Guidelines for minimal information on cellular senescence experimentation in vivo.

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.

The Multifaceted Actions of PVP-Curcumin for Treating Infections.

Curcumin is a natural compound that is considered safe and may have potential health benefits; however, its poor stability and water insolubility limit its therapeutic applications. Different strategies aim to increase its water solubility. Here, we tested the compound PVP-curcumin as a photosensitizer for antimicrobial photodynamic therapy (aPDT) as well as its potential to act as an adjuvant in antibiotic drug therapy. Gram-negative E. coli K12 and Gram-positive S. capitis were subjected to aPDT using various PVP-curcumin concentrations (1-200 µg/mL) and 475 nm blue light (7.5-45 J/cm2). Additionally, results were compared to aPDT using 415 nm blue light. Gene expression of recA and umuC were analyzed via RT-qPCR to assess effects on the bacterial SOS response. Further, the potentiation of Ciprofloxacin by PVP-curcumin was investigated, as well as its potential to prevent the emergence of antibiotic resistance. Both bacterial strains were efficiently reduced when irradiated with 415 nm blue light (2.2 J/cm2) and 10 µg/mL curcumin. Using 475 nm blue light, bacterial reduction followed a biphasic effect with higher efficacy in S. capitis compared to E. coli K12. PVP-curcumin decreased recA expression but had limited effect regarding enhancing antibiotic treatment or impeding resistance development. PVP-curcumin demonstrated effectiveness as a photosensitizer against both Gram-positive and Gram-negative bacteria but did not modulate the bacterial SOS response.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Elimination of damaged mitochondria during UVB-induced senescence is orchestrated by NIX-dependent mitophagy.

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress-induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX-dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB-irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB-irradiated NIX-depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB-induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF.

Aged-vascular niche hinders osteogenesis of mesenchymal stem cells through paracrine repression of Wnt-axis.

Age-induced decline in osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) potentiates osteoporosis and increases the risk for bone fractures. Despite epidemiology studies reporting concurrent development of vascular and bone diseases in the elderly, the underlying mechanisms for the vascular-bone cross-talk in aging are largely unknown. In this study, we show that accelerated endothelial aging deteriorates bone tissue through paracrine repression of Wnt-driven-axis in BMSCs. Here, we utilize physiologically aged mice in conjunction with our transgenic endothelial progeria mouse model (Hutchinson-Gilford progeria syndrome; HGPS) that displays hallmarks of an aged bone marrow vascular niche. We find bone defects associated with diminished BMSC osteogenic differentiation that implicate the existence of angiocrine factors with long-term inhibitory effects. microRNA-transcriptomics of HGPS patient plasma combined with aged-vascular niche analyses in progeria mice reveal abundant secretion of Wnt-repressive microRNA-31-5p. Moreover, we show that inhibition of microRNA-31-5p as well as selective Wnt-activator CHIR99021 boosts the osteogenic potential of BMSCs through de-repression and activation of the Wnt-signaling, respectively. Our results demonstrate that the vascular niche significantly contributes to osteogenesis defects in aging and pave the ground for microRNA-based therapies of bone loss in elderly.

Mesenchymal Stem Cell Conditioned Medium Modulates Inflammation in Tenocytes: Complete Conditioned Medium Has Superior Therapeutic Efficacy than Its Extracellular Vesicle Fraction.

Tendinopathy, a prevalent overuse injury, lacks effective treatment options, leading to a significant impact on quality of life and socioeconomic burden. Mesenchymal stem/stromal cells (MSCs) and their secretome, including conditioned medium (CM) and extracellular vesicles (EVs), have shown promise in tissue regeneration and immunomodulation. However, it remains unclear which components of the secretome contribute to their therapeutic effects. This study aimed to compare the efficacy of CM, EVs, and the soluble protein fraction (PF) in treating inflamed tenocytes. CM exhibited the highest protein and particle concentrations, followed by PF and EVs. Inflammation significantly altered gene expression in tenocytes, with CM showing the most distinct separation from the inflamed control group. Treatment with CM resulted in the most significant differential gene expression, with both upregulated and downregulated genes related to inflammation and tissue regeneration. EV treatment also demonstrated a therapeutic effect, albeit to a lesser extent. These findings suggest that CM holds superior therapeutic efficacy compared with its EV fraction alone, emphasizing the importance of the complete secretome in tendon injury treatment.

