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Purpose: To determine the effect of inspiratory muscle training (IMT) on respiratory and peripheral muscle strength, functional exercise capacity, health-related quality of life (HRQoL), fatigue, depression, and cardiac functions in patients with stable angina. Methods: A randomized, controlled, single-blinded study. Twenty patients (59.95 ± 7.35 y, LVEF - 58.77 ± 7.49) with stable angina received IMT at the lowest load (10 cmH2O), and 20 patients (55.85 ± 7.60 y, LVEF - 62.26 ± 7.75) received training at 30% of maximal inspiratory pressure (MIP) seven days/8 weeks. Respiratory muscle strength (MIP; maximal expiratory pressure, MEP), peripheral muscle strength, pulmonary functions, functional exercise capacity (6-min walking test; exercise test), fatigue, HRQoL, depression, and cardiac functions were evaluated before and after. Results: A statistical difference was found between groups in terms of respiratory and peripheral muscle strength, pulmonary functions, functional exercise capacity (p<0.05). The results of fatigue, depression, HRQoL, and cardiac functions were similar between the groups (p>0.05). Conclusions: This study is the first to demonstrate the positive effects of IMT in patients with stable angina. IMT is a safe and effective method and is recommended to be added to cardiopulmonary rehabilitation programs and guidelines, as it results in increased peripheral muscle strength and functional exercise capacity in stable angina patients. [ABSTRACT FROM AUTHOR]

Gamma radiation is widely used to sterilize bone allografts but may impair their strength. While radioprotectant use may reduce radiation damage they may compromise sterility by protecting pathogens. We assessed the radioprotective potential of various agents (L-cysteine, N-acetyl-L-cysteine, L-cysteine-ethyl-ester and L-cysteine-methyl-ester) to identify those which do not protect spores of Bacillus subtilis. We hypothesized charge of these agents will affect their ability to radioprotect spores. We also determined ability of these radioprotectants and a radiosensitizer (nitroimidazole-linked phenanthridinium) to selectively sensitize spores to radiation damage by intercalating into the nucleic acid of spores. Spores were treated either directly in solutions of these agents or treated after being embedded and sealed in bone to assess the ability of these agents to diffuse into bone. L-cysteine and L-cysteine-ethyl-ester did not provide radioprotection. Positively charged L-cysteine-methyl-ester protected the spores, whereas positively charged L-cysteine-ethyl-ester did not, indicating charge does not determine the extent of radioprotection. The spores were sensitized to radiation damage when irradiated in nitroimidazole-linked phenanthridinium solution and sensitization disappeared after rinsing, suggesting nitroimidazole-linked phenanthridinium was unable to intercalate into the nucleic acid of the spores. Some cysteine-derived radioprotectants do not shield bacterial spores against gamma radiation and may be suitable for curbing the radiation damage to bone grafts while achieving sterility. [ABSTRACT FROM AUTHOR]

It has been suggested that the vibration transmission across a fracture is affected by the stages of healing of the fracture callus. This study aims to correlate the change in vibration transmission with mechanical stiffness of the callus measured by three-point bending. The right tibiae of male, three-month old local albino rabbits were osteotomized and stabilized by intramedullary fixation following open reduction. The intramedullary rods were removed on the 15th, 28th, 42nd and 90th days postoperatively and the tibiae were excised for vibration, three-point bending and bone mineral density analysis by quantitative computerized tomography (QCT). Optimum time for clinical weight bearing was determined by checking the convergence of the vibration parameters of the fractured tibia to those of the unfractured contralateral. The conclusions obtained from curvature analysis, based on vibration experiments, were in considerable correlation (Spearman's rank correlation coefficient r = 0.93, p = 0.003) with the conclusions obtained from the three-point bending test data which reflected the mechanical condition of the bone by direct means. However, no correlation between bone mineral density change and vibration transmission was noted. [ABSTRACT FROM AUTHOR]

