introduction and Objective

The aim of this study was to evaluate whether CT-based pre-operative planning, integrated with intra-operative navigation could improve glenoid baseplate fixation and positioning by increasing screw length, reducing number of screws required to obtain fixation and increasing the use of augmented baseplate to gain the desired positioning. Reverse total shoulder arthroplasty (RSA) successfully restores shoulder function in different conditions. Glenoid baseplate fixation and positioning seem to be the most important factors influencing RSA survival. When scapular anatomy is distorted (primitive or secondary), optimal baseplate positioning and secure screw purchase can be challenging.

Tendon injuries are common and current therapies often are unsuccessful. Cell-based therapy using mesenchymal stem cells (MSCs) seems to be the most promising approach to heal tendon. Moreover, providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices (GMP). Adipose-derived stem cells (n=4) were cultured in 6-well plates coated with type-I collagen in a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL). At passage 4, ASCs were induced to tendon lineage for 14 days using 100ng/ml CTGF, 10ng/ml TGFβ3, 50ng/ml BMP12 and 50µg/ml ascorbic acid in the SF (SF-TENO) or in the hPL (hPL-TENO) medium. Cells cultured without any supplements are used as control. Morphological appearance, cell viability and FACS were performed in undifferentiated cells to evaluate the xenogenic-free culture conditions; the gene and protein expression were performed by RT-PCR and immunofluorescence to evaluate to expression of stem cell- and tendon-related markers upon cell differentiation. SF-CTRL and hPL-CTRL showed similar viability and MSC's surface proteins and expressed the stemness markers NANOG, OCT4 and Ki67. Moreover, both SF-TENO and hPL-TENO expressed significant higher levels of SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 genes already at 3d (p<0.05) respect to CTRLs. Scleraxis and collagen were also detected in both SF-TENO and hPL-TENO at protein level in higher amount than CTRLs. In conclusion, ASCs exposed to CTGF, BMP12, TGFb3 and AA in both serum and xenogenic-free media possess similar tenogenic differentiation ability moving forward the GMP-compliant approaches for the clinical use of ASCs.

Little information exists when using cell viability assays to evaluate cells within whole tissue, particularly specific types such as the intervertebral disc (IVD). When comparing the reported methodologies and the protocols issued by manufacturers, the processing, working times, and dye concentrations vary significantly, making the assay's reproducibility a costly and time-consuming trial and error process. This study aims to develop a detailed step-by-step cell viability assay protocol for evaluating IVD tissue. IVDs were harvested from bovine tails (n=8) and processed at day 0 and after 7 days of culture. Nucleus pulposus (NP) and the annulus fibrosus (AF) 3 mm cuts were incubated at room temperature (26˚C) with a Viability/Cytotoxicity Kit containing Calcein AM and Ethidium Ethidium homodimer-1 for 2 hr, followed by flash freezing in liquid nitrogen. Thirty µm sections were placed in glass slides and sealed with nail varnish or Antifade Mounting Medium. The IVD tissue was imaged within the next 4h after freezing using an inverted confocal laser-scanning microscope equipped with 488 and 543 nm laser lines. Cell viability at day 0 (NP: 92±9.6 % and AF:80±14.0%) and day 7 (NP: 91±7.9% and AF:76±20%) was successfully maintained and evaluated. The incubation time required is dependent on the working temperatures and tissue thickness. The calcein-AM dye will not be retained in the cells for more than four hours. The specimen preparation and culturing protocol have demonstrated good cell viability at day 0 and after seven days of culture. Processing times and sample preparation play an essential role as the cell viability components in most kits hydrolyse or photobleach quickly. A step-by-step replicable protocol for evaluating the cell viability in IVD will facilitate the evaluation of cell and toxicity-related outcomes of biomechanical testing protocols and IVD regenerative therapies