Circulating microRNAs in young individuals with long-duration type 1 diabetes in comparison with healthy controls.

MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional control of gene expression and might be used as biomarkers for diabetes-related complications. The aim of this case-control study was to explore potential differences in circulating miRNAs in young individuals with long-duration type 1 diabetes (T1D) compared to healthy controls, and how identified miRNAs are expressed across different tissues. Twelve adolescents, age 15.0-17.9 years, with T1D duration of more than 8 years (mean 11.1 years), were enrolled from the Swedish diabetes quality registry. An age-matched control group was recruited. Circulating miRNAs (n = 187) were analyzed by quantitative PCR. We observed that 27 miRNAs were upregulated and one was downregulated in T1D. Six of these miRNAs were tissue-enriched (blood cells, gastrointestinal, nerve, and thyroid tissues). Six miRNAs with the largest difference in plasma, five up-regulated (hsa-miR-101-3p, hsa-miR-135a-5p, hsa-miR-143-3p, hsa-miR-223-3p and hsa-miR-410-3p (novel for T1D)) and one down-regulated (hsa-miR-495-3p), with P-values below 0.01, were selected for further in-silico analyses. AKT1, VEGFA and IGF-1 were identified as common targets. In conclusion, 28 of the investigated miRNAs were differently regulated in long-duration T1D in comparison with controls. Several associations with cancer were found for the six miRNAs with the largest difference in plasma.

PRPF19 modulates morphology and growth behavior in a cell culture model of human skin.

The skin provides one of the most visual aging transformations in humans, and premature aging as a consequence of oxidative stress and DNA damage is a frequently seen effect. Cells of the human skin are continuously exposed to endogenous and exogenous DNA damaging factors, which can cause DNA damage in all phases of the cell cycle. Increased levels of DNA damage and/or defective DNA repair can, therefore, accelerate the aging process and/or lead to age-related diseases like cancer. It is not yet clear if enhanced activity of DNA repair factors could increase the life or health span of human skin cells. In previous studies, we identified and characterized the human senescence evasion factor (SNEV)/pre-mRNA-processing factor (PRPF) 19 as a multitalented protein involved in mRNA splicing, DNA repair pathways and lifespan regulation. Here, we show that overexpression of PRPF19 in human dermal fibroblasts leads to a morphological change, reminiscent of juvenile, papillary fibroblasts, despite simultaneous expression of senescence markers. Moreover, conditioned media of this subpopulation showed a positive effect on keratinocyte repopulation of wounded areas. Taken together, these findings indicate that PRPF19 promotes cell viability and slows down the aging process in human skin.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells.

Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed-batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale-up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next-generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21-mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor-like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine-enriched cell culture media.

Biomechanical properties of a suture anchor system from human allogenic mineralized cortical bone matrix for rotator cuff repair.

BACKGROUND: Suture anchors (SAs) made of human allogenic mineralized cortical bone matrix are among the newest developments in orthopaedic and trauma surgery. Biomechanical properties of an allogenic mineralized suture anchor (AMSA) are not investigated until now. The primary objective was the biomechanical investigation of AMSA and comparing it to a metallic suture anchor (MSA) and a bioabsorbable suture anchor (BSA) placed at the greater tuberosity of the humeral head of cadaver humeri. Additionally, we assessed the biomechanical properties of the SAs with bone microarchitecture parameters. METHODS: First, bone microarchitecture of 12 fresh frozen human cadaver humeri from six donors was analyzed by high-resolution peripheral quantitative computed tomography. In total, 18 AMSAs, 9 MSAs, and 9 BSAs were implanted at a 60° angle. All three SA systems were systematically implanted alternating in three positions within the greater tuberosity (position 1: anterior, position 2: central, position 3: posterior) with a distance of 15 mm to each other. Biomechanical load to failure was measured in a uniaxial direction at 135°. RESULTS: Mean age of all specimens was 53.6 ± 9.1 years. For all bone microarchitecture measurements, linear regression slope estimates were negative which implies decreasing values with increasing age of specimens. Positioning of all three SA systems at the greater tuberosity was equally distributed (p = 0.827). Mean load to failure rates were higher for AMSA compared to MSA and BSA without reaching statistical significance between the groups (p = 0.427). Anchor displacement was comparable for all three SA systems, while there were significant differences regarding failure mode between all three SA systems (p < 0.001). Maximum load to failure was reached in all cases for AMSA, in 44.4% for MSA, and in 55.6% for BSA. Suture tear was observed in 55.6% for MSA and in 22.2% for BSA. Anchor breakage was solely seen for BSA (22.2%). No correlations were observed between bone microarchitecture parameters and load to failure rates of all three suture anchor systems. CONCLUSIONS: The AMSA showed promising biomechanical properties for initial fixation strength for RCR. Since reduced BMD is an important issue for patients with chronic rotator cuff lesions, the AMSA is an interesting alternative to MSA and BSA. Also, the AMSA could improve healing of the enthesis.

Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells.

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.

Osteoarthritis Progression after ACL Reconstruction Was Significantly Higher Than That of the Healthy Contralateral Knees: Long-Term Follow Up Study of Mean 16.4 Years.

BACKGROUND: This study aimed to assess long-term progression of osteoarthritis (OA) after isolated anterior cruciate ligament (ACL) reconstruction in athletes compared to the healthy contralateral side. METHODS: The study included 15 patients and 30 knees with a mean age of 40 years (range, 35-46) years, none of whom had had revision surgery or an injury to the contralateral side. The mean follow-up period was 16.4 years (range, 13-22). Clinical and radiographic assessment included the Tegner activity scale (TAS), International Knee Documentation Committee (IKDC) score, Knee injury and Osteoarthritis Outcome Score (KOOS), and Kellgren and Lawrence (KL) grade. The long-term results of the injured knees were compared with the status of the healthy contralateral side and compared with previously published mid-term results of the same cohort of patients. RESULTS: Patients generally remained clinically asymptomatic or mildly symptomatic at final follow-up, which is reflected by a KOOS pain score of 33 points (maximum 36 points) and an IKDC total subjective score of 87% (maximum 100%). There was a significant difference between mid-term and final follow-up in terms of the function score of the IKDC subjective questionnaire (p = 0.031), compartment findings and donor site morbidity of the IKDC functional examination (both p = 0.034), and the total KOOS score (p = 0.047). The KL score indicated significant progression of OA from mid-term to final follow-up in the injured knees (p = 0.004) and healthy contralateral knees (p = 0.014). Mean OA grades of the injured knees were significantly higher compared with the healthy contralateral side (p = 0.006) at final follow-up, and two patients showed moderate to severe signs of OA in the injured knee. CONCLUSIONS: Although most patients remained clinically asymptomatic or mildly symptomatic, long-term progression of OA after isolated ACL reconstruction in athletes was significantly higher compared with the healthy contralateral knee.

An in vitro strategy using multiple human induced pluripotent stem cell-derived models to assess the toxicity of chemicals: A case study on paraquat.

Most OECD guidelines for chemical risk assessment include tests performed on animals, raising financial, ethical and scientific concerns. Thus, the development of human-based models for toxicity testing is highly encouraged. Here, we propose an in vitro multi-organ strategy to assess the toxicity of chemicals. Human induced pluripotent stem cells (hiPSCs)-derived models of the brain, blood-brain barrier, kidney, liver and vasculature were generated and exposed to paraquat (PQ), a widely employed herbicide with known toxic effects in kidneys and brain. The models showed differential cytotoxic sensitivity to PQ after acute exposure. TempO-Seq analysis with a set of 3565 probes revealed the deregulation of oxidative stress, unfolded protein response and estrogen receptor-mediated signaling pathways, in line with the existing knowledge on PQ mechanisms of action. The main advantages of this strategy are to assess chemical toxicity on multiple tissues/organs in parallel, exclusively in human cells, eliminating the interspecies bias, allowing a better evaluation of the differential sensitivity of the models representing the diverse organs, and increasing the chance to identify toxic compounds. Furthermore, although we focused on the mechanisms of action of PQ shared by the different models, this strategy would also allow for organ-specific toxicity testing, by including more cell type-specific probes for TempO-Seq analyses. In conclusion, we believe this strategy will participate in the further improvement of chemical risk assessment for human health.