Femoral fractures are observed in selective-bred commercial turkeys; however, the etiology of such fractures is unknown. The current study investigated the whole bone morphological, material-level mechani-cal, and bone ash properties to determine the effect of selective breeding on bone strength. Femora from 3 divergent strains of turkeys, a commercial line, a dif-ferent selectively bred heavy line (F-line), and a lighter age or weight matched random-bred line (RBC2/R-EQ, respectively), were compared. Bone geometric proper-ties were measured with micro-CT and bone mechani-cal properties were measured using 3-point bending tests. Whole bone ash quantities were also recorded. Statistics were run using a general linear model multi-variate ANOVA (GLM ANOVA). Results showed that at similar ages, the faster growing birds (commercial and F-line) had femurs twice the size of the RBC2 line as measured by cross-sectional area as early as 8 wk into the study. The femurs of the commercial and F-lines also exhibited as much as 20% greater mechanical strength than femurs from the RBC2 line at 16 and 20 wk of age as measured by properties such as elas-tic modulus and ultimate tensile strength. However, at similar BW, the slower growing R-EQ line had higher mechanical properties than the other lines, with the elastic modulus being 40% greater and the ultimate tensile strength being 37% greater at weights equiva-lent to those of the commercial and F-lines at 12 wk of age. Moreover, it was observed that the morpho-logical properties (i.e., cross-sectional area, moments of inertia) are largely governed by BW, as there is little difference in the amount gained per week of age across the different lines. Conversely, the mechanical proper-ties, as well as the related ash content, appear to be governed at least in part by time. Therefore, whereas modulation of bone geometry is the key responder for changes in BW, sufficient time for matrix mineraliza-tion or maturation or both to occur is also essential for mechanical competence of bone. [ABSTRACT FROM AUTHOR]

