Pure and syndromic optic atrophy explained by deep intronic OPA1 mutations and an intralocus modifier.

The genetic diagnosis in inherited optic neuropathies often remains challenging, and the emergence of complex neurological phenotypes that involve optic neuropathy is puzzling. Here we unravel two novel principles of genetic mechanisms in optic neuropathies: deep intronic OPA1 mutations, which explain the disease in several so far unsolved cases; and an intralocus OPA1 modifier, which explains the emergence of syndromic 'optic atrophy plus' phenotypes in several families. First, we unravelled a deep intronic mutation 364 base pairs 3' of exon 4b in OPA1 by in-depth investigation of a family with severe optic atrophy plus syndrome in which conventional OPA1 diagnostics including gene dosage analyses were normal. The mutation creates a new splice acceptor site resulting in aberrant OPA1 transcripts with retained intronic sequence and subsequent translational frameshift as shown by complementary DNA analysis. In patient fibroblasts we demonstrate nonsense mediated messenger RNA decay, reduced levels of OPA1 protein, and impairment of mitochondrial dynamics. Subsequent site-specific screening of >360 subjects with unexplained inherited optic neuropathy revealed three additional families carrying this deep intronic mutation and a base exchange four nucleotides upstream, respectively, thus confirming the clinical significance of this mutational mechanism. Second, in all severely affected patients of the index family, the deep intronic mutation occurred in compound heterozygous state with an exonic OPA1 missense variant (p.I382M; NM_015560.2). The variant alone did not cause a phenotype, even in homozygous state indicating that this long debated OPA1 variant is not pathogenic per se, but acts as a phenotypic modifier if it encounters in trans with an OPA1 mutation. Subsequent screening of whole exomes from >600 index patients identified a second family with severe optic atrophy plus syndrome due to compound heterozygous p.I382M, thus confirming this mechanism. In summary, we provide genetic and functional evidence that deep intronic mutations in OPA1 can cause optic atrophy and explain disease in a substantial share of families with unsolved inherited optic neuropathies. Moreover, we show that an OPA1 modifier variant explains the emergence of optic atrophy plus phenotypes if combined in trans with another OPA1 mutation. Both mutational mechanisms identified in this study-deep intronic mutations and intragenic modifiers-might represent more generalizable mechanisms that could be found also in a wide range of other neurodegenerative and optic neuropathy diseases.

Influence of left- and right-side total hip arthroplasty on the ability to perform an emergency stop while driving a car.

OBJECTIVE: To show the possible effect of left- and right-side total hip arthroplasty (THA) on the ability to perform an emergency stop when driving a car. DESIGN: Inception cohort. SETTING: A driving simulator using an actual car cabin, specifically developed for the experiment, was used for testing driving ability. PARTICIPANTS: Patients (N=40; 20 left-side THA/20 right-side THA) were tested preoperatively and in increments of 8 days and 6, 12, and 52 weeks after surgery. INTERVENTIONS: Left- and right-side THA. MAIN OUTCOME MEASURES: Reaction time, movement time, total brake response time (TBRT), and maximum brake force. RESULTS: Eight days postoperatively, measurements on driving performance indicated a slight worsening for all outcome parameters in patients after left-side THA and considerably more worsening in patients after right-side THA. For both patient groups, significant improvements in outcome measures were noted during the 1-year follow-up. Brake force declined significantly in patients with left-side THA (P=.012) and in patients after right-side THA (P<.001). A total of 35% of the patients with right-side THA and 15% with left-side THA could not meet the 600 ms TBRT threshold 6 weeks postoperatively. CONCLUSIONS: Most patients who underwent right-side THA reached their preoperative baseline 6 weeks after surgery. Most of the patients with left-side THA showed no TBRT limitations 8 days postoperatively. Because of the patients' highly individual rehabilitation course and considering the possible consequences of the premature resumption of driving a motor vehicle, individual examination and recommendation are necessary.