Improved biomechanics in experimental chronic rotator cuff repair after shockwaves is not reflected by bone microarchitecture.

PURPOSE: The aim of this study was to investigate the effect of extracorporeal shockwave therapy (ESWT) on bone microstructure as well as the bone-tendon-interface and the musculo-tendinous transition zone to explain the previously shown improved biomechanics in a degenerative rotator cuff tear animal model. This study hypothesized that biomechanical improvements related to ESWT are a result of improved bone microstructure and muscle tendon properties. METHODS: In this controlled laboratory study unilateral supraspinatus (SSP) tendon detachment was performed in 48 male Sprague-Dawley rats. After a degeneration period of three weeks, SSP tendon was reconstructed transosseously. Rats were randomly assigned into three groups (n = 16 per group): control (noSW); intraoperative shockwave treatment (IntraSW); intra- and postoperative shockwave treatment (IntraPostSW). Eight weeks after SSP repair, all rats were sacrificed and underwent bone microstructure analysis as well as histological and immunohistochemical analyses. RESULTS: With exception of cortical porosity at the tendon area, bone microstructure analyses revealed no significant differences between the three study groups regarding cortical and trabecular bone parameters. Cortical Porosity at the Tendon Area was lowest in the IntraPostSW (p≤0.05) group. Histological analyses showed well-regenerated muscle and tendon structures in all groups. Immunohistochemistry detected augmented angiogenesis at the musculo-tendinous transition zone in both shockwave groups indicated by CD31 positive stained blood vessels. CONCLUSION: In conclusion, bone microarchitecture changes are not responsible for previously described improved biomechanical results after shockwave treatment in rotator cuff repair in rodents. Immunohistochemical analysis showed neovascularization at the musculo-tendinous transition zone within ESWT-treated animals. Further studies focusing on neovascularization at the musculo-tendinous transition zone are necessary to explain the enhanced biomechanical and functional properties observed previously. CLINICAL RELEVANCE: In patients treated with a double-row SSP tendon repair, an improvement in healing through ESWT, especially in this area, could prevent a failure of the medial row, which is considered a constantly observed tear pattern.

Label-free characterization of an extracellular vesicle-based therapeutic.

Interest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers. Furthermore, analysis of QCM dissipation versus frequency allowed us to quantitatively determine the ratio of marker-specific EVs versus non-vesicular particles (NVPs) - a parameter that cannot be obtained by any other technique so far. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and NVPs in our EV preparations. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV therapeutics and opens the door to improved quality control.

Age-related alterations and senescence of mesenchymal stromal cells: Implications for regenerative treatments of bones and joints.

The most common clinical manifestations of age-related musculoskeletal degeneration are osteoarthritis and osteoporosis, and these represent an enormous burden on modern society. Mesenchymal stromal cells (MSCs) have pivotal roles in musculoskeletal tissue development. In adult organisms, MSCs retain their ability to regenerate tissues following bone fractures, articular cartilage injuries, and other traumatic injuries of connective tissue. However, their remarkable regenerative ability appears to be impaired through aging, and in particular in age-related diseases of bones and joints. Here, we review age-related alterations of MSCs in musculoskeletal tissues, and address the underlying mechanisms of aging and senescence of MSCs. Furthermore, we focus on the properties of MSCs in osteoarthritis and osteoporosis, and how their changes contribute to onset and progression of these disorders. Finally, we consider current treatments that exploit the enormous potential of MSCs for tissue regeneration, as well as for innovative cell-free extracellular-vesicle-based and anti-aging treatment approaches.

A Microfluidic Multisize Spheroid Array for Multiparametric Screening of Anticancer Drugs and Blood-Brain Barrier Transport Properties.

Physiological-relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclinical studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple-culture model of the blood-brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compound penetration across an advanced BBB model. The reproducible on-chip generation of 360 spheroids of five dimensions on a well-plate format using an integrated microlens technology is demonstrated. While spheroid size-related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size-dependent compound uptake studies in a perfused BBB model. Generally, smaller BBB-spheroids reveal an 80% higher compound penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size-related modulation on paracellular transport properties.