The Role of Alumni Networks and Career Advising in Early Career Stability of Urologists: Results of a Multi-institutional Study.

OBJECTIVE: To characterize training program and early career factors that impact decision-making and job retention following graduation in a diverse population of urologists. MATERIALS AND METHODS: We performed a computer-based survey distributed to residency graduates from 25 urology training programs. Five focus institutions were identified with a goal >30% response rate. The survey included questions about training program specifics and post-training employment characteristics. RESULTS: We obtained 180 responses from urology residency graduates of 25 programs. Overall, 72% (N = 129) remain in their initial post-training position at a median of 6years postgraduation (Interquartile Range (IQR) 3-10). On Cox-regression analysis stronger trainee-rated formal career advising was associated with lower risk of changing jobs (HR 0.77, 0.60-0.99, P = .048). Location/proximity to family was the most consistently cited as the top reason for selecting a job (41%). Sixty-three respondents (35%) joined practices employing graduates of the same residency program. Cox regression analysis showed that joining a practice with alumni of the same program was associated with lower risk of changing jobs from one's initial post-training position (HR 0.39, 95% CI 0.17-0.91, P = .03). CONCLUSION: In this multi-institutional study of urologists, we observed a high rate of job retention out to a median of 6years following completion of training, with formal career advising and joining alumni in practice being associated with job retention. Collectively, our data highlights that training programs should emphasize advising programs and alumni networking in guiding their graduates in the job search process.

Wrap It! Preventive Antimicrobial Treatment Shows No Negative Effects on Tenocytes and Tendons-A Comprehensive Approach.

Although the rate of infection after the reconstruction of a ruptured anterior cruciate ligament (ACL) is low, prophylactic incubation of the graft with vancomycin (Vanco-wrap or vancomycin soaking) is routinely performed. A cytotoxic effect of vancomycin is reported for several cell types, and the prophylactic treatment might prevent infection but harm the tissue and cells. AIM: A comprehensive study was performed to investigate the effect of vancomycin on tendon tissue and isolated tenocytes using cell viability, molecular and mechanical analysis. MATERIAL AND METHODS: Rat tendons or isolated tenocytes were incubated in increasing concentrations of vancomycin (0-10 mg/mL) for different times, and cell viability, gene expression, histology and Young's modulus were analyzed. RESULTS: The clinically used concentration of vancomycin (5 mg/mL for 20 min) had no negative effect on cell viability in the tendons or the isolated tenocytes, while incubation with the toxic control significantly reduced cell viability. Increasing the concentration and prolonging the incubation time had no negative effect on the cells. The expression of Col1a1, Col3a1 and the tenocyte markers mohawk, scleraxis and tenomodulin was not affected by the various vancomycin concentrations. The structural integrity as measured through histological and mechanical testing was not compromised. CONCLUSION: The results proved the safe application of the Vanco-wrap on tendon tissue. LEVEL OF EVIDENCE: IV.

Transdermal carbon monoxide delivery.

Overuse injuries or acute trauma in joints often lead to painful tendinopathy, and pharmacological treatment effects are limited. The site of the disease is hard to reach with drugs, both systemically and through the skin. Therapeutic gases may close this gap, as they permeate easier through tissues than conventional small molecules. We present a patch device releasing the anti-inflammatory gas carbon monoxide (CO) through the skin to the subcutaneous tendons and tissues. CO is chemically generated upon device activation and its design maximizes CO exposure to the underlying skin and protects the patient from all side and degradation products. The patch delivered CO successfully through the intact skin, granting lasting, subcutaneous CO exposure for up to 16 h. Furthermore, the released CO induced the proliferation of fibroblasts and the polarization of monocytes into anti-inflammatory M2 macrophages. In conclusion, the CO-releasing device might open an entirely new treatment option against tendinopathies in case of a positive outcome of future in vivo studies.

Behavioral Voluntary and Social Bioassays Enabling Identification of Complex and Sex-Dependent Pain-(-Related) Phenotypes in Rats with Bone Cancer.