Osteoarthritis of the knee or hip significantly impairs driving ability (cross-sectional survey).

BACKGROUND: Advising patients about when they can drive after surgery is common practice after arthroplasty of the knee or hip. In the literature, the preoperative braking performance values of the patients are frequently taken as the "safe" landmark. We hypothesised that osteoarthritis (OA), the most frequent reason for arthroplasty, already compromises the ability to perform an emergency stop. We expected that both Reaction Time (RT) and Movement Time (MT) as components of the Total Brake Response Time (TBRT), would be prolonged in patients with OA of the knee or hip in comparison with healthy subjects. We also expected maximum pressure levels on the brake pedal to be reduced in such cases. METHODS: A real car cabin was equipped with pressure sensors on the accelerator and brake pedals to measure RT, MT, TBRT and maximum Brake Force (BF) under realistic spatial constraints. Patients with OA of the knee (right n = 18, left n = 15) or hip (right n = 20, left n = 19) were compared with a healthy control group (n = 21). RESULTS: All measured values for TBRT in the control group remained below 600 ms. OA of the right hip or knee significantly prolonged the braking performance (right hip: TBRT p = 0.025, right knee: TBRT p < 0.001), whereas OA of the left hip did not impair driving ability (TBRT p = 0.228). Intriguingly, OA of the left knee prolonged RT and MT to the same degree as OA on the contralateral side (RT p = 0.001, MT p < 0.001). CONCLUSIONS: This study demonstrates that depending on the localisation of OA, driving capability can be impaired; OA can significantly increase the total braking distance. To ensure safe traffic participation the safety margin for TBRT should be strictly set, under our experimental conditions, at around 600 ms. Moreover, therapeutic approaches to OA, such as physiotherapy, and patients receiving surgery of the left knee should take into account that left knee OA can also impair driving ability. CLINICAL TRIAL REGISTRATION NUMBER: Project number of the ethics committee of the University of Tübingen: 268/2009BO2; 267/2009BO2.

Implementation of Outpatient Parenteral Antimicrobial Therapy (OPAT) in Patients with Complicated Periprosthetic Joint Infections. / Etablierung der ambulanten intravenösen Antibiotikatherapie (APAT) bei Patienten mit komplizierten Periimplantatinfektionen.

Periprosthetic joint infections (PJI) are a serious complication of arthroplasty with high morbidity. With growing bacterial resistance and limited disposability of oral antibiotics with sufficient bioavailability, the need for intravenous antibiotic application is raising. This causes long-term hospital stays and rising costs. In the course of transferring procedures into an outpatient setting as well as coping with pressures on hospital capacity, outpatient parenteral antimicrobial therapy (OPAT) can build a bridge for the treatment of such infections.In a single centre analysis, 47 cases treated with OPAT were studied in relation to pathogen, antimicrobial resistance, indication for OPAT and follow up. Furthermore, the patients received an anonymised questionnaire with 4 clusters of interest in terms of internal quality assessment on the success and evaluation of this therapeutic procedure. Special attention was paid to the descriptive analysis of patients with periprosthetic joint infections (n = 30).Between May 2021 and October 2022 out of 47 patients with OPAT, 30 cases with periprosthetic joint infections were identified. For infected hip- and knee arthroplasties, a remarkable spectrum of pathogens was found. In hip infections highly resistant strains of Staphylococcus epidermidis and Enterococci were detected. In knee infections, the pathogens were more susceptible, but however highly virulent Staphylococcus aureus and Streptococci. Difficult to treat, mixed infections were found in both locations. The indication for OPAT was based in half of the cases on the high level of antimicrobial resistance, with availability of only parenteral applicable antibiotics. Further indications were mixed infections and difficult to treat pathogens, with flucloxacillin therapy as well as OPAT as the last therapeutic option. The questionnaire showed 96% patient satisfaction in terms of organisation and acceptance of this kind of therapy. Complications or unexpected outpatient/ hospital treatments were very rare in connection with OPAT. Two thirds of patients reported completion of the treatment. In the clinical follow up (average of 5.7 months), 96.6% of cases were declared free of infection. In one patient the infection persisted.OPAT is a safe and reliable therapeutic option for outpatients to continue parenteral antimicrobial treatment in joint infections. Due to increasing pressure on hospitals in terms of costs and capacity, this therapy offers an alternative to inpatient treatment. The indication for OPAT should be set individually, risk adjusted and not generalised for all patients. The outpatient sector needs financial and structural support for comprehensive roll-out of this treatment in Germany. A further focus should be on the prevention of periprosthetic joint infections. With the knowledge of the expected pathogens and the surgical resources, the standards should be adapted. The choice of the antibiotic should be specified and the intervals of application be shortened, according to the surgical course, in order to yield high levels of agent concentration in the surgical area. Further investigations are required to test the superiority of OPAT versus the oral administration of antibiotics in long-term observations as well as to define the necessary duration of OPAT.

Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties. METHODS: BM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells. RESULTS: BM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRß, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6ß, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, ß1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs. CONCLUSIONS: By identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.

Mesenchymal stromal/stem cells markers in the human bone marrow.

BACKGROUND AIMS: Mesenchymal stromal/stem cells (MSCs) can be isolated from human bone marrow (BM), expanded ex vivo and identified via numerous surface antigens. Despite the importance of these cells in regenerative therapy programs, it is unclear whether the cell membrane signature defining MSC preparations ex vivo is determined during culture or may reflect an in vivo counterpart. BM-MSC phenotype in vivo requires further investigation. METHODS: To characterize cells in their natural BM environment, we performed multi-parametric immunohistochemistry on trabecular bone biopsy specimens from multiple donors and described cells by different morphology and micro-anatomic localization in relationship to a precise pattern of MSC antigen expression. RESULTS: Microscopically examined high-power field marrow sections revealed an overlapping in vivo expression of antigens characterizing ex vivo expanded BM-MSCs, including CD10, CD73, CD140b, CD146, GD2 and CD271. Expanding this panel to proteins associated with pluripotency, such as Oct4, Nanog and SSEA-4, we were able to identify different cellular populations in the human trabecular bone and BM expressing different progenitor cell markers. CONCLUSIONS: Targeting several multipotency and pluripotency markers, we found that the BM contains identifiable and distinct progenitor cells further justifying their introduction for a wide range of applications in regenerative medicine.

The health-related quality of life of sarcoma patients treated with neoadjuvant versus adjuvant radiotherapy - Results of a multi-center observational study.

AIM: The sequence of radiotherapy and resection in patients with soft tissue sarcomas is usually discussed on an individual basis. Better understanding of potential differences of health-related quality of life (QoL) between patients undergoing adjuvant (ART) versus neoadjuvant radiotherapy (NART) is therefore helpful for clinical decision making. METHODS: Adult sarcoma patients from 39 hospitals completed the European Organisation for Research and Treatment of Cancer Quality of Life Core Questionnaire (EORTC QLQ-C30). Differences in global QoL, physical functioning, role functioning, fatigue, pain, and insomnia between ART versus NART were investigated with multivariate regression, adjusting for age, gender, chemotherapy, grading, stage, tumor location, recurrence/distant metastasis, sarcoma type, time since last treatment, and treatment status using validated thresholds. RESULTS: A total of 1110 patients participated. Of them, 340 had received radiotherapy (NART: n = 95, 28%; ART: n = 245, 72%). Global QoL was 59.3 on average after NART and 60.5 after ART (Badj = 1.0, p = 0.74). Physical functioning was 65.9 compared to 70.5 (Badj = 4.2; p = 0.16), role function 48.8 vs. 56.7 (Badj = 7.0, p = 0.08), fatigue 47.5 vs. 45.4 (Badj = -1.2; p = 0.71), pain 40.2 vs. 34.1 (Badj = -6.8; p = 0.08), and insomnia 33.7 vs. 41.6 (Badj = 5.5, p = 0.16). Among patients with NART, clinically relevant QoL impairments were less frequent 2 years after treatment compared to < 2 years thereafter (n = 6 vs. n = 4 on average). CONCLUSION: There is little evidence for QoL differences in most domains and overall QoL between the two irradiation groups. However, patients after NART might experience worse role functioning and pain but fewer problems with insomnia compared to patients after ART.

No red cell alloimmunization or change of clinical outcome after using fresh frozen cancellous allograft bone for acetabular reconstruction in revision hip arthroplasty: a follow up study.

BACKGROUND: Possible immunization to blood group or other antigens and subsequent inhibition of remodeling or incorporation after use of untreated human bone allograft was described previously. This study presents the immunological, clinical and radiological results of 30 patients with acetabular revisions using fresh frozen non-irradiated bone allograft. METHODS: AB0-incompatible (donor-recipient) bone transplantation was performed in 22 cases, Rh(D) incompatible transplantation in 6 cases. The mean follow up of 23 months included measuring Harris hip score and radiological examination with evaluation of remodeling of the bone graft, implant migration and heterotopic ossification. In addition, all patients were screened for alloimmunization to Rh blood group antigens. RESULTS: Compared to the whole study group, there were no differences in clinical or radiological measurements for the groups with AB0- or Rh(D)-incompatible bone transplantation. The mean Harris Hip Score was 80.6. X-rays confirmed total remodeling of all allografts with no acetabular loosening. At follow up, blood tests revealed no alloimmunization to Rh blood group donor antigens. CONCLUSIONS: The use of fresh frozen non-irradiated bone allograft in acetabular revision is a reliable supplement to reconstruction. The risk of alloimmunization to donor-blood group antigens after AB0- or Rh-incompatible allograft transplantation with a negative long-term influence on bone-remodeling or the clinical outcome is negligible.

The development of whole blood titanium levels after instrumented spinal fusion - is there a correlation between the number of fused segments and titanium levels?

BACKGROUND: Most modern spinal implants contain titanium and remain in the patient's body permanently. Local and systemic effects such as tissue necrosis, osteolysis and malignant cell transformation caused by implants have been described. Increasing tissue concentration and whole blood levels of ions are necessary before a disease caused by a contaminant develops. The aim of the present study was the measurement of whole blood titanium levels and the evaluation of a possible correlation between these changes and the number of fused segments. METHODS: A prospective study was designed to determine changes in whole blood titanium levels after spinal fusion and to analyze the correlation to the number of pedicle screws, cross connectors and interbody devices implanted.Blood samples were taken preoperatively in group I (n = 15), on the first, second and 10th day postoperatively, as well as 3 and 12 months after surgery.Group II (n = 16) served as a control group of volunteers who did not have any metal implants in the body. Blood samples were taken once in this group.The number of screw-rod-connections and the length of the spinal fusion were determined using radiographic pictures. This study was checked and approved by the ethical committee of the University of Tuebingen. RESULTS: The mean age in group I was 47 ± 22 years (range 16 - 85 years). There were three male (20%) and twelve female (80%) patients. The median number of fused segments was 5 (range 1 to 11 segments).No statistically significant increase in the titanium level was seen 12 months after surgery (mean difference: -7.2 µg/l, 95% CI: -26.9 to 12.5 µg/l, p = 0.446). By observing the individual titanium levels, 4 out of 15 patients demonstrated an increase in titanium levels 12 months after surgery.No statistically significant correlation between fused segments (r = -0.188, p = 0.503) length of instrumentation (r = -0.329, p = 0.231), number of interbody devices (r = -0.202, p = 0.291) and increase of titanium levels over the observation period was seen. CONCLUSIONS: Instrumented spinal fusion does not lead to a statistically significant increase in whole blood titanium levels. There seems to be no correlation between the number of pedicle screws, cross connectors and interbody devices implanted and the increase of serum titanium levels.

Fibrin sealants in orthopaedic surgery: practical experiences derived from use of QUIXIL® in total knee arthroplasty.

BACKGROUND: Total knee arthroplasty is associated with a significant postoperative blood loss even without any form of perioperative anticoagulation. METHODS: The potential role of QUIXIL(®), a fibrin sealant used in orthopaedic surgery to control blood loss and avoid blood transfusions in patients undergoing total knee arthroplasty was evaluated in a prospective randomized trial with twenty-four patients diagnosed with primary osteoarthritis of the knee. RESULTS: Results showed that application of 2 ml QUIXIL(®) adds costs to treatment without reducing the number of transfused red blood cell counts and postoperative haemoglobin loss. However, significant lower levels of postoperative fluid loss (P = 0.026) was detected in QUIXIL(®) treated patients. CONCLUSION: Regarding cost effectiveness and benefit no indication for the use of 2 ml QUIXIL(®) fibrin sealant in standard knee arthroplasty could be proofed statistically.

Expression of blood group genes by mesenchymal stem cells.

Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate- and protein-based membrane structures, defined by blood group antigens, we investigated human bone marrow-derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase-1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2(dim+) H(+) MSCs retain a better 'stemness'. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications.

Arthrofibrosis after TKA - Influence factors on the absolute flexion and gain in flexion after manipulation under anaesthesia.

BACKGROUND: Stiffness with decreased range of motion (ROM) has been described as a frustrating complication after TKA. If all methods of physiotherapeutic treatment have been exhausted trying to develop ROM, manipulation under anaesthesia (MUA) can be discussed. The aim of the present study was to show the effect of MUA and to determine the influence of BMI, number of previous surgical procedures, pre-MUA ROM and timing of MUA for the results after MUA in regard to absolute flexion and gain in flexion. METHODS: 858 patients underwent TKA at our institution between 2004 and 2009. 39 of these patients underwent MUA because of postoperative knee stiffness. The data were retrospective analysed for the influence of BMI, pre-MUA flexion (/≤ 30 days after TKA) and number of previous surgery on the results after MUA (absolute Flexion/gain in flexion). RESULTS: The prevalence for stiffness after TKA was 4.54%. There was a statistically significant improvement in flexion not only directly after MUA but also 6 weeks after MUA. Patients with two or more previous operations before TKA showed statistically significant worse results six weeks after MUA in absolute flexion and gain in flexion(p = 0.039) than patients with one or two previous operations. No statistical significance in absolute flexion (p = 0.655) and gain in flexion (p = 0.328) after MUA between "early" and "late" was detected. The stiffer knees with a flexion below 70° showed significantly worse results (p = 0.044) in absolute flexion six weeks after MUA, but they also had statistical statistically better results with regard to gain in flexion (p ≤ 0.001). CONCLUSION: MUA is a good instrument for improving ROM after TKA. The time between TKA and MUA seems less important, so different types of physiotherapeutic treatment could be tried before the procedure is started. MUA in patients with many previous operations and a flexion of less than 70° before MUA is not as effective as in other patients, but they also benefit from MUA.

Chromium and cobalt intoxication mimicking mitochondriopathy.

A 53-year old male with a history of progressive visual impairment, hearing loss, peripheral neuropathy, poorly controlled diabetes mellitus, cardiomyopathy, and weight loss was referred to the rare disease center due to the suspicion of mitochondrial cytopathy. In line with mitochondrial dysfunction, lactate in CSF was increased. Genetic testing by whole-exome sequencing and mitochondrial DNA did not reveal a likely cause. The case remained unsolved until he developed pain in his right hip, where he had received total hip arthroplasty 12 years earlier. An orthopedic evaluation revealed substantial shrinkage of the head of the hip prosthesis. Due to metal-on-metal wear, debris chromium and cobalt levels in serum were massively increased and significantly improved with multisystemic impairment after exchanging the defective implant.

Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles.

BACKGROUND: For clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed. RESULTS: Human MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-gamma in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs. CONCLUSIONS: In our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.

Tumour stromal cells derived from paediatric malignancies display MSC-like properties and impair NK cell cytotoxicity.

BACKGROUND: Tumour growth and metastatic infiltration are favoured by several components of the tumour microenvironment. Bone marrow-derived multipotent mesenchymal stromal cells (MSC) are known to contribute to the tumour stroma. When isolated from healthy bone marrow, MSC exert potent antiproliferative effects on immune effector cells. Due to phenotypic and morphological similarities of MSC and tumour stromal cells (TStrC), we speculated that immunotherapeutic approaches may be hampered if TStrC may still exhibit immunomodulatory properties of MSC. METHODS: In order to compare immunomodulatory properties of MSC and tumour stromal cells (TStrC), we established and analyzed TStrC cultures from eleven paediatric tumours and MSC preparations from bone marrow aspirates. Immunophenotyping, proliferation assays and NK cell cytotoxicity assays were employed to address the issue. RESULTS: While TStrC differed from MSC in terms of plasticity, they shared surface expression of CD105, CD73 and other markers used for MSC characterization. Furthermore, TStrC displayed a strong antiproliferative effect on peripheral blood mononuclear cells (PBMC) in coculture experiments similar to MSC. NK cell cytotoxicity was significantly impaired after co-culture with TStrC and expression of the activating NK cell receptors NKp44 and NKp46 was reduced. CONCLUSIONS: Our data show that TStrC and MSC share important phenotypic and functional characteristics. The inhibitory effect of TStrC on PBMC and especially on NK cells may facilitate the immune evasion of paediatric tumours.

In vitro measurement of intraarticular pressure in the ankle joint.

Ankle joint affections and injuries are common problems in sports traumatology and in the daily routine of arthroscopic surgeons. However, there is little knowledge regarding intraarticular loads. Pressures on the ankle were determined in a dynamic model on 8 cadaver specimens, applying forces to tendons of the foot over the stance phase under vertical loading. A characteristic course of loading in the tibiotalar joint with a rapid increase upon heel contact was observed. It increased gradually to reach a maximum after 70% of the stance phase, during the push-off phase. The major torque in the ankle joint is located anterolaterally. A dynamic loading curve of the ankle joint can be demonstrated. These observations explain phenomena such as the appearance of osteophytes on the anterior tibia in the case of ankle osteoarthritis and the relatively low incidence of posterior tibial edge fragments in the case of trimalleolar ankle fracture. Furthermore, the medial side of the talus is less loaded compared to the lateral side, which appears relevant to the treatment of osteochondrosis dissecans.

Longtime Outcome After Intraosseous Application of Autologous Mesenchymal Stromal Cells in Pediatric Patients and Young Adults with Avascular Necrosis After Steroid or Chemotherapy.

Avascular necrosis (AVN) is a severe complication of immunosuppressant therapy or chemotherapy. A beneficial AVN therapy with core decompression (CD) and intraosseous infusion of mesenchymal stromal cells (MSCs) has been described in adult patients, but there are only few data on MSC applications in pediatric and young adult patients (PYAP). Between 2006 and 2015, 14 AVN lesions of 10 PYAP (6 females) with a median age of 16.9 years (range 8.5-25.8 years) received CD and intraosseous application of autologous MSCs. Data of these patients were analyzed regarding efficacy, safety, and feasibility of this procedure as AVN therapy and compared with a control group of 13 AVN lesions of 11 PYAP (5 females) with a median age of 17.9 years (range 13.5-27.5 years) who received CD only. During the follow-up analysis [MSC group: median 3.1 (1.6-5.8) years after CD; CD group: median 2.0 (1.5-8.5) years after CD], relative lesion sizes (as assessed by magnetic resonance imaging) compared with the initial lesion volume, were significantly lower (P < 0.05) in the MSC group (volume reduction to a median of 18.5%) when compared with the CD group (58.0%). One lesion in the MSC group comprised a complete remission. Size progression was not observed in either group. Clinical improvement (pain, mobility) was not significantly different between the two groups. None of the patients experienced treatment-related adverse effects. CD and additional MSC application was regarded safe, effective, feasible, and superior in reducing the lesion size when compared with CD only. Prospective, randomized clinical trials are needed to further evaluate these findings.

Establishment and Characterization of a Sclerosing Spindle Cell Rhabdomyosarcoma Cell Line with a Complex Genomic Profile.

Sclerosing spindle cell rhabdomyosarcoma (SSRMS) is a rare rhabdomyosarcomas (RMS) subtype. Especially cases bearing a myogenic differentiation 1 (MYOD1) mutation are characterized by a high recurrence and metastasis rate, often leading to a fatal outcome. SSRMS cell lines are valuable in vitro models for studying disease mechanisms and for the preclinical evaluation of new therapeutic approaches. In this study, a cell line established from a primary SSRMS tumor of a 24-year-old female after multimodal chemotherapeutic pretreatment has been characterized in detail, including immunohistochemistry, growth characteristics, cytogenetic analysis, mutation analysis, evaluation of stem cell marker expression, differentiation potential, and tumorigenicity in mice. The cell line which was designated SRH exhibited a complex genomic profile, including several translocations and deletions. Array-comparative genomic hybridization (CGH) revealed an overall predominating loss of gene loci. The mesenchymal tumor origin was underlined by the expression of mesenchymal markers and potential to undergo adipogenic and osteogenic differentiation. Despite myogenic marker expression, terminal myogenic differentiation was inhibited, which might be elicited by the MYOD1 hotspot mutation. In vivo tumorigenicity could be confirmed after subcutaneous injection into NOD/SCID/γcnull mice. Summarized, the SRH cell line is the first adult SSRMS cell line available for preclinical research on this rare RMS subtype.

Modulating endothelial adhesion and migration impacts stem cell therapies efficacy.

BACKGROUND: Limited knowledge of stem cell therapies` mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. METHODS: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. FINDINGS: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. INTERPRETATION: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. FUNDING: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health.

Labeling of human mesenchymal stromal cells with superparamagnetic iron oxide leads to a decrease in migration capacity and colony formation ability.

BACKGROUND AIMS: Labeling of stem cells is crucial to allow tracking of stem cell homing and engraftment after transplantation. In this study we evaluated the influence of cell labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC). METHODS: The study was approved by the institutional review board of the University of Tubingen, Germany. Seven populations of bone marrow (BM)-derived human mesenchymal stem cells (MSC) were labeled with SPIO alone or in combination with various TA. Directly after labeling and two passages after labeling migration assays, quantification of colony-forming units and quantitative evaluation of the differentiation potential were performed. Quantification of the cellular total iron load (TIL), determination of the cellular viability and electron microscopy were also performed. RESULTS: Labeling of mesenchymal stem cells with SPIO with or without TA did not affect cell viability and differentiation potential significantly. SPIO in combination with TA coated the cellular surface directly after labeling but was incorporated into the cells after two passages. Labeling of mesenchymal stem cells with TA led to a significant decrease of migration capacity. This effect was abolished after two passages. Labeling with and without TA led to a significant decrease in colony formation ability. This effect could also be observed after two passages. CONCLUSIONS: The observed decrease of migration capacity and colony-formation ability was not associated with either TIL or localization of particles of iron oxide. SPIO labeling with and without TA had functional effects on human mesenchymal stem cells by decreasing the migration capacity and colony-formation ability of the stem cells.