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With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

Knee osteoarthritis, the most prevalent degenerative joint disorder worldwide, is driven by chronic low-grade inflammation and subsequent cartilage degradation. Clinical data on the role of the Hoffa or infrapatellar fat pad in knee osteoarthritis are, however, scarce. The infrapatellar fat pad is a richly innervated intracapsular, extrasynovial adipose tissue, and an abundant source of adipokines and proinflammatory and catabolic cytokines, which may contribute to chronic synovial inflammation, cartilage destruction, and subchondral bone remodeling during knee osteoarthritis. How the infrapatellar fat pad interacts with neighboring tissues is poorly understood. Here, we review available literature with regard to the infrapatellar fat pad's interactions with cartilage, synovium, bone, menisci, ligaments, and nervous tissue during the development and progression of knee osteoarthritis. Signaling cascades are described with a focus on immune cell populations, pro- and anti-inflammatory cytokines, adipokines, mesenchymal stromal cells, and molecules derived from conditioned media from the infrapatellar fat pad. Understanding the complex interplay between the infrapatellar fat pad and its neighboring articular tissues may help to better understand and treat the multifactorial pathogenesis of osteoarthritis.

ZusammenfassungRegenerative Therapien in der Orthopädie und Unfallchirurgie (O&U) werden zur Behandlung akuter Verletzungen oder degenerativer Gewebeschäden eingesetzt. Insbesondere die Therapie verschiedener Arthroseformen und Tendinopathien mittels Platelet-rich Plasma (PRP) nimmt mittlerweile einen zentralen Platz in der ambulanten Versorgung ein. Es fehlt bis dato allerdings an robusten Daten zur Wirksamkeit und Produktcharakterisierung. Mesenchymale Zelltherapien werden ebenfalls zur Geweberegeneration durch parakrine Immunmodulation eingesetzt und befinden sich aktuell noch in präklinischen und klinischen Forschungsstadien. In den kommenden Jahren wird neben der Sicherheit insbesondere die langfristige Wirksamkeit von Plasma- und Zelltherapien im Fokus der Wissenschaft stehen, um regenerative Therapien klinisch und evidenzbasiert in der O&U zu verankern.

Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFβ and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFβ, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.

Patients suffering from musculoskeletal diseases must cope with a diminished quality of life and an increased burden on medical expenses. The interaction of immune cells and mesenchymal stromal cells during bone regeneration is one of the key requirements for the restoration of skeletal integrity. While stromal cells of the osteo-chondral lineage support bone regeneration, an excessive accumulation of cells of the adipogenic lineage is thought to promote low-grade inflammation and impair bone regeneration. Increasing evidence indicates that pro-inflammatory signaling from adipocytes is responsible for various chronic musculoskeletal diseases. This review aims to summarize the features of bone marrow adipocytes by phenotype, function, secretory features, metabolic properties and their impact on bone formation. In detail, the master regulator of adipogenesis and prominent diabetes drug target, peroxisome proliferator-activated receptor γ (PPARG), will be debated as a potential therapeutic approach to enhance bone regeneration. We will explore the possibilities of using clinically established PPARG agonists, the thiazolidinediones (TZDs), as a treatment strategy to guide the induction of a pro-regenerative, metabolically active bone marrow adipose tissue. The impact of this PPARG induced bone marrow adipose tissue type on providing the necessary metabolites to sustain osteogenic-as well as beneficial immune cells during bone fracture healing will be highlighted.

Objective Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes. Design For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed. Results All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content. Conclusions Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual’s immune profile and the concentration method appear to impact the final PRP product. Trial registration This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFβ signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.

In vitro transcribed (IVT-)mRNA has entered center stage for vaccine development due to its immune co-stimulating properties. Given the widely demonstrated safety of IVT-mRNA-based vaccines, we aimed to adopt IVT-mRNA encoding VEGF for secretory phenotype modulation of therapeutic cells. However, we observed that the immunogenicity of IVT-mRNA impairs the endogenous secretion of pro-angiogenic mediators from transfected mesenchymal stromal cells, instead inducing anti-angiogenic chemokines. This inflammatory secretome modulation limits the application potential of unmodified IVT-mRNA for cell therapy manufacturing, pro-angiogenic therapy and regenerative medicine. To uncouple immunogenicity from the protein expression functionality, we immuno-engineered IVT-mRNA with different chemically modified ribonucleotides. 5-Methoxy-uridine-modification of IVT-mRNA rescued the endogenous secretome pattern of transfected cells and prolonged secretion of IVT-mRNA-encoded VEGF. We found that high secretion of IVT-mRNA-encoded protein further depends on optimized cell adhesion. Cell encapsulation in a collagen-hyaluronic acid hydrogel increased secretion of IVT-mRNA-encoded VEGF and augmented the endogenous secretion of supporting pro-angiogenic mediators, such as HGF. Integrating minimally immunogenic mRNA technology with predesigned matrix-derived cues allows for the synergistic combination of multiple dimensions of cell manipulation and opens routes for biomaterial-based delivery of mRNA-engineered cell products. Such multimodal systems could present a more biologically relevant way to therapeutically address complex multifactorial processes such as tissue ischemia, angiogenesis, and regeneration.

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3′ UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%. Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two alpha-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3 ' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

AIMS Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% of cases, GCTB is characterised by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant-cell-rich to giant-cell-poor. The diagnosis of GCTB can be challenging, and several other lesions need to be excluded, e.g. aneurysmal bone cysts, non-ossifying fibromas, chondroblastomas, brown tumours, and giant-cell-rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology and histology of three H3F3A-mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. METHODS AND RESULTS Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphological features of all three reported lesions were fibrocytic, oval cells in a background of osteoid and an absence of multinuclear giant cells as confirmed with CD68 immunohistochemistry. We used immunohistochemistry and Sanger sequencing to demonstrate positivity for the H3.3 p.G34W mutation. Differential diagnoses were systematically excluded on the basis of histomorphology, immunohistochemistry, and fluorescence in-situ hybridisation. The imaging (radiography, computed tomography, and magnetic resonance imaging) for all three cases is presented and discussed. CONCLUSIONS We believe that these GCTBs without giant cells expand one end of the heterogeneous range of GCTB. Because of the lack of giant cells, correct diagnosis of GCTB is challenging or even impossible on histological grounds alone. In these cases, detection of the characteristic H3F3A mutation (G34W-specific antibody RM263 or sequencing) is extremely helpful for diagnosing those lesions without giant cells as giant cell tumours of bone.

Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFβ signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.

Background Bacillus subtilis is a Gram-positive bacterium used as a cell factory for protein production. Over the last decades, the continued optimization of production strains has increased yields of enzymes, such as amylases, and made commercial applications feasible. However, current yields are still significantly lower than the theoretically possible yield based on the available carbon sources. In its natural environment, B. subtilis can respond to unfavorable growth conditions by differentiating into motile cells that use flagella to swim towards available nutrients. Results In this study, we analyze existing transcriptome data from a B. subtilis α-amylase production strain at different time points during a 5-day fermentation. We observe that genes of the fla/che operon, essential for flagella assembly and motility, are differentially expressed over time. To investigate whether expression of the flagella operon affects yield, we performed CRISPR-dCas9 based knockdown of the fla/che operon with sgRNA target against the genes flgE, fliR, and flhG, respectively. The knockdown resulted in inhibition of mobility and a striking 2–threefold increase in α-amylase production yield. Moreover, replacing flgE (required for flagella hook assembly) with an erythromycin resistance gene followed by a transcription terminator increased α-amylase yield by about 30%. Transcript levels of the α-amylase were unaltered in the CRISPR-dCas9 knockdowns as well as the flgE deletion strain, but all manipulations disrupted the ability of cells to swim on agar. Conclusions We demonstrate that the disruption of flagella in a B. subtilis α-amylase production strain, either by CRISPR-dCas9-based knockdown of the operon or by replacing flgE with an erythromycin resistance gene followed by a transcription terminator, increases the production of α-amylase in small-scale fermentation.

Objective: Platelet-rich plasma (PRP) is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples.Design: For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Results: All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood (p < 0.0001). Further, the pro-inflammatory cytokines IFN-γ (p = 0.039) and TNF-α (p = 0.013) were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products.Conclusions: Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy. Both the individual’s immune profile and the concentration method appear to impact the final PRP product.Trial registration: This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4th November 2021 (registration number DRKS00026175).

Aims The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells. Methods HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 106 PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%. Conclusion The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF. Cite this article: Bone Jt Open 2022;3(4):340–347.

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A-mutated neoplastic mononucleated fibroblast-like cell population, and H3F3A wild-type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome-wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell-rich lesions comprising three H3F3B-mutated chondroblastomas, three USP6-rearranged aneurysmal bone cysts, three non-ossifying fibromas, two hyperparathyroidism-associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A-mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell-containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p 0.001). This hypermethylation correlates with the lower gene expression at the mRNA level for these three genes in the cell lines, the lack of p16 and p21 in these cell lines, and the lower expression of p16 and p21 in GCTB. Overall, our analysis reveals characteristic DNA methylation patterns of giant cell tumours of bone and chondroblastomas and shows that cell lines of giant cell tumours of bone are a valid model for further analysis of H3F3A-mutated tumour cells. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

Particles released from cobalt-chromium-molybdenum (CoCrMo) alloys are considered common elicitors of chronic inflammatory adverse effects. There is a lack of data demonstrating particle numbers, size distribution and elemental composition of bone marrow resident particles which would allow for implementation of clinically relevant test strategies in bone marrow models at different degrees of exposure. The aim of this study was to investigate metal particle exposure in human periprosthetic bone marrow of three types of arthroplasty implants. Periprosthetic bone marrow sections from eight patients exposed to CoCrMo particles were analyzed via spatially resolved and synchrotron-based nanoscopic X-ray fluorescence imaging. These analyses revealed lognormal particle size distribution patterns predominantly towards the nanoscale. Analyses of particle numbers and normalization to bone marrow volume and bone marrow cell number indicated particle concentrations of up to 1 × 1011 particles/ml bone marrow or 2 × 104 particles/bone marrow cell, respectively. Analyses of elemental ratios of CoCrMo particles showed that particularly the particles’ Co content depends on particle size. The obtained data point towards Co release from arthroprosthetic particles in the course of dealloying and degradation processes of larger particles within periprosthetic bone marrow. This is the first study providing data based on metal particle analyses to be used for future in vitro and in vivo studies of possible toxic effects in human bone marrow following exposure to arthroprosthetic CoCrMo particles of different concentration, size, and elemental composition.

The influence of a SARS-CoV-2 infection on inflammatory bowel disease (IBD) has not yet been well characterized and it is unclear whether this requires an adaptation of the immunosuppressive therapy.A national register was established for the retrospective documentation of clinical parameters and changes in immunosuppressive therapy in SARS-CoV-2 infected IBD patients.In total, only 3 of 185 IBD patients (1.6 %) were tested for SARS-CoV-2 infection because of abdominal symptoms. In the course of COVID-19 disease, 43.5 % developed diarrhea, abdominal pain or hematochezia (risk of hospitalization with vs. without abdominal symptoms: 20.0 % vs. 10.6 %, p 0.01). With active IBD at the time of SARS-CoV-2 detection, there was an increased risk of hospitalization (remission 11.2 %, active IBD 23.3 % p 0.05). IBD-specific therapy remained unchanged in 115 patients (71.4 %); the most common change was an interruption of systemic therapy (16.2 %).New abdominal symptoms often appeared in SARS-CoV-2 infected IBD patients. However, these only rarely led to SARS-CoV-2 testing. A high IBD activity at the time of SARS-CoV-2 detection was associated with an increased risk of hospitalization.Der Einfluss einer SARS-CoV-2-Infektion auf chronisch entzündliche Darmerkrankungen (CED) ist bislang nicht gut charakterisiert, und es ist unklar, ob diese eine Anpassung der immunsuppressiven Therapie erfordert.Für die retrospektive Dokumentation klinischer Parameter und Veränderungen einer immunsuppressiven Therapie von mit SARS-CoV-2 infizierten CED-Patienten wurde ein nationales Melderegister etabliert.Insgesamt wurden nur 3 von 185 CED-Patienten (1,6 %) wegen abdomineller Symptome auf eine SARS-CoV-2-Infektion getestet. Im COVID-19-Krankheitsverlauf entwickelten 43,5 % Durchfall, abdominelle Schmerzen oder Hämatochezie (Hospitalisierungsrisiko mit vs. ohne abdominelle Symptome: 20,0 % vs. 10,6 %, p 0,01). Bei aktiver CED zum Zeitpunkt des SARS-CoV-2-Nachweises bestand ein erhöhtes Hospitalisierungsrisiko (Remission 11,2 %, aktive CED 23,3 % p 0,05). Die CED-spezifische Therapie blieb bei 115 Patienten (71,4 %) unverändert; die häufigste Änderung bestand in einer Unterbrechung der systemischen Therapie (16,2 %).Bei mit SARS-CoV-2 infizierten CED-Patienten traten häufig neue abdominelle Symptome bei Infektion auf. Diese führten aber nur selten zur SARS-CoV-2-Testung. Eine hohe CED-Aktivität zum Zeitpunkt des SARS-CoV-2-Nachweises war mit einem erhöhten Hospitalisierungsrisiko assoziiert.

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.

Intravascular infusion is the most popular route for therapeutic multipotent mesenchymal stromal/stem cell (MSC) delivery in hundreds of clinical trials. Meta-analysis has demonstrated that bone marrow MSC infusion is safe. It is not clear if this also applies to diverse new cell products derived from other sources, such as adipose and perinatal tissues. Different MSC products display varying levels of highly procoagulant tissue factor (TF) and may adversely trigger the instant blood-mediated inflammatory reaction (IBMIR). Suitable strategies for assessing and controlling hemocompatibility and optimized cell delivery are crucial for the development of safer and more effective MSC therapies.

A large part of our current understanding of gene regulation in Gram-positive bacteria is based on Bacillus subtilis , as it is one of the most well studied bacterial model systems. The rapid growth in data concerning its molecular and genomic biology is distributed across multiple annotation resources. Consequently, the interpretation of data from further B. subtilis experiments becomes increasingly challenging in both low- and large-scale analyses. Additionally, B. subtilis annotation of structured RNA and non-coding RNA (ncRNA), as well as the operon structure, is still lagging behind the annotation of the coding sequences. To address these challenges, we created the B. subtilis genome atlas, BSGatlas, which integrates and unifies multiple existing annotation resources. Compared to any of the individual resources, the BSGatlas contains twice as many ncRNAs, while improving the positional annotation for 70 % of the ncRNAs. Furthermore, we combined known transcription start and termination sites with lists of known co-transcribed gene sets to create a comprehensive transcript map. The combination with transcription start/termination site annotations resulted in 717 new sets of co-transcribed genes and 5335 untranslated regions (UTRs). In comparison to existing resources, the number of 5′ and 3′ UTRs increased nearly fivefold, and the number of internal UTRs doubled. The transcript map is organized in 2266 operons, which provides transcriptional annotation for 92 % of all genes in the genome compared to the at most 82 % by previous resources. We predicted an off-target-aware genome-wide library of CRISPR–Cas9 guide RNAs, which we also linked to polycistronic operons. We provide the BSGatlas in multiple forms: as a website (https://rth.dk/resources/bsgatlas/), an annotation hub for display in the UCSC genome browser, supplementary tables and standardized GFF3 format, which can be used in large scale -omics studies. By complementing existing resources, the BSGatlas supports analyses of the B. subtilis genome and its molecular biology with respect to not only non-coding genes but also genome-wide transcriptional relationships of all genes.

A large part of our current understanding of gene regulation in Gram-positive bacteria is based on Bacillus subtilis , as it is one of the most well studied bacterial model systems. The rapid growth in data concerning its molecular and genomic biology is distributed across multiple annotation resources. Consequently, the interpretation of data from further B. subtilis experiments becomes increasingly challenging in both low- and large-scale analyses. Additionally, B. subtilis annotation of structured RNA and non-coding RNA (ncRNA), as well as the operon structure, is still lagging behind the annotation of the coding sequences. To address these challenges, we created the B. subtilis genome atlas, BSGatlas, which integrates and unifies multiple existing annotation resources. Compared to any of the individual resources, the BSGatlas contains twice as many ncRNAs, while improving the positional annotation for 70 % of the ncRNAs. Furthermore, we combined known transcription start and termination sites with lists of known co-transcribed gene sets to create a comprehensive transcript map. The combination with transcription start/termination site annotations resulted in 717 new sets of co-transcribed genes and 5335 untranslated regions (UTRs). In comparison to existing resources, the number of 5′ and 3′ UTRs increased nearly fivefold, and the number of internal UTRs doubled. The transcript map is organized in 2266 operons, which provides transcriptional annotation for 92 % of all genes in the genome compared to the at most 82 % by previous resources. We predicted an off-target-aware genome-wide library of CRISPR–Cas9 guide RNAs, which we also linked to polycistronic operons. We provide the BSGatlas in multiple forms: as a website (https://rth.dk/resources/bsgatlas/), an annotation hub for display in the UCSC genome browser, supplementary tables and standardized GFF3 format, which can be used in large scale -omics studies. By complementing existing resources, the BSGatlas supports analyses of the B. subtilis genome and its molecular biology with respect to not only non-coding genes but also genome-wide transcriptional relationships of all genes.

Zusammenfassung Einleitung Der Einfluss einer SARS-CoV-2-Infektion auf chronisch entzündliche Darmerkrankungen (CED) ist bislang nicht gut charakterisiert, und es ist unklar, ob diese eine Anpassung der immunsuppressiven Therapie erfordert. Methodik Für die retrospektive Dokumentation klinischer Parameter und Veränderungen einer immunsuppressiven Therapie von mit SARS-CoV-2 infizierten CED-Patienten wurde ein nationales Melderegister etabliert. Ergebnisse Insgesamt wurden nur 3 von 185 CED-Patienten (1,6 %) wegen abdomineller Symptome auf eine SARS-CoV-2-Infektion getestet. Im COVID-19-Krankheitsverlauf entwickelten 43,5 % Durchfall, abdominelle Schmerzen oder Hämatochezie (Hospitalisierungsrisiko mit vs. ohne abdominelle Symptome: 20,0 % vs. 10,6 %, p Diskussion Bei mit SARS-CoV-2 infizierten CED-Patienten traten häufig neue abdominelle Symptome bei Infektion auf. Diese führten aber nur selten zur SARS-CoV-2-Testung. Eine hohe CED-Aktivität zum Zeitpunkt des SARS-CoV-2-Nachweises war mit einem erhöhten Hospitalisierungsrisiko assoziiert.

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harsh in vivo environments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.

Loosening of orthodontic and orthopedic implants is a critical and common clinical problem. To minimize the numbers of revision surgeries due to peri-implant inflammation or insufficient osseointegration, developments of new implant manufacturing strategies are indicated. Ultrafast laser surface texturing is a promising contact-free technology to modify the physicochemical properties of surfaces toward an anti-infectious functionalization. This work aims to texture Ti6Al4V surfaces with ultraviolet (UV) and green (GR) radiation for the manufacturing of laser-induced periodic surface structures (LIPSS). The assessment of these surface modifications addresses key aspects of topography, morphology and chemical composition. Human primary mesenchymal stromal cells (hMSCs) were cultured on laser-textured and polished Ti6Al4V to characterize the surfaces in terms of their in vitro biocompatibility, cytotoxicity, and metal release. The outcomes of the in vitro experiment show the successful culture of hMSCs on textured Ti6Al4V surfaces developed within this work. Cells cultured on LIPSS surfaces were not compromised in terms of their viability if compared to polished surfaces. Yet, the hMSC culture on UV-LIPSS show significantly lower lactate dehydrogenase and titanium release into the supernatant compared to polished. Thus, the presented surface modification can be a promising approach for future applications in orthodontics and orthopedics.

Vascularization of the fracture site and cell-mediated deposition of the mineralized matrix are crucial determinants for successful bone regeneration after injury. Ceramic biomaterials such as bioactive glasses (BAGs) that release bioactive ions have shown promising results in bone defect regeneration. However, it remains unclear how the dosage and composition of bioactive ions influence the angiogenic and osteogenic behavior of primary human mesenchymal stromal cells (MSCs). Here, we show that exposure to ionic dissolution products from 1393 and 45S5 BAGs can evoke distinct angiogenic and osteogenic responses from primary MSCs in a dose- and composition-dependent manner. Significantly higher concentrations of the pro-angiogenic factors VEGF, HGF, PIGF, angiopoietin, and angiogenin were detected in conditioned media (CM) from MSCs exposed to 45S5, but not 1393, BAGs. Application of this CM to human umbilical vein endothelial cells (HUVECs) resulted in robust 2D tube formation in vitro. Osteogenic differentiation of MSCs was assessed by gene expression analysis and mineralization assays. Low concentrations (0.1% w/v) of 1393 BAGs significantly enhanced the gene expression of RUNX2 and ALP and induced an earlier onset of matrix mineralization compared to all other groups. We further tested whether simultaneous exposure to both BAGs would improve both angiogenic secretion and osteogenic differentiation of MSCs, and did not find evidence to support this hypothesis. Our results provide evidence of BAG composition-dependent enhancement of primary human MSCs' regenerative function, besides also underlining the importance of an in vitro evaluation of the dose-response relationship to translate BAG based approaches into safe and effective clinical therapies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2827-2837, 2018., 2018.

Background No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma-related muscle injuries using local intraoperative application of allogeneic placenta-derived, mesenchymal-like adherent cells (PLX-PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. Methods Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo-controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX-PAD or a placebo (n = 7) into the injured gluteus medius muscles. Results We did not observe any relevant PLX-PAD-related adverse events at the 2-year follow-up. Improved gluteus medius strength was noted as early as Week 6 in the treatment-groups. Surprisingly, until Week 26, the low-dose group outperformed the high-dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3 , 150 M: 237.4 ± 27.2 cm3 ]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low-dose treatment reduced the surgery-related immunological stress reaction more than high-dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T-cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late-onset immune reactivity after high-dose treatment corresponded to reduced functional improvement. Conclusions Allogeneic PLX-PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress-related immunological reactions.

Porous tantalum components are widely used for complex acetabular reconstructions in revision hip arthroplasty. Multiple other metal alloys such as titanium-aluminum-vanadium or cobalt-chromium-molybdenum are principally used in artificial joint setups. We report a case of tantalum component failure being both cause and effect of a multiple metal exposure. Our aims were to assess and to characterize associated particle exposure and biological consequences. Metal level quantification revealed substantial in vivo exposure to particulate and dissociated tantalum, zirconium, chromium, cobalt, molybdenum, titanium, aluminum and vanadium in periprosthetic compartments. Aside from micron-sized particles, nanoparticles of a broad size range and elemental composition were obtained. Histological exams verified a spectrum of necrotic changes in the periprosthetic tissues. In the presented case tantalum release was accompanied by concomitance of particles originating from other utilized metals. We conclude that an overall in vivo exposure assessment is mandatory for realistic appraisal of metal toxicity and associated risks.

Controlled revascularization and inflammation are key elements regulating endogenous regeneration after (bone) tissue trauma. Peripheral blood-derived cell subsets, such as regulatory T-helper cells and circulating (endothelial) progenitor cells, respectively, can support endogenous tissue healing, whereas effector T cells that are associated with an aged immune system can hinder bone regeneration. CD31 is expressed by diverse leukocytes and is well recognized as a marker of circulating endothelial (precursor) cells; however, CD31 is absent from the surface of differentiated effector T cells. Thus, we hypothesized that by separating the inhibitory fractions from the supportive fractions of circulating cells within the peripheral blood (PB) using the CD31 marker, bone regeneration in biologically compromised conditions, such as those observed in aged patients, could be improved. In support of our hypothesis, we detected an inverse correlation between CD31+ cells and effector T cells in the hematomas of human fracture patients, dependent on the age of the patient. Furthermore, we demonstrated the regenerative capacity of human PB-CD31+ cells in vitro. These findings were translated to a clinically relevant rat model of impaired bone healing. The transplantation of rat PB-CD31+ cells advanced bone tissue restoration in vivo and was associated with an early anti-inflammatory response, the stimulation of (re)vascularization, and reduced fibrosis. Interestingly, the depletion or enrichment of the highly abundant CD31+/14+ monocytes from the mixed CD31+ cell population diminished tissue regeneration at different levels, suggesting combined effects within the PB-CD31+ subsets. In summary, an intraoperative enrichment of PB-CD31+ cells might be a novel option to facilitate endogenous regeneration under biologically impaired situations by supporting immunomodulation and vascularization. © 2016 American Society for Bone and Mineral Research.

In education, learners’ autonomy and agency have been emphasized across various domains. However, ability to self-regulate their learning by setting a goal, monitoring, regulating and evaluating their learning progress is not easy. With wearable sensor technology, various physiological and contextual data can be detected and collected. To provide learners with a context-aware personal learning support, we have researched physiological sensor data (EDA and ECG) by providing emotional stimulants to 70 students from two higher education institutes. We have analyzed our collected data using multiple methods (qualitative, quantitative, machine learning and fuzzy logic approaches) and found a relation between physiological sensor data and emotion that seems promising. Consecutively, we have investigated a learning support system for self-regulated learning and proposed three ideas with prototypes. Our future work will entail implementation of research findings to develop a learning companion system to support learners’ self-regulated learning.

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics.

Delayed type hypersensitivity (DTH) reactions are considered infrequent complications in arthroplasty, but have been recognized to be associated with devastating morbidity and substantial decrease in quality of life of affected patients. Chronic inflammation of artificial joints and associated loss of peri-implant bone often require revision surgery. Methods for the diagnosis of implant-related DTH are available but infrequently considered to the full extent. Sequential diagnostics based on exclusion of septic complications, local and systemic metal level determination, lymphocyte transformation testing (LTT), and local T cell subset analysis are required for an unequivocal DTH diagnosis. Here, we report on a patient with a history of chronic rheumatoid arthritis and an unfavorable outcome of unilateral knee arthroplasty. This case illustrates pitfalls and difficulties in the course of recurrent inflammation following joint replacement. In the early course, suspicion of low-grade bacterial infection led to three two-stage revisions. Afterwards, the joint was proven to be sterile. However, metal level quantification revealed release of especially cobalt and chromium from the joint, LTT indicated persisting cobalt and nickel sensitization and subset analysis of T cells from the synovium suggested DTH as a root cause for the inflammatory symptoms. This report aims to recommend the depicted diagnostic algorithm as an adequate tool for future DTH detection. Yet, systemic to local subset ratios for effector memory and regulatory T cells should be derived from sufficient patient numbers to establish it as a diagnostic marker. Moreover, future prospects regarding implant-related DTH diagnostics are discussed. Therapeutic options for the portrayed patient are proposed, considering pharmaceutical, cell-therapeutic and surgical aspects. Patients who experience peri-implant inflammation but do not have obvious mechanical or infectious problems remain a diagnostic challenge and are at high risk of being treated inadequately. Since potentially sensitizing materials are regularly used in arthroplasty, it is essential to detect cases of acute DTH-derived inflammation of an artificial joint at early postoperative stages. This would reduce the severity of inflammation-related long-term consequences for affected patients and may avoid unnecessary revision surgery.

Placenta-derived decidua stromal cells (DSCs) are being investigated as an alternative to other sources of mesenchymal stromal cells (MSCs) for cellular therapy. DSCs are more effective in treating acute inflammatory diseases in human and this is our preclinical safety study of human DSCs in Sprague-Dawley rats and Balb/c mice. Human DSCs were cultured and expanded from fetal membranes obtained from placentas following cesarean section. In rats, 0.5 × 106 cells/kg were injected intravenously (n = 4) or intra-aortal (n = 4). In mice, DSCs were given intravenously at doses ranging from 4-40 × 106 cells/kg (total of n = 120 mice). In vivo tracking of human cells in mice was performed by using transduced DSC with luciferin gene, and in rats by using 18F-FDG PET. Clotting parameters were determined in vitro and in vivo. All intra-arterially DSC-treated rats had normal motility and behavior and histological examination was normal for liver, spleen kidneys and thigh muscles. Mice treated with DSCs showed no immediate or long-term side effects. None of the mice died or showed acute toxicity or adverse reactions 3 and 30 days after DSC infusion. Murine blood biochemistry profiles related to liver, kidney, heart, and inflammatory indices was not influenced by DSC infusion and complete blood counts were normal. In vivo tracking of infused DSCs detected a signal in the lungs for up to 4 days post infusion. Compared to bone marrow derived MSCs, the DSCs had better viability, smaller size, but stronger clotting in human blood and plasma. Both MSC- and DSC-induced coagulation and complement activation markers, thrombin-anti-thrombin complex (TAT) and C3a, and in vitro clotting parameters were decreased by heparin supplementation. In conclusion, DSCs are safe with almost no side effects even with doses 40 times higher than are used clinically, particularly when supplemented with low-dose heparin.

The genome of Bacillus subtilis continues to provide exiting genomic insights. However, the growing collective genomic knowledge about this micro-organism is spread across multiple annotation resources. Thus, the full annotation is not directly accessible neither for specific genes nor for large-scale high-throughput analyses. Furthermore, access to annotation of non-coding RNA genes (ncRNAs) and polycistronic mRNAs is difficult. To address these challenges we introduce the Bacillus subtilis genome atlas, BSGatlas, in which we integrate and unify multiple existing annotation resources. Our integration provides twice as many ncRNAs than the individual resources, improves the positional annotation for 70% of the combined ncRNAs, and makes it possible to infer specific ncRNA types. Moreover, we unify known transcription start sites, termination, and transcriptional units (TUs) as a comprehensive transcript map. This transcript map implies 815 new TUs and 6, 164 untranslated regions (UTRs), which is a five-fold increase over existing resources. We furthermore, find 2, 309 operons covering the transcriptional annotation for 93% of all genes, corresponding to an improvement by 11%. The BSGatlas is available in multiple formats. A user can either download the entire annotation in the standardized GFF3 format, which is compatible with most bioinformatics tools for omics and high-throughput studies, or view the annotation in an online browser at http://rth.dk/resources/bsgatlas.ImportanceThe Bacillus subtilis genome has been studied in numerous context and consequently multiple efforts have been made in providing a complete annotation. Unfortunately, a number of resources are no longer maintained, and (i) the collective annotation knowledge is dispersed over multiple resources, of which each has a different focus of what type of annotation information they provide. (ii) Thus, it is difficult to easily and at a large scale obtain information for a genomic region or genes of interest. (iii) Furthermore, all resources are essentially incomplete when it comes to annotating non-coding and structured RNA, and transcripts in general. Here, we address all three problems by first collecting existing annotations of genes and transcripts start and termination sites; afterwards resolving discrepancies in annotations and combining them, which doubled the number of ncRNAs; inferring full transcripts and 2,309 operons from the combined knowledge of known transcript boundaries and meta-information; and critically providing it all in a standardized UCSC browser. That interface and its powerful set of functionalities allow users to access all the information in a single resource as well as enables them to include own data on top the full annotation.

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion-based 3D printing is used to fabricate photo-crosslinkable gelatin-based scaffolds with pore sizes in the range of 200-600 mu m. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC-based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.

Cell encapsulation in confining 3D hydrogels typically prevents encapsulated cells from spreading and establishing cell-cell contacts. Interactions with neighboring cells or with the extracellular matrix (ECM) influence the paracrine activity of mesenchymal stromal cells (MSCs), but how these interactions are regulated by structural properties of biomaterial scaffolds remains insufficiently explored. Here, we describe the use of extrusion-based 3D printing to fabricate acellular, gelatin-based scaffolds with programmed strut spacings of 400 (small), 500 (medium), and 600 μm (large). These scaffolds showed similar effective Young's moduli in the range of 2-5 kPa, and varied based on average pore size which ranged from ∼200 μm (small pore: SP) through ∼302 μm (medium pore: MP) to ∼382 μm (large pore: LP). When seeded with MSCs, pore size guided cell distribution on the scaffolds, with smaller pores preventing cell infiltration, medium ones causing cells to aggregate in between struts, and large ones causing cells to flow through after attachment on the struts. These changes in cell distribution regulated cell-cell and cell-matrix interactions at the gene level, as assessed by pathway focused PCR arrays. Medium pore size scaffolds stimulated the highest paracrine secretion of a panel of angiogenic cytokines. This enhancement of paracrine activity substantially improved endothelial cell migration in a chemotaxis assay, increased single cell migration kinetics such as velocity, and stimulated the formation of robust tubular structures. Together, these findings not only provide new insights on cellular interactions in scaffold environments but also demonstrate how 3D biomaterial design can instruct and enhance the regenerative paracrine activities of MSCs.

Arthroplasty ranks among the greatest achievements of surgical medicine, with total hip replacement termed “the operation of the century.” Despite its wide success, arthroplasty bears risks, such as local reactions to implant derived wear and corrosion products. Prevalence of allergies across Western society increases and along the number of reported hypersensitivity reactions to orthopedic implant materials. In this context the main focus is on delayed hypersensitivity (DTH). This mechanism is mainly attributed to T cells and an overreaction of the adaptive immune system. Arthroplasty implant materials are in direct contact with bone marrow (BM), which is discussed as a secondary lymphoid organ. However, the mechanisms of sensitization toward implant wear remain elusive. Nickel and cobalt ions can form haptens with native peptides to activate immune cell receptors and are therefore common T helper allergens in cutaneous DTH. The rising prevalence of metal-related allergy in the general population and evidence for the immune-modulating function of BM allow for the assumption hypersensitivity reactions could occur in peri-implant BM. There is evidence that pro-inflammatory factors released during DTH reactions enhance osteoclast activity and inhibit osteoblast function, an imbalance characteristic for osteolysis. Even though some mechanisms are understood, hypersensitivity has remained a diagnosis of exclusion. This review aims to summarize current views on the pathomechanism of DTH in arthroplasty with emphasis on BM and discusses recent advances and future directions for basic research and clinical diagnostics.

There is increasing evidence that T lymphocytes play a key role in controlling endogenous regeneration. Regeneration appears to be impaired in case of local accumulation of CD8+ effector T cells (TEFF), impairing endogenous regeneration by increasing a primary “useful” inflammation toward a damaging level. Thus, rescuing regeneration by regulating the heightened pro-inflammatory reaction employing regulatory CD4+ T (TReg) cells could represent an immunomodulatory option to enhance healing. Hypothesis was that CD4+ TReg might counteract undesired effects of CD8+ TEFF. Using adoptive TReg transfer, bone healing was consistently improved in mice possessing an inexperienced immune system with low amounts of CD8+ TEFF. In contrast, mice with an experienced immune system (high amounts of CD8+ TEFF) showed heterogeneous bone repair with regeneration being dependent upon the individual TEFF/TReg ratio. Thus, the healing outcome can only be improved by an adoptive TReg therapy, if an unfavorable TEFF/TReg ratio can be reshaped; if the individual CD8+ TEFF percentage, which is dependent on the individual immune experience can be changed toward a favorable ratio by the TReg transfer. Remarkably, also in patients with impaired fracture healing the TEFF/TReg ratio was higher compared to uneventful healers, validating our finding in the mouse osteotomy model. Our data demonstrate for the first time the key-role of a balanced TEFF/TReg response following injury needed to reach successful regeneration using bone as a model system. Considering this strategy, novel opportunities for immunotherapy in patients, which are at risk for impaired healing by targeting TEFF cells and supporting TReg cells to enhance healing are possible.

Bone defect repair can benefit from local delivery of mesenchymal stromal cells (MSCs). However, local harsh environmental conditions after injury may necessitate a cell therapy strategy that shields MSCs initially and releases them locally over time. This may be possible by using biomaterials that exhibit stimuli-responsive degradability, such as oxidized alginate hydrogels that undergo hydrolytic degradation. However, it remains unknown whether varying encapsulation periods compromise MSC osteogenic differentiation capacity after release. To address this, we cultured MSCs in 3D alginate beads with tunable degradability before characterizing the function of released cells. Alginates were oxidized to different degrees (2%, 3%, and 4%) to achieve distinct rates of degradation (days to weeks), then functionalized with RGD peptides to enable cell adhesion, and modified additionally with 6-aminofluorescin to enable fluorescence-based detection. Bead morphology, degradation kinetics, cell morphology, and cell release kinetics were monitored over time. Cells that were released from the beads were stimulated to differentiate into the osteogenic lineage. Our results indicate that MSCs released from all bead groups retained a strong ability to deposit mineralized matrix under osteogenic differentiation conditions. These findings provide the basis for designing and implementing biomaterial-based strategies for the in-situ temporal delivery of potent MSCs at bone defect sites.

A focused low-intensity pulsed ultrasound (FLIPUS) was used to investigate the effects of stimulation period, acoustic intensity and donor age on the osteogenic differentiation potential of rat mesenchymal stromal cells (rMSCs). rMSCs from 3- and 12-mo-old female Sprague Drawly rats were isolated from bone marrow and stimulated 20 min/d with either 11.7 or 44.5 mW/cm2 (spatial average temporal average intensity) for 7 or 14 d. Osteogenic differentiation markers, i.e., Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and degree of matrix calcification were analyzed. On day 7 of stimulation, OCN gene expression was enhanced 1.9-fold in cells from young rats when stimulated with low intensity. The low intensity also led to a 40% decrease in RUNX2 expression on day 7 in aged cells, whereas high intensity enhanced expression of RUNX2 on day 14. FLIPUS treatment with low intensity resulted in a 15% increase in extracellular matrix mineralization in young but not old rMSCs. These differences suggest the necessity of a donor-age related optimization of stimulation parameters.

Severe injury to the skeletal muscle often results in the formation of scar tissue, leading to a decline in functional performance. Traditionally, tissue engineering strategies for muscle repair have focused on substrates that promote myogenic differentiation of transplanted cells. In the current study, the reported data indicates that mesenchymal stromal cells (MSCs) transplanted via porous alginate cryogels promote muscle regeneration by secreting bioactive factors that profoundly influence the function of muscle progenitor cells. These cellular functions, which include heightened resistance of muscle progenitor cells to apoptosis, migration to site of injury, and prevention of premature differentiation are highly desirable in the healing cascade after acute muscle trauma. Furthermore, stimulation of MSCs with recombinant growth factors IGF-1 and VEGF165 was found to significantly enhance their paracrine effects on muscle progenitor cells. Multifunctional alginate cryogels were then utilized as synthetic niches that facilitate local stimulation of seeded MSCs by providing a sustained release of growth factors. In a clinically relevant injury model, the modulation of MSC paracrine signaling via engineered niches significantly improved muscle function by remodeling scar tissue and promoting the formation of new myofibers, outperforming standalone cell or growth factor delivery.