Cancer-induced bone pain (CIBP) is a common and devastating symptom with limited treatment options in patients, significantly affecting their quality of life. The use of rodent models is the most common approach to uncovering the mechanisms underlying CIBP; however, the translation of results to the clinic may be hindered because the assessment of pain-related behavior is often based exclusively on reflexive-based methods, which are only partially indicative of relevant pain in patients. To improve the accuracy and strength of the preclinical, experimental model of CIBP in rodents, we used a battery of multimodal behavioral tests that were also aimed at identifying rodent-specific behavioral components by using a home-cage monitoring assay (HCM). Rats of all sexes received an injection with either heat-deactivated (sham-group) or potent mammary gland carcinoma Walker 256 cells into the tibia. By integrating multimodal datasets, we assessed pain-related behavioral trajectories of the CIBP-phenotype, including evoked and non-evoked based assays and HCM. Using principal component analysis (PCA), we discovered sex-specific differences in establishing the CIBP-phenotype, which occurred earlier (and differently) in males. Additionally, HCM phenotyping revealed the occurrence of sensory-affective states manifested by mechanical hypersensitivity in sham when housed with a tumor-bearing cagemate (CIBP) of the same sex. This multimodal battery allows for an in-depth characterization of the CIBP-phenotype under social aspects in rats. The detailed, sex-specific, and rat-specific social phenotyping of CIBP enabled by PCA provides the basis for mechanism-driven studies to ensure robustness and generalizability of results and provide information for targeted drug development in the future.

[Modern imaging of the pulmonary hilum : Anatomy, pathologies, pitfalls]. / Moderne Bildgebung des Lungenhilus : Anatomie, Pathologien, Pitfalls.

BACKGROUND: Modern medical imaging is a key component of efficient in- and out-patient precision medicine. Conventional radiography and computer tomography scans (CT) are among the most frequent radiologic exams. Medical imaging plays a key role in target-oriented medicine. OBJECTIVES: The purpose of this article is to review the anatomy of the hilum of the lung and its most frequent associated pathologies since it is an important gateway of elementary structures of the thorax. Important signs and patterns for image interpretation in different modalities are also reviewed. RESULTS: Thorough knowledge of anatomy, signs, and patterns of pathologies especially in conventional radiography and pitfalls of the more sensitive cross-sectional imaging is essential to support target-oriented patient care. CONCLUSION: Conventional radiography is affordable and readily available. It is very suitable for pathology screening, i.e., at the hilum of the lung. Cross-sectional imaging specifies diagnostics due to superior anatomic discrimination.

Performance of virtual non-contrast images generated on clinical photon-counting detector CT for emphysema quantification: proof of concept.

OBJECTIVE: To evaluate the performance of virtual non-contrast images (VNC) compared to true non-contrast (TNC) images in photon-counting detector computed tomography (PCD-CT) for the evaluation of lung parenchyma and emphysema quantification. METHODS: 65 (mean age 73 years; 48 male) consecutive patients who underwent a three-phase (non-contrast, arterial and venous) chest/abdomen CT on a first-generation dual-source PCD-CT were retrospectively included. Scans were performed in the multienergy (QuantumPlus) mode at 120 kV with 70 ml intravenous contrast agent at an injection rate of 4 ml s-1. VNC were reconstructed from the arterial (VNCart) and venous phase (VNCven). TNC and VNC images of the lung were assessed quantitatively by calculating the global noise index (GNI) and qualitatively by two independent, blinded readers (overall image quality and emphysema assessment). Emphysema quantification was performed using a commercially available software tool at a threshold of -950 HU for all data sets. TNC images served as reference standard for emphysema quantification. Low attenuation values (LAV) were compared in a Bland-Altman plot. RESULTS: GNI was similar in VNCart (103.0 ± 30.1) and VNCven (98.2 ± 22.2) as compared to TNC (100.9 ± 19.0, p = 0.546 and p = 0.272, respectively). Subjective image quality (emphysema assessment and overall image quality) was highest for TNC (p = 0.001), followed by VNCven and VNCart. Both, VNCart and VNCven showed no significant difference in emphysema quantification as compared to TNC (p = 0.409 vs. p = 0.093; respectively). CONCLUSION: Emphysema evaluation is feasible using virtual non-contrast images from PCD-CT. ADVANCES IN KNOWLEDGE: Emphysema quantification is feasible and accurate using VNC images in PCD-CT. Based on these findings, additional TNC scans for emphysema quantification could be omitted in the future.

Human Sex Matters: Y-Linked Lysine Demethylase 5D Drives Accelerated Male Craniofacial Osteogenic Differentiation.

Female sex is increasingly associated with a loss of bone mass during aging and an increased risk of developing nonunion fractures. Hormonal factors and cell-intrinsic mechanisms are suggested to drive these sexual dimorphisms, although underlying molecular mechanisms are still a matter of debate. Here, we observed a decreased capacity of calvarial bone recovery in female rats and a profound sexually dimorphic osteogenic differentiation in human adult neural crest-derived stem cells (NCSCs). Next to an elevated expression of pro-osteogenic regulators, global transcriptomics revealed Lysine Demethylase 5D (KDM5D) to be highly upregulated in differentiating male NCSCs. Loss of function by siRNA or pharmacological inhibition of KDM5D significantly reduced the osteogenic differentiation capacity of male NCSCs. In summary, we demonstrated craniofacial osteogenic differentiation to be sexually dimorphic with the expression of KDM5D as a prerequisite for accelerated male osteogenic differentiation, emphasizing the analysis of sex-specific differences as a crucial parameter for treating bone defects.

Impact of Contrast Enhancement and Virtual Monoenergetic Image Energy Levels on Emphysema Quantification: Experience With Photon-Counting Detector Computed Tomography.

PURPOSE: The aim of this study was to evaluate the impact of contrast enhancement and different virtual monoenergetic image energies on automatized emphysema quantification with photon-counting detector computed tomography (PCD-CT). MATERIAL AND METHODS: Sixty patients who underwent contrast-enhanced chest CT on a first-generation, clinical dual-source PCD-CT were retrospectively included. Scans were performed in the multienergy (QuantumPlus) mode at 120 kV with weight-adjusted intravenous contrast agent. Virtual noncontrast (VNC) images as well as virtual monoenergetic images (VMIs) from 40 to 80 keV obtained in 10-keV intervals were reconstructed. Computed tomography attenuation was measured in the aorta. Noise was measured in subcutaneous fat and defined as the standard deviation of attenuation. Contrast-to-noise with region of interest in the ascending aorta and signal-to-noise ratio in the subcutaneous fat were calculated. Subjective image quality (and emphysema assessment, lung parenchyma evaluation, and vessel evaluation) was rated by 2 blinded radiologists. Emphysema quantification (with a threshold of -950 HU) was performed by a commercially available software. Virtual noncontrast images served as reference standard for emphysema quantification. RESULTS: Noise and contrast-to-noise ratio showed a strong negative correlation (r = -0.98; P < 0.01) to VMI energies. The score of subjective assessment was highest at 70 keV for lung parenchyma and 50 keV for pulmonary vessel evaluation (P < 0.001). The best trade-off for the assessment of emphysema while maintaining reasonable contrast for pulmonary vessel evaluation was determined between 60 and 70 keV. Overall, contrast-enhanced imaging led to significant and systematic underestimation of emphysema as compared with VNC (P < 0.001). This underestimation decreased with increasing VMI-energy (r = 0.98; P = 0.003). Emphysema quantification showed significantly (P < 0.05) increased emphysema volumes with increasing VMI energies, except between 60-70 keV and 70-80 keV. The least difference in emphysema quantification between contrast-enhanced scans and VNC was found at 80 keV. CONCLUSION: Computed tomography emphysema quantification was significantly affected by intravenous contrast administration and VMI-energy level. Virtual monoenergetic image at 80 keV yielded most comparable results to VNC. The best trade-off in qualitative as well as in quantitative image quality evaluation was determined at 60/70 keV.

Influence of the Peripheral Nervous System on Murine Osteoporotic Fracture Healing and Fracture-Induced Hyperalgesia.

Osteoporotic fractures are often linked to persisting chronic pain and poor healing outcomes. Substance P (SP), α-calcitonin gene-related peptide (α-CGRP) and sympathetic neurotransmitters are involved in bone remodeling after trauma and nociceptive processes, e.g., fracture-induced hyperalgesia. We aimed to link sensory and sympathetic signaling to fracture healing and fracture-induced hyperalgesia under osteoporotic conditions. Externally stabilized femoral fractures were set 28 days after OVX in wild type (WT), α-CGRP- deficient (α-CGRP -/-), SP-deficient (Tac1-/-) and sympathectomized (SYX) mice. Functional MRI (fMRI) was performed two days before and five and 21 days post fracture, followed by µCT and biomechanical tests. Sympathectomy affected structural bone properties in the fracture callus whereas loss of sensory neurotransmitters affected trabecular structures in contralateral, non-fractured bones. Biomechanical properties were mostly similar in all groups. Both nociceptive and resting-state (RS) fMRI revealed significant baseline differences in functional connectivity (FC) between WT and neurotransmitter-deficient mice. The fracture-induced hyperalgesia modulated central nociception and had robust impact on RS FC in all groups. The changes demonstrated in RS FC in fMRI might potentially be used as a bone traumata-induced biomarker regarding fracture healing under pathophysiological musculoskeletal conditions. The findings are of clinical importance and relevance as they advance our understanding of pain during osteoporotic fracture healing and provide a potential imaging biomarker for fracture-related hyperalgesia and its temporal development. Overall, this may help to reduce the development of chronic pain after fracture thereby improving the treatment of osteoporotic fractures.

The small-molecule protein ligand interface stabiliser E7820 induces differential cell line specific responses of integrin α2 expression.

BACKGROUND: The mechanism of small-molecule stabilised protein-protein interactions is of growing interest in the pharmacological discovery process. A plethora of different substances including the aromatic sulphonamide E7820 have been identified to act by such a mechanism. The process of E7820 induced CAPERα degradation and the resultant transcriptional down regulation of integrin α2 expression has previously been described for a variety of different cell lines and been made responsible for E7820's antiangiogenic activity. Currently the application of E7820 in the treatment of various malignancies including pancreas carcinoma and breast cancer is being investigated in pre-clinical and clinical trials. It has been shown, that integrin α2 deficiency has beneficial effects on bone homeostasis in mice. To transfer E7820 treatment to bone-related pathologies, as non-healing fractures, osteoporosis and bone cancer might therefore be beneficial. However, at present no data is available on the effect of E7820 on osseous cells or skeletal malignancies. METHODS: Pre-osteoblastic (MC3T3 and Saos-2) cells and endothelial (eEnd2 cells and HUVECs) cells, each of human and murine origin respectively, were investigated. Vitality assay with different concentrations of E7820 were performed. All consecutive experiments were done at a final concentration of 50 ng/ml E7820. The expression and production of integrin α2 and CAPERα were investigated by quantitative real-time PCR and western blotting. Expression of CAPERα splice forms was differentiated by semi-quantitiative reverse transcriptase PCR. RESULTS: Here we present the first data showing that E7820 can increase integrin α2 expression in the pre-osteoblast MC3T3 cell line whilst also reproducing canonical E7820 activity in HUVECs. We show that the aberrant activity of E7820 in MC3T3 cells is likely due to differential activity of CAPERα at the integrin α2 promoter, rather than due to differential CAPERα degradation or differential expression of CAPERα spliceforms. CONCLUSION: The results presented here indicate that E7820 may not be suitable to treat certain malignancies of musculoskeletal origin, due to the increase in integrin α2 expression it may induce. Further investigation of the differential functioning of CAPERα and the integrin α2 promoter in cells of various origin would however be necessary to more clearly differentiate between cell lines that will positively respond to E7820 from those that will not.

Spongostan™ Leads to Increased Regeneration of a Rat Calvarial Critical Size Defect Compared to NanoBone® and Actifuse.

Bone substitute materials are becoming increasingly important in oral and maxillofacial surgery. Reconstruction of critical size bone defects is still challenging for surgeons. Here, we compared the clinically applied organic bone substitute materials NanoBone® (nanocrystalline hydroxyapatite and nanostructured silica gel; n = 5) and Actifuse (calcium phosphate with silicate substitution; n = 5) with natural collagen-based Spongostan™ (hardened pork gelatin containing formalin and lauryl alcohol; n = 5) in bilateral rat critical-size defects (5 mm diameter). On topological level, NanoBone is known to harbour nanopores of about 20 nm diameter, while Actifuse comprises micropores of 200-500 µm. Spongostan™, which is clinically applied as a haemostatic agent, combines in its wet form both nano- and microporous topological features by comprising 60.66 ± 24.48 µm micropores accompanied by nanopores of 32.97 ± 1.41 nm diameter. Micro-computed tomography (µCT) used for evaluation 30 days after surgery revealed a significant increase in bone volume by all three bone substitute materials in comparison to the untreated controls. Clearly visual was the closure of trepanation in all treated groups, but granular appearance of NanoBone® and Actifuse with less closure at the margins of the burr holes. In contrast, transplantion of Spongostan™ lead to complete filling of the burr hole with the highest bone volume of 7.98 ccm and the highest bone mineral density compared to all other groups. In summary, transplantation of Spongostan™ resulted in increased regeneration of a rat calvarial critical size defect compared to NanoBone and Actifuse, suggesting the distinct nano- and microtopography of wet Spongostan™ to account for this superior regenerative capacity. Since Spongostan™ is a clinically approved product used primarily for haemostasis, it may represent an interesting alternative in the reconstruction of defects in the maxillary region.

The heparan sulfate proteoglycan Syndecan-1 influences local bone cell communication via the RANKL/OPG axis.

The heparan sulfate proteoglycan Syndecan-1, a mediator of signals between the extracellular matrix and cells involved is able to interact with OPG, one of the major regulators of osteoclastogenesis. The potential of osteoblasts to induce osteoclastogenesis is characterized by a switch of OPG (low osteoclastogenic potential) towards RANKL production (high osteoclastogenic potential). In the present study, we investigated the influence of endogenous Syndecan-1 on local bone-cell-communication via the RANKL/OPG-axis in murine osteoblasts and osteoclasts in wild type and Syndecan-1 lacking cells. Syndecan-1 expression and secretion was increased in osteoblasts with high osteoclastogenic potential. Syndecan-1 deficiency led to increased OPG release by osteoblasts that decreased the availability of RANKL. In co-cultures of Syndecan-1 deficient osteoblasts with osteoclast these increased OPG in supernatant caused decreased development of osteoclasts. Syndecan-1 and RANKL level were increased in serum of aged WT mice, whereas Syndecan-1 deficient mice showed high serum OPG concentration. However, bone structure of Syndecan-1 deficient mice was not different compared to wild type. In conclusion, Syndecan-1 could be regarded as a new modulator of bone-cell-communication via RANKL/OPG axis. This might be of high impact during bone regeneration or bone diseases like cancer where Syndecan-1 expression is known to be even more prevalent.

Microsurgical reconstruction affects the outcome in a translational mouse model for Achilles tendon healing.

BACKGROUND: Animal models are one of the first steps in translation of basic science findings to clinical practice. For tendon healing research, transgenic mouse models are important to advance therapeutic strategies. However, the small size of the structures complicates surgical approaches, histological assessment, and biomechanical testing. In addition, available models are not standardized and difficult to compare. How surgery itself affects the healing outcome has not been investigated yet. The focus of the study was to develop a procedure that includes a transection and microsurgical reconstruction of the Achilles tendon but, unlike other models, preserves the sciatic nerve. We wanted to examine how distinct parts of the technique influenced healing. METHODS: For this animal model study, we used 96 wild-type male C57BL/6 mice aged 8-12 weeks. We evaluated different suture techniques and macroscopically confirmed the optimal combination of suture material and technique to minimize tendon gap formation. A key element is the detailed, step-by-step illustration of the surgery. In addition, we assessed histological (Herovici and Alcian blue staining) outcome parameters at 1-16 weeks postoperatively. Microcomputed tomography (micro-CT) was performed to measure the bone volume of heterotopic ossifications (HOs). Biomechanical analyses were carried out using a viscoelastic protocol on the biomechanical testing machine LM1. RESULTS: A modified 4-strand suture combined with a cerclage for immobilization without transection of the sciatic nerve reliably eliminated gap formation. The maximal dorsal extension of the hindlimb at the upper ankle joint from the equinus position (limited by the immobilization cerclage) increased over time postoperatively (operation: 28.8 ± 2.2°; 1 week: 54 ± 36°; 6 weeks: 80 ± 11.7°; 16 weeks: 96 ± 15.8°, p > 0.05). Histological staining revealed a maturation of collagen fibres within 6 weeks, whereas masses of cartilage were visible throughout the healing period. Micro-CT scans detected the development of HOs starting at 4 weeks and further progression at 6 and 16 weeks (bone volume, 4 weeks: 0.07604 ± 0.05286 mm3; 6 weeks: 0.50682 ± 0.68841 mm3; 16 weeks: 2.36027 ± 0.85202 mm3, p > 0.001). In-depth micro-CT analysis of the different surgical elements revealed that an injury of the tendon is a key factor for the development of HOs. Immobilization alone does not trigger HOs. Biomechanical properties of repaired tendons were greatly altered and remained inferior 6 weeks after surgery. CONCLUSION: With this study, we demonstrated that the microsurgical technique greatly influences the short- and longer-term healing outcome. When the sciatic nerve is preserved, the best surgical reconstruction of the tendon defect is achieved by a 4-strand core suture in combination with a tibiofibular cerclage for postoperative immobilization. The cerclage promotes a gradual increase in the range of motion of the upper ankle joint, comparable with an early mobilization rehabilitation protocol. HO, as a key mechanism for poor tendon healing, is progressive and can be monitored early in the model. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The study enhances the understanding of model dependent factors of healing. The described reconstruction technique provides a reproducible and translational rodent model for future Achilles tendon healing research. In combination with transgenic strains, it can be facilitated to advance therapeutic strategies to improve the clinical results of tendon injuries.

Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin α2ß1-Deficient Mice.

Integrins are a family of transmembrane proteins, involved in substrate recognition and cell adhesion in cross-talk with the extra cellular matrix. In this study, we investigated the influence of integrin α2ß1 on tendons, another collagen type I-rich tissue of the musculoskeletal system. Morphological, as well as functional, parameters were analyzed in vivo and in vitro, comparing wild-type against integrin α2ß1 deficiency. Tenocytes lacking integrin α2ß1 produced more collagen in vitro, which is similar to the situation in osseous tissue. Fibril morphology and biomechanical strength proved to be altered, as integrin α2ß1 deficiency led to significantly smaller fibrils as well as changes in dynamic E-modulus in vivo. This discrepancy can be explained by a higher collagen turnover: integrin α2ß1-deficient cells produced more matrix, and tendons contained more residual C-terminal fragments of type I collagen, as well as an increased matrix metalloproteinase-2 activity. A greatly decreased percentage of non-collagenous proteins may be the cause of changes in fibril diameter regulation and increased the proteolytic degradation of collagen in the integrin-deficient tendons. The results reveal a significant impact of integrin α2ß1 on collagen modifications in tendons. Its role in tendon pathologies, like chronic degradation, will be the subject of future investigations.

Modulation of Transient Receptor Potential Channels 3 and 6 Regulates Osteoclast Function with Impact on Trabecular Bone Loss.

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss.

Bone Regeneration: A Novel Osteoinductive Function of Spongostan by the Interplay between Its Nano- and Microtopography.

Scaffold materials for bone regeneration are crucial for supporting endogenous healing after accidents, infections, or tumor resection. Although beneficial impacts of microtopological or nanotopological cues in scaffold topography are commonly acknowledged, less consideration is given to the interplay between the microscale and nanoscale. Here, micropores with a 60.66 ± 24.48 µm diameter ordered by closely packed collagen fibers are identified in pre-wetted Spongostan, a clinically-approved collagen sponge. On a nanoscale level, a corrugated surface of the collagen sponge is observable, leading to the presence of 32.97 ± 1.41 nm pores. This distinct micro- and nanotopography is shown to be solely sufficient for guiding osteogenic differentiation of human stem cells in vitro. Transplantation of Spongostan into a critical-size calvarial rat bone defect further leads to fast regeneration of the lesion. However, masking the micro- and nanotopographical cues using SiO2 nanoparticles prevents bone regeneration in vivo. Therefore, we demonstrate that the identified micropores allow migration of stem cells, which are further driven towards osteogenic differentiation by scaffold nanotopography. The present findings emphasize the necessity of considering both micro- and nanotopographical cues to guide intramembranous ossification, and might provide an optimal cell- and growth-factor-free scaffold for bone regeneration in clinical settings.

Cryopreservation of tendon tissue using dimethyl sulfoxide combines conserved cell vitality with maintained biomechanical features.

Biomechanical research on tendon tissue evaluating new treatment strategies to frequently occurring clinical problems regarding tendon degeneration or trauma is of expanding scientific interest. In this context, storing tendon tissue deep-frozen is common practice to collect tissue and analyze it under equal conditions. The commonly used freezing medium, phosphate buffered saline, is known to damage cells and extracellular matrix in frozen state. Dimethyl sulfoxide, however, which is used for deep-frozen storage of cells in cell culture preserves cell vitality and reduces damage to the extracellular matrix during freezing. In our study, Achilles tendons of 26 male C57/Bl6 mice were randomized in five groups. Tendons were deep frozen in dimethyl sulfoxide or saline undergoing one or four freeze-thaw-cycles and compared to an unfrozen control group analyzing biomechanical properties, cell viability and collagenous structure. In electron microscopy, collagen fibrils of tendons frozen in saline appeared more irregular in shape, while dimethyl sulfoxide preserved the collagenous structure during freezing. In addition, treatment with dimethyl sulfoxide preserved cell viability visualized with an MTT-Assay, while tendons frozen in saline showed no remaining metabolic activity, indicating total destruction of cells during freezing. The biomechanical results revealed no differences between tendons frozen once in saline or dimethyl sulfoxide. However, tendons frozen four times in saline showed a significantly higher Young's modulus over all strain rates compared to unfrozen tendons. In conclusion, dimethyl sulfoxide preserves the vitality of tendon resident cells and protects the collagenous superstructure during the freezing process resulting in maintained biomechanical properties of the tendon.

CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

It is widely accepted that central and effector memory CD4+ T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4+ T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4+ T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4+ T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4+ T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4+ T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones.

International consensus criteria for the diagnosis of Raynaud's phenomenon.

Vasoconstriction accompanied by changes in skin color is a normal physiologic response to cold. The distinction between this normal physiology and Raynaud's phenomenon (RP) has yet to be well characterized. In anticipation of the 9th International Congress on Autoimmunity, a panel of 12 RP experts from 9 different institutes and four different countries were assembled for a Delphi exercise to establish new diagnostic criteria for RP. Relevant investigators with highly cited manuscripts in Raynaud's-related research were identified using the Web of Science and invited to participate. Surveys at each stage were administered to participants via the on-line SurveyMonkey software tool. The participants evaluated the level of appropriateness of statements using a scale of 1 (extremely inappropriate) through 9 (extremely appropriate). In the second stage, panel participants were asked to rank rewritten items from the first round that were scored as "uncertain" for the diagnosis of RP, items with significant disagreement (Disagreement Index > 1), and new items suggested by the panel. Results were analyzed using the Interpercentile Range Adjusted for Symmetry (IPRAS) method. A 3-Step Approach to diagnose RP was then developed using items the panelists "agreed" were "appropriate" diagnostic criteria. In the final stage, the panel was presented with the newly developed diagnostic criteria and asked to rate them against previous models. Following the first two iterations of the Delphi exercise, the panel of 12 experts agreed that 36 of the items were "appropriate", 12 items had "uncertain" appropriateness, and 13 items were "inappropriate" to use in the diagnostic criteria of RP. Using an expert committee, we developed a 3-Step Approach for the diagnosis of RP and 5 additional criteria for the diagnosis of primary RP. The committee came to an agreement that the proposed criteria were "appropriate and accurate" for use by physicians to diagnose patients with RP.