Both high-cycle fatigue life and fatigue crack propagation resistance of human cortical bone allograft are radiation dose-dependent between 0 and 25 kGy such that higher doses exhibit progressively shorter lifetimes. Recently, we have shown that collagen chain fragmentation and stable crosslink accumulation may contribute to the radiation dose-dependent loss in fatigue crack propagation resistance of human cortical bone. To our knowledge, the influence of these mechanisms on high-cycle fatigue life of cortical bone have not been established. Sequential irradiation has also been shown to mitigate the loss of fatigue life of tendons, however, whether this mitigates losses in fatigue life of cortical bone has not been explored. Our objectives were to evaluate the influence of radiation-induced collagen chain fragmentation and crosslinking on the high-cycle fatigue life of cortical bone in the dose range of 0-15 kGy, and to evaluate the capability of sequential irradiation at 15 kGy to mitigate the loss of high-cycle fatigue life and radiation-induced collagen damage. High-cycle fatigue life specimens from four male donor femoral pairs were divided into 5 treatment groups (0 kGy, 5 kGy, 10 kGy, 15 kGy, and 15 kGy sequentially irradiated) and subjected to high-cycle fatigue life testing with a custom rotating-bending apparatus at a cyclic stress of 35 MPa. Following fatigue testing, collagen was isolated from fatigue specimens, and collagen chain fragmentation and crosslink accumulation were quantified using SDS-PAGE and a fluorometric assay, respectively. Both collagen chain fragmentation (p = 0.006) and non-enzymatic crosslinking (p < 0.001) influenced high-cycle fatigue life, which decreased with increasing radiation dose from 0 to 15 kGy (p = 0.016). Sequential irradiation at 15 kGy did not offer any mitigation in high-cycle fatigue life (p = 0.93), collagen chain fragmentation (p = 0.99), or non-enzymatic crosslinking (p ≥ 0.10) compared to a single radiation dose of 15 kGy. Taken together with our previous findings on the influence of collagen damage on fatigue crack propagation resistance, collagen chain fragmentation and crosslink accumulation both contribute to radiation-induced losses in notched and unnotched fatigue life of cortical bone. To maximize the functional lifetime of radiation sterilized structural cortical bone allografts, pathways other than sequential radiation should be explored to mitigate collagen matrix damage., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dylan Crocker reports financial support was provided by National Institute of Arthritis and Musculoskeletal and Skin Diseases. Megan Oest reports financial support was provided by National Institute of Arthritis and Musculoskeletal and Skin Diseases. Clare Rimnac reports financial support was provided by Musculoskeletal Transplant Foundation Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Macrophages are involved in several critical activities associated with tissue repair and regeneration. Current approaches in regenerative medicine are focusing on leveraging the innate immune response to accelerate tissue regeneration and improve long-term healing outcomes. Of particular interest in this regard are the currently known, four main M2 macrophage subtypes: M2 interleukin (IL)-4,IL-13 , M2 IC , M2 IL-10 , M2 non-selective adenosine receptor agonists (NECA) (M2 IL-4,IL-13  → M2 NECA ). In this study, rat bone marrow-derived macrophages (M 0 ) were polarized to each of the four subtypes M2 IL-4,IL-13  → M2 NECA and cultured for 72 h in vitro. Luminex assay results highlighted increased production of tissue inhibitor of metalloproteinases-1 (TIMP-1) for M2 IL-4,IL-13 , higher amounts of transforming growth factor-beta 1 (TGF-β1) for M2 IL-10 , and elevated vascular endothelial growth factor A (VEGF-A) from M2 NECA . Co-culture experiments performed with M2 IL-10 macrophages and L929 fibroblasts highlighted the increased production of soluble collagen within the media as well as higher amounts of collagen in the extracellular matrix. Human umbilical vein endothelial cells (HUVECs) were co-cultured with M2 NECA macrophages, which demonstrated an increase in intercellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM), as well as increased formation of endothelial tubes. The findings of this study emphasize a critical demand for further characterization and analyses of distinct M 2 subtypes and careful selection of specific macrophage populations for regeneration of specific tissue types. The current, broad classification of "M 2 " may be sufficient in many general tissue engineering applications, but, as conditions are constantly in flux within the microenvironment in vivo, a higher degree of specificity and control over the initial M 2 subtype could result in more consistent long-term outcomes where macrophages are utilized as part of an overall regenerative strategy., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Fatigue crack propagation resistance and high-cycle S-N fatigue life of cortical bone allograft tissue are both negatively impacted in a radiation dose-dependent manner from 0 to 25 kGy. The standard radiation sterilization dose of 25-35 kGy has been shown to induce cleavage of collagen molecules into smaller peptides and accumulation of stable crosslinks within the collagen matrix, suggesting that these mechanisms may influence radiation-induced losses in cyclic fracture resistance. The objective of this study was to determine the radiation dose-dependency of collagen chain fragmentation and crosslink accumulation within the dose range of 0-25 kGy. Previously, cortical bone compact tension specimens from two donor femoral pairs were divided into four treatment groups (0 kGy, 10 kGy, 17.5 kGy, and 25 kGy) and underwent cyclic loading fatigue crack propagation testing. Following fatigue testing, collagen was isolated from one compact tension specimen in each treatment group from both donors. Radiation-induced collagen chain fragmentation was assessed using SDS-PAGE (n = 5), and accumulation of pentosidine, pyridinoline, and non-specific advanced glycation end products were assessed using a fluorometric assay (n = 4). Collagen chain fragmentation increased progressively in a dose-dependent manner (p < 0.001). Crosslink accumulation at all radiation dose levels increased relative to the 0 kGy control but did not demonstrate dose-dependency (p < 0.001). Taken together with our previous findings on fatigue crack propagation behavior, these data suggest that while collagen crosslink accumulation may contribute to reduced notched fatigue behavior with irradiation, dose-dependent losses in fatigue crack propagation resistance are mainly influenced by radiation-induced chain fragmentation., (© 2024. The Author(s).)

Soft-bodied animals, such as worms and snakes, use many muscles in different ways to traverse unstructured environments and inspire tools for accessing confined spaces. They demonstrate versatility of locomotion which is essential for adaptation to changing terrain conditions. However, replicating such versatility in untethered soft-bodied robots with multimodal locomotion capabilities have been challenging due to complex fabrication processes and limitations of soft body structures to accommodate hardware such as actuators, batteries and circuit boards. Here, we present MetaCrawler, a 3D printed metamaterial soft robot designed for multimodal and omnidirectional locomotion. Our design approach facilitated an easy fabrication process through a discrete assembly of a modular nodal honeycomb lattice with soft and hard components. A crucial benefit of the nodal honeycomb architecture is the ability of its hard components, nodes, to accommodate a distributed actuation system, comprising servomotors, control circuits, and batteries. Enabled by this distributed actuation, MetaCrawler achieves five locomotion modes: peristalsis, sidewinding, sideways translation, turn-in-place, and anguilliform. Demonstrations showcase MetaCrawler's adaptability in confined channel navigation, vertical traversing, and maze exploration. This soft robotic system holds the potential to offer easy-to-fabricate and accessible solutions for multimodal locomotion in applications such as search and rescue, pipeline inspection, and space missions., (Creative Commons Attribution license.)

Tracheal stenosis is commonly caused by injury, resulting in inflammation and fibrosis. Inhibiting inflammation and promoting epithelization can reduce recurrence after initial successful treatment of tracheal stenosis. Steroids play an important role in tracheal stenosis management. This study in vitro evaluated effectiveness of a polydopaminated polycaprolactone stent coated with dexamethasone-eluting poly(lactic-co-glycolic) acid microparticles (μPLGA) for tracheal stenosis management. Polydopamination was characterized by Raman spectroscopy and promoted epithelialization while dexamethasone delivery reduced macrophage activity, assessed by individual cell area measurements and immunofluorescent staining for inducible nitric oxide synthase (iNOS). Dexamethasone release was quantified by high-performance liquid chromatography over 30 days. Activation-related increase in cell area and iNOS production by RAW 264.7 were both reduced significantly (p < .05) through dexamethasone release. Epithelial cell spreading was higher on polydopaminated polycaprolactone (PCL) than PCL-alone (p < .05). Force required for stent migration was measured by pullout tests of PCL-μPLGA stents from cadaveric rabbit and porcine tracheas (0.425 ± 0.068 N and 1.082 ± 0.064 N, respectively) were above forces estimated to occur during forced respiration. Biomechanical support provided by stents to prevent airway collapse was assessed by comparing compressive circumferential stiffness, and stiffness of the stent was about 1/10th of the rabbit trachea (0.156 ± 0.023 N/mm vs. 1.420 ± 0.194 N/mm, respectively). A dexamethasone-loaded PCL-μPLGA stent platform can deliver dexamethasone and exhibits sufficient mechanical properties to anchor within the trachea and polydopamination of PCL is conducive to epithelial layer formation. Therefore, a polydopaminated PCL-μPLGA stent is a promising candidate for in vivo evaluation for treatment of tracheal restenosis., (© 2024 Wiley Periodicals LLC.)

Computational models of mature bone have been used to predict fracture; however, analogous study of immature diaphyseal fracture has not been conducted due to sparse experimental mechanical data. A model of immature bone fracture may be used to aid in the differentiation of accidental and non-accidental trauma fractures in young, newly ambulatory children (0-3 years). The objective of this study was to characterize the evolution of tissue-level mechanical behavior, composition, and microstructure of maturing cortical porcine bone with uniaxial tension, Raman spectroscopy, and light microscopy as a function of maturation. We asked: 1) How do the monotonic uniaxial tensile properties change with maturation and displacement rate; 2) How does the composition and microstructure change with maturation; and 3) Is there a correlation between composition and tensile properties with maturation? Elastic modulus (p < 0.001), fracture stress (p < 0.001), and energy absorption (p < 0.014) increased as a function of maturation at the quasistatic rate by 110%, 86%, and 96%, respectively. Fracture stress also increased by 90% with maturation at the faster rate (p = 0.001). Fracture stress increased as a function of increasing displacement rate by 28% (newborn p = 0.048; 1-month p = 0.004; 3-month p= < 0.001), and fracture strain decreased by 68% with increasing displacement rate (newborn p = 0.002; 1-month p = 0.036; 3-month p < 0.001). Carbonate-to-phosphate ratio was positively linearly related to elastic modulus, and fracture stress was positively related to carbonate-to-phosphate ratio and matrix maturation ratio. The results of this study support that immature bone is strain-rate dependent and becomes more brittle at faster rates, contributing to the foundation upon which a computational model can be built to evaluate immature bone fracture., Competing Interests: Declaration of competing interest Ozan Akkus is Chief Scientific Officer of CollaMedix Inc. Emily Szabo, Jay Bensusan, and Clare Rimnac have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Oxygen (O 2 ) binds to hemoglobin (Hb) in the lungs and is then released (dissociated) in the tissues. The Bohr effect is a physiological mechanism that governs the affinity of Hb for O 2 based on pH, where a lower pH results in a lower Hb-O 2 affinity and higher Hb-O 2 dissociation. Hb-O 2 affinity and dissociation are crucial for maintaining aerobic metabolism in cells and tissues. Despite its vital role in human physiology, Hb-O 2 dissociation measurement is underutilized in basic research and in clinical laboratories, primarily due to the technical complexity and limited throughput of existing methods. We present a rapid Hb-O 2 dissociation measurement approach by leveraging the Bohr effect and detecting the optical shift in the Soret band that corresponds to the light absorption by the heme group in Hb. This new method reduces Hb-O 2 dissociation measurement time from hours to minutes. We show that Hb deoxygenation can be accelerated chemically at the optimal pH of 6.9. We show that time and pH-controlled deoxygenation of Hb results in rapid and distinct conformational changes in its tertiary structure. These molecular conformational changes are manifested as significant, detectable shifts in Hb's optical absorption spectrum, particularly in the characteristic Soret band (414 nm). We extensively validated the method by testing human blood samples containing normal Hb and Hb variants. We show that rapid Hb-O 2 dissociation can be used to screen for and detect Hb-O 2 affinity disorders and to evaluate the function and efficacy of Hb-modifying therapies. The ubiquity of optical absorption spectrophotometers positions this approach as an accessible, rapid, and accurate Hb-O 2 dissociation measurement method for basic research and clinical use. We anticipate this method's broad adoption will democratize the diagnosis and prognosis of Hb disorders, such as sickle cell disease. Further, this method has the potential to transform the research and development of new targeted and genome-editing-based therapies that aim to modify or improve Hb-O 2 affinity.

Background: Chronic coronary syndrome (CCS) is one of the most life-restricting coronary artery diseases, and symptom relief is the main goal in CCS patients who suffer from angina., Objectives: To assess the potential benefits of device-guided breathing in CCS patients with angina in this randomized, controlled, single-blinded study., Methods: Fifty-one patients with CCS received device-guided breathing for 7 days/8 weeks. Exercise capacity [exercise stress test], cardiac function [transthoracic echocardiography], and angina severity [Canadian Cardiovascular Society Classification] were evaluated initially and after the training. Device-guided breathing was performed at the lowest resistance of the device (POWERbreathe® Classic LR) for the control group (n = 17). The low load training group (LLTG; n = 18) and high load training group (HLTG; n = 16) were trained at 30% and 50% of maximal inspiratory pressure. Baseline characteristics were compared using one-way ANOVA and Kruskal-Wallis test. Categorical data were compared using the chi-square test. ANCOVA was performed to compare changes between three groups. A p value < 0.05 was considered statistically significant., Results: Metabolic equivalent values were significantly improved in both HLTG and LLTG groups (p < 0.001, p = 0.003). The Duke treadmill score significantly improved and shifted to low-risk both in the HLTG (p < 0.001) and LLTG (p < 0.001) groups. Angina severity significantly alleviated after the training in both HLTG and LLTG groups (p < 0.001, p = 0.002)., Conclusions: An 8-week long program of short-term respiratory muscle training provided positive gains in exercise capacity and angina severity in CCS patients with angina. The effects of long-term training programs on CCS patients should be investigated clinically because of the possibility of helping to decrease the need for invasive treatments., Competing Interests: The authors stated that there is no conflict of interest.

Cortical bone allograft sterilized with a standard γ-radiation dose of 25-35kGy has demonstrated reduced static and cyclic fracture resistance compared with unirradiated bone. To mitigate radiation damage, we recently observed a dose-dependent response of high-cycle fatigue behavior of human cortical bone from 0 to 25 kGy, with lower doses exhibiting logarithmically longer fatigue lives. The objectives of this study were as follows: (1) to determine whether fracture toughness, work-to-fracture, and fatigue crack propagation resistance of human cortical bone are also radiation dose-dependent, and (2) to determine the associations of radiation dose and a Raman biomarker for collagen disorder with fracture properties. Compact tension specimens were machined from two donor femoral pairs and allocated to four treatment groups: 0 (unirradiated control), 10, 17.5, and 25 kGy. Fracture toughness specimens were monotonically loaded to failure and the critical stress intensity factor (K C ) was determined. Work-to-fracture was calculated from the load versus displacement integral up to fracture. Fatigue crack propagation specimens were cyclically loaded under constant room-temperature irrigation and fatigue crack growth rate (da/dN) and cyclic stress intensity (∆K) were calculated. Fracture toughness, work-to-fracture, and fatigue crack propagation resistance decreased 18%, 33%, and 15-fold from 0 to 25 kGy, respectively (p < 0.05). Radiation dose was more predictive of fracture properties than collagen disorder. These findings support that quasi-static and fatigue fracture properties of cortical bone are radiation dose-dependent within this dose range. The structural alterations arising from irradiation that cause these losses in fracture resistance remain to be elucidated., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Introduction: The sheep was evaluated as a potential model for preclinical evaluation of urethral slings in vivo based on: (1) anatomical measurements of the sheep vagina and (2) histological tissue integration and host response to polypropylene (PP) slings., Methods: Eight female, multiparous sheep were utilized. Three of 8 animals underwent surgery mimicking human tension-free vaginal tape protocols for midurethral slings and were euthanized at 6 months. The following measurements were obtained: vaginal length, maximum vaginal width with retraction, symphysis pubis length, and distance from the pubic bone to incision. Explanted sling samples from sheep and human were stained with hematoxylin and eosin for host reaction assessment., Results: Geometric measurements were similar between humans and sheep. Sheep vaginal anatomy allowed sling placement similar to procedures in human surgeries, and all sheep recovered without problems. Comparative histology between the sheep and human indicated similar host reaction and collagen deposition around implants, confirming suitability of the sheep model for biomaterial response assessment., Conclusion: Sheep vaginal length is comparable to humans. Tissue integration and host response to PP slings showed chronic inflammation with rich collagen deposition around the material in both sheep and human specimens, highlighting the sheep as a potential animal model for preclinical testing of midurethral slings., (© 2022 S. Karger AG, Basel.)

Stress urinary incontinence (SUI) impacts ~1/3 of women over age 50. Negative publicity around PP meshes used in pelvic prolapse repair drives the need for identifying alternative biomaterials for SUI repair. Our study evaluated in vivo response to collagen sling implanted in an ovine model. Electrocompacted collagen threads were filament wound as slings and crosslinked in genipin. Collagen slings were implanted suburethrally mimicking the transvaginal tape technique. Main study groups were: Collagen sling (n = 3, 6 months) and PP sling (n = 3, 6 months). Collagen sling was also tested at 3-weeks (n = 1) to observe early-stage tissue response and 1-year (n = 2) to assess biomaterial longevity in a preliminary capacity. Collagen slings healed to a fibrous ligament texture at 6 months and maintained such texture to 1 year. Histological scoring indicated biocompatible responses to collagen slings with no adverse events. All study groups exhibited complete tissue ingrowth and interstitial de novo collagen deposition at all time points. Collagen threads induced orderly de novo collagen deposition that was aligned along long axes of threads. Tissue infiltrated collagen slings that were explanted at 6 and 12 months presented similar structural strength with native tissues such as vagina and fascia, and PP (Lynx) slings (p > .05). With the limitation of low number of animals per time point in hindsight, this preliminary study justifies evaluation of collagen slings in a larger sample size of animals, particularly to assess persistence of ligamentous tissue response over longer durations than 1-year., (© 2022 Wiley Periodicals LLC.)

A rotator cuff tