Rare coding variants in NOX4 link high ROS levels to psoriatic arthritis mutilans.

Psoriatic arthritis mutilans (PAM) is the rarest and most severe form of psoriatic arthritis, characterized by erosions of the small joints and osteolysis leading to joint disruption. Despite its severity, the underlying mechanisms are unknown, and no susceptibility genes have hitherto been identified. We aimed to investigate the genetic basis of PAM by performing massive parallel sequencing in sixty-one patients from the PAM Nordic cohort. We found rare variants in the NADPH oxidase 4 (NOX4) in four patients. In silico predictions show that the identified variants are potentially damaging. NOXs are the only enzymes producing reactive oxygen species (ROS). NOX4 is specifically involved in the differentiation of osteoclasts, the cells implicated in bone resorption. Functional follow-up studies using cell culture, zebrafish models, and measurement of ROS in patients uncovered that these NOX4 variants increase ROS levels both in vitro and in vivo. We propose NOX4 as the first candidate susceptibility gene for PAM. Our study links high levels of ROS caused by NOX4 variants to the development of PAM, offering a potential therapeutic target.

Correlates of protection and viral load trajectories in omicron breakthrough infections in triple vaccinated healthcare workers.

Vaccination offers protection against severe COVID-19 caused by SARS-CoV-2 omicron but is less effective against infection. Characteristics such as serum antibody titer correlation to protection, viral abundance and clearance of omicron infection in vaccinated individuals are scarce. We present a 4-week twice-weekly SARS-CoV-2 qPCR screening in 368 triple vaccinated healthcare workers. Spike-specific IgG levels, neutralization titers and mucosal spike-specific IgA-levels were determined at study start and qPCR-positive participants were sampled repeatedly for two weeks. 81 (cumulative incidence 22%) BA.1, BA.1.1 and BA.2 infections were detected. High serum antibody titers are shown to be protective against infection (p < 0.01), linked to reduced viral load (p < 0.01) and time to viral clearance (p < 0.05). Pre-omicron SARS-CoV-2 infection is independently associated to increased protection against omicron, largely mediated by mucosal spike specific IgA responses (nested models lr test p = 0.02 and 0.008). Only 10% of infected participants remain asymptomatic through the course of their infection. We demonstrate that high levels of vaccine-induced spike-specific WT antibodies are linked to increased protection against infection and to reduced viral load if infected, and suggest that the additional protection offered by pre-omicron SARS-CoV-2 infection largely is mediated by mucosal spike-specific IgA.

Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis.

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Novel RPL13 Variants and Variable Clinical Expressivity in a Human Ribosomopathy With Spondyloepimetaphyseal Dysplasia.

Spondyloepimetaphyseal dysplasias (SEMDs) are a heterogeneous group of disorders with variable growth failure and skeletal impairments affecting the spine and long bone epiphyses and metaphyses. Here we report on four unrelated families with SEMD in which we identified two monoallelic missense variants and one monoallelic splice site variant in RPL13, encoding the ribosomal protein eL13. In two out of four families, we observed autosomal dominant inheritance with incomplete penetrance and variable clinical expressivity; the phenotypes of the mutation-positive subjects ranged from normal height with or without hip dysplasia to severe SEMD with severe short stature and marked skeletal dysplasia. In vitro studies on patient-derived dermal fibroblasts harboring RPL13 missense mutations demonstrated normal eL13 expression, with proper subcellular localization but reduced colocalization with eL28 (p < 0.001). Cellular functional defects in fibroblasts from mutation-positive subjects indicated a significant increase in the ratio of 60S subunits to 80S ribosomes (p = 0.007) and attenuated global translation (p = 0.017). In line with the human phenotype, our rpl13 mutant zebrafish model, generated by CRISPR-Cas9 editing, showed cartilage deformities at embryonic and juvenile stages. These findings extend the genetic spectrum of RPL13 mutations causing this novel human ribosomopathy with variable skeletal features. Our study underscores for the first time incomplete penetrance and broad phenotypic variability in SEMD-RPL13 type and confirms impaired ribosomal function. Furthermore, the newly generated rpl13 mutant zebrafish model corroborates the role of eL13 in skeletogenesis. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Biomarkers in WNT1 and PLS3 Osteoporosis: Altered Concentrations of DKK1 and FGF23.

Recent advancements in genetic research have uncovered new forms of monogenic osteoporosis, expanding our understanding of the molecular pathways regulating bone health. Despite active research, knowledge on the pathomechanisms, disease-specific biomarkers, and optimal treatment in these disorders is still limited. Mutations in WNT1, encoding a WNT/ß-catenin pathway ligand WNT1, and PLS3, encoding X chromosomally inherited plastin 3 (PLS3), both result in early-onset osteoporosis with prevalent fractures and disrupted bone metabolism. However, despite marked skeletal pathology, conventional bone markers are usually normal in both diseases. Our study aimed to identify novel bone markers in PLS3 and WNT1 osteoporosis that could offer diagnostic potential and shed light on the mechanisms behind these skeletal pathologies. We measured several parameters of bone metabolism, including serum dickkopf-1 (DKK1), sclerostin, and intact and C-terminal fibroblast growth factor 23 (FGF23) concentrations in 17 WNT1 and 14 PLS3 mutation-positive subjects. Findings were compared with 34 healthy mutation-negative subjects from the same families. Results confirmed normal concentrations of conventional metabolic bone markers in both groups. DKK1 concentrations were significantly elevated in PLS3 mutation-positive subjects compared with WNT1 mutation-positive subjects (p < .001) or the mutation-negative subjects (p = .002). Similar differences were not seen in WNT1 subjects. Sclerostin concentrations did not differ between any groups. Both intact and C-terminal FGF23 were significantly elevated in WNT1 mutation-positive subjects (p = .039 and p = .027, respectively) and normal in PLS3 subjects. Our results indicate a link between PLS3 and DKK1 and WNT1 and FGF23 in bone metabolism. The normal sclerostin and DKK1 levels in patients with impaired WNT signaling suggest another parallel regulatory mechanism. These findings provide novel information on the molecular networks in bone. Extended studies are needed to investigate whether these biomarkers offer diagnostic value or potential as treatment targets in osteoporosis. © 2020 American Society for Bone and Mineral Research.

New Insights Into Monogenic Causes of Osteoporosis.

Osteoporosis, characterized by deteriorated bone microarchitecture and low bone mineral density, is a chronic skeletal disease with high worldwide prevalence. Osteoporosis related to aging is the most common form and causes significant morbidity and mortality. Rare, monogenic forms of osteoporosis have their onset usually in childhood or young adulthood and have specific phenotypic features and clinical course depending on the underlying cause. The most common form is osteogenesis imperfecta linked to mutations in COL1A1 and COL1A2, the two genes encoding type I collagen. However, in the past years, remarkable advancements in bone research have expanded our understanding of the intricacies behind bone metabolism and identified novel molecular mechanisms contributing to skeletal health and disease. Especially high-throughput sequencing techniques have made family-based studies an efficient way to identify single genes causative of rare monogenic forms of osteoporosis and these have yielded several novel genes that encode proteins partaking in type I collagen modification or regulating bone cell function directly. New forms of monogenic osteoporosis, such as autosomal dominant osteoporosis caused by WNT1 mutations or X-linked osteoporosis due to PLS3 mutations, have revealed previously unidentified bone-regulating proteins and clarified specific roles of bone cells, expanded our understanding of possible inheritance mechanisms and paces of disease progression, and highlighted the potential of monogenic bone diseases to extend beyond the skeletal tissue. The novel gene discoveries have introduced new challenges to the classification and diagnosis of monogenic osteoporosis, but also provided promising new molecular targets for development of pharmacotherapies. In this article we give an overview of the recent discoveries in the area of monogenic forms of osteoporosis, describing the key cellular mechanisms leading to skeletal fragility, the major recent research findings and the essential challenges and avenues in future diagnostics and treatments.

Novel fibronectin mutations and expansion of the phenotype in spondylometaphyseal dysplasia with "corner fractures".

Heterozygous pathogenic variants in the FN1 gene, encoding fibronectin (FN), have recently been shown to be associated with a skeletal disorder in some individuals affected by spondylometaphyseal dysplasia with "corner fractures" (SMD-CF). The most striking feature characterizing SMD-CF is irregularly shaped metaphyses giving the appearance of "corner fractures". An array of secondary features, including developmental coxa vara, ovoid vertebral bodies and severe scoliosis, may also be present. FN is an important extracellular matrix component for bone and cartilage development. Here we report five patients affected by this subtype of SMD-CF caused by five novel FN1 missense mutations: p.Cys123Tyr, p.Cys169Tyr, p.Cys213Tyr, p.Cys231Trp and p.Cys258Tyr. All individuals shared a substitution of a cysteine residue, disrupting disulfide bonds in the FN type-I assembly domains located in the N-terminal assembly region. The abnormal metaphyseal ossification and "corner fracture" appearances were the most remarkable clinical feature in these patients. In addition, generalized skeletal fragility with low-trauma bilateral femoral fractures was identified in one patient. Interestingly, the distal femoral changes in this patient healed with skeletal maturation. Our report expands the phenotypic and genetic spectrum of the FN1-related SMD-CF and emphasizes the importance of FN in bone formation and possibly also in the maintenance of bone strength.

Radiostereometric analysis of the initial stability of internally fixed femoral neck fractures under differential loading.

We examined the feasibility of radiostereometric analysis (RSA) in the assessment of the initial stability of internally fixed femoral neck fractures. The study included 16 patients (mean age 73 years). During surgery, multiple RSA-beads were inserted on both sides of the fracture. Radiographs for RSA were taken in the supine position within the first 3 days and 6, 12, 24, and 52 weeks after surgery. To detect any inducible motion at the fracture-site, radiographs for RSA were taken with the patient resting or applying a load through the fracture. Fracture loading was achieved by the patient pressing the ipsilateral foot as much as tolerated on a force plate while providing a counterforce through both hands. Micromotion exceeding the precision values of RSA (≥0.3 mm for the translation vector and/or ≥1.2 degrees for the rotation vector) was considered significant. Permanent three-dimensional fracture-site displacement was also recorded. Voluntary loading induced fracture-site micromotion, which exhibited a dichotomous distribution. In patients with uncomplicated fracture union, inducible micromotion was detectable only at baseline-if at all. Conversely, fractures that developed a nonunion were characterized by the continuation of inducible micromotion beyond baseline. Permanent fracture-site displacement was, on average, nearly an order of magnitude greater than the inducible micromotion. Fracture unions were characterized by the cessation of permanent fracture-site displacement by 12 weeks. Nonunions presented as outliers in permanent fracture-site displacement. Large-scale studies are warranted to evaluate whether the detection of inducible micromotion beyond baseline could serve as an indicator of insufficient fixation stability. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society.

Good stability of a cementless, anatomically designed femoral stem in aging women: a 9-year RSA study of 32 patients.

Background and purpose - We previously reported a transient, bone mineral density (BMD)-dependent early migration of anatomically designed hydroxyapatite-coated femoral stems with ceramic-ceramic bearing surfaces (ABG-II) in aging osteoarthritic women undergoing cementless total hip arthroplasty. To evaluate the clinical significance of the finding, we performed a follow-up study for repeated radiostereometric analysis (RSA) 9 years after surgery. Patients and methods - Of the 53 female patients examined at 2 years post-surgery in the original study, 32 were able to undergo repeated RSA of femoral stem migration at a median of 9 years (7.8-9.3) after surgery. Standard hip radiographs were obtained, and the subjects completed the Harris Hip Score and Western Ontario and McMaster Universities Osteoarthritis Index outcome questionnaires. Results - Paired comparisons revealed no statistically significant migration of the femoral stems between 2 and 9 years post-surgery. 1 patient exhibited minor but progressive RSA stem migration. All radiographs exhibited uniform stem osseointegration. No stem was revised for mechanical loosening. The clinical outcome scores were similar between 2 and 9 years post-surgery. Interpretation - Despite the BMD-related early migration observed during the first 3 postoperative months, the anatomically designed femoral stems in aging women are osseointegrated, as evaluated by RSA and radiographs, and exhibit good clinical function at 9 years.

Dynamic Changes in Brain Mesenchymal Perivascular Cells Associate with Multiple Sclerosis Disease Duration, Active Inflammation, and Demyelination.

Vascular changes, including blood brain barrier destabilization, are common pathological features in multiple sclerosis (MS) lesions. Blood vessels within adult organs are reported to harbor mesenchymal stromal cells (MSCs) with phenotypical and functional characteristics similar to pericytes. We performed an immunohistochemical study of MSCs/pericytes in brain tissue from MS and healthy persons. Post-mortem brain tissue from patients with early progressive MS (EPMS), late stage progressive MS (LPMS), and healthy persons were analyzed for the MSC and pericyte markers CD146, platelet-derived growth factor receptor beta (PDGFRß), CD73, CD271, alpha-smooth muscle actin, and Ki67. The MS samples included active, chronic active, chronic inactive lesions, and normal-appearing white matter. MSC and pericyte marker localization were detected in association with blood vessels, including subendothelial CD146+ PDGFRß+ Ki67+ cells and CD73+ CD271+ PDGFRß+ Ki67- cells within the adventitia and perivascular areas. Both immunostained cell subpopulations were termed mesenchymal perivascular cells (MPCs). Quantitative analyses of immunostainings showed active lesions containing increased regions of CD146+ PDGFRß+ Ki67+ and CD73+ CD271+ PDGFRß+ Ki67- MPC subpopulations compared to inactive lesions. Chronic lesions presented with decreased levels of CD146+ PDGFRß+ Ki67+ MPC cells compared to control tissue. Furthermore, LPMS lesions displayed increased numbers of blood vessels harboring greatly enlarged CD73+ CD271+ adventitial and perivascular areas compared to control and EPMS tissue. In conclusion, we demonstrate the presence of MPC subgroups in control human brain vasculature, and their phenotypic changes in MS brain, which correlated with inflammation, demyelination and MS disease duration. Our findings demonstrate that brain-derived MPCs respond to pathologic mechanisms involved in MS disease progression and suggest that vessel-targeted therapeutics may benefit patients with progressive MS. Stem Cells Translational Medicine 2017;6:1840-1851.

Type 1 Diabetes Mellitus Donor Mesenchymal Stromal Cells Exhibit Comparable Potency to Healthy Controls In Vitro.

: Bone marrow mesenchymal stromal cells (BM-MSCs) have been characterized and used in many clinical studies based on their immunomodulatory and regenerative properties. We have recently reported the benefit of autologous MSC systemic therapy in the treatment of type 1 diabetes mellitus (T1D). Compared with allogeneic cells, use of autologous products reduces the risk of eliciting undesired complications in the recipient, including rejection, immunization, and transmission of viruses and prions; however, comparable potency of autologous cells is required for this treatment approach to remain feasible. To date, no analysis has been reported that phenotypically and functionally characterizes MSCs derived from newly diagnosed and late-stage T1D donors in vitro with respect to their suitability for systemic immunotherapy. In this study, we used gene array in combination with functional in vitro assays to address these questions. MSCs from T1D donors and healthy controls were expanded from BM aspirates. BM mononuclear cell counts and growth kinetics were comparable between the groups, with equivalent colony-forming unit-fibroblast capacity. Gene microarrays demonstrated differential gene expression between healthy and late-stage T1D donors in relation to cytokine secretion, immunomodulatory activity, and wound healing potential. Despite transcriptional differences, T1D MSCs did not demonstrate a significant difference from healthy controls in immunosuppressive activity, migratory capacity, or hemocompatibility. We conclude that despite differential gene expression, expanded MSCs from T1D donors are phenotypically and functionally similar to healthy control MSCs with regard to their immunomodulatory and migratory potential, indicating their suitability for use in autologous systemic therapy. SIGNIFICANCE: The potential for mesenchymal stromal cells (MSCs) as a cell-based therapy in the treatment of immunologic disorders has been well established. Recent studies reported the clinical potential for autologous MSCs as a systemic therapy in the treatment of type I diabetes mellitus (T1D). The current study compared the genotypic and phenotypic profiles of bone marrow-derived MSCs from T1D and healthy donors as autologous (compared with allogeneic) therapy provides distinct advantages, such as reduced risk of immune reaction and transmission of infectious agents. The findings of the current study demonstrate that despite moderate differences in T1D MSCs at the gene level, these cells can be expanded in culture to an extent corresponding to that of MSCs derived from healthy donors. No functional difference in terms of immunosuppressive activity, blood compatibility, or migratory capacity was evident between the groups. The study findings also show that autologous MSC therapy holds promise as a T1D treatment and should be evaluated further in clinical trials.

Is Model-based Radiostereometric Analysis Suitable for Clinical Trials of a Cementless Tapered Wedge Femoral Stem?

BACKGROUND: In clinical trials of THA, model-based radiostereometric analysis (RSA) techniques may be less precise than conventional marker-based RSA for measurement of femoral stem rotation. We verified the accuracy and clinical precision of RSA based on computer-aided design models of a cementless tapered wedge femoral stem. QUESTIONS: We asked: (1) Is the accuracy of model-based RSA comparable to that of marker-based RSA? (2) What is the clinical precision of model-based RSA? METHODS: Model-based RSA was performed using combined three-dimensional computer-aided design models of the stem and head provided by the implant manufacturer. The accuracy of model-based RSA was compared with that of marker-based RSA in a phantom model using micromanipulators for controlled translation in three axes (x, y, z) and rotation around the y axis. The clinical precision of model-based RSA was evaluated by double examinations of patients who had arthroplasties (n = 24) in an ongoing trial. The clinical precision was defined as being at an acceptable level if the number of patients needed for a randomized trial would not differ from a trial done with conventional marker-based RSA (15-25 patients per group). RESULTS: The accuracy of model-based RSA was 0.03 mm for subsidence (translation along the y axis) (95% CI for the difference between RSA measurements and actual displacement measured with micrometers, -0.03-0.00) and 0.39° for rotation around the y axis (95% CI, -0.41 to -0.06). The accuracy of marker-based RSA was 0.06 mm for subsidence (95% CI, -0.04-0.01; p = 0.728 compared with model-based RSA) and 0.18° for the y axis rotation (95% CI, -0.23 to -0.07; p = 0.358). The clinical precision of model-based RSA was 0.14 mm for subsidence (95% CI for the difference between double examinations, -0.02-0.04) and 0.79° for the y axis rotation (95% CI, -0.16-0.18). CONCLUSIONS: The accuracy of model-based RSA for measurement of the y axis rotation was not quite as high as that of marker-based RSA, but its clinical precision is at an acceptable level. CLINICAL RELEVANCE: Model-based RSA may be suitable for clinical trials of cementless tapered wedge femoral stem designs.

Zoledronic acid in vivo increases in vitro proliferation of rat mesenchymal stromal cells.

Background and purpose - Bisphosphonates are widely used in the treatment of bone loss, but they might also have positive effects on osteoblastic cells and bone formation. We evaluated the effect of in vivo zoledronic acid (ZA) treatment and possible concomitant effects of ZA and fracture on the ex vivo osteogenic capacity of rat mesenchymal stromal cells (MSCs). Methods - A closed femoral fracture model was used in adult female rats and ZA was administered as a single bolus or as weekly doses up to 8 weeks. Bone marrow MSCs were isolated and cultured for in vitro analyses. Fracture healing was evaluated by radiography, micro-computed tomography (µCT), and histology. Results - Both bolus and weekly ZA increased fracture-site bone mineral content and volume. MSCs from weekly ZA-treated animals showed increased ex vivo proliferative capacity, while no substantial effect on osteoblastic differentiation was observed. Fracture itself did not have any substantial effect on cell proliferation or differentiation at 8 weeks. Serum biochemical markers showed higher levels of bone formation in animals with fracture than in intact animals, while no difference in bone resorption was observed. Interestingly, ex vivo osteoblastic differentiation of MSCs was found to correlate with in vivo serum bone markers. Interpretation - Our data show that in vivo zoledronic acid treatment can influence ex vivo proliferation of MSCs, indicating that bisphosphonates can have sustainable effects on cells of the osteoblastic lineage. Further research is needed to investigate the mechanisms.

In vitro osteogenic capacity of bone marrow MSCs from postmenopausal women reflect the osseointegration of their cementless hip stems.

Age-related dysfunction of mesenchymal stromal cells (MSCs) is suggested as a main cause of altered bone repair with aging. We recently showed that in postmenopausal women undergoing cementless total hip arthroplasty (THA) aging, low bone mineral density (BMD) and age-related geometric changes of the proximal femur are risk factors for increased early migration and delayed osseointegration of the femoral stems. Extending these analyses, we have here explored how the in vitro osteogenic capacity of bone marrow MSCs from these patients reflects implant osseointegration, representing the patient's in vivo bone healing capacity. A total of 19 postmenopausal women with primary hip osteoarthritis (mean age 65 years, range 50-78) and well-defined bone quality underwent successful preoperative in vitro analysis of osteogenic capacity of iliac crest bone marrow MSCs as well as two-year radiostereometric (RSA) follow-up of femoral stem migration after cementless THA. In patients with MSCs of low osteogenic capacity, the magnitude of cumulative stem subsidence after the settling period of three months was greater (p = 0.028) and the time point for translational osseointegration was significantly delayed (p = 0.030) compared to patients with MSCs of high osteogenic capacity. This study suggests that patients with MSCs of low in vitro osteogenic capacity may display increased stem subsidence after the settling period of 3 months and thereby delayed osseointegration. Our study presents a novel approach for studying the biological progress of hip implant osseointegration and to verify the impact of decreased MSCs function, especially in patients with age-related dysfunction of MSCs and bone healing capacity.

Increased migration of uncemented acetabular cups in female total hip arthroplasty patients with low systemic bone mineral density. A 2-year RSA and 8-year radiographic follow-up study of 34 patients.

BACKGROUND AND PURPOSE: Low bone mineral density (BMD) may jeopardize the initial component stability and delay osseointegration of uncemented acetabular cups in total hip arthroplasty (THA). We measured the migration of uncemented cups in women with low or normal BMD. PATIENTS AND METHODS: We used radiostereometric analysis (RSA) to measure the migration of hydroxyapatite-coated titanium alloy cups with alumina-on-alumina bearings in THA of 34 female patients with a median age of 64 (41-78) years. 10 patients had normal BMD and 24 patients had low systemic BMD (T-score ≤ -1) based on dual-energy X-ray absorptiometry (DXA). Cup migration was followed with RSA for 2 years. Radiographic follow-up was done at a median of 8 (2-10) years. RESULTS: Patients with normal BMD did not show a statistically significant cup migration after the settling period of 3 months, while patients with low BMD had a continuous proximal migration between 3 and 12 months (p = 0.03). These differences in cup migration persisted at 24 months. Based on the perceived risk of cup revision, 14 of the 24 cases were "at risk" (proximal translation of 0.2 to 1.0 mm) in the low-BMD group and 2 of the 10 cases were "at risk" in the normal-BMD group (odds ratio (OR) = 8.0, 95% CI: 1.3-48). The radiographic follow-up showed no radiolucent lines or osteolysis. 2 cups have been revised for fractures of the ceramic bearings, but none for loosening. INTERPRETATION: Low BMD contributed to cup migration beyond the settling period of 3 months, but the migrating cups appeared to osseointegrate eventually.

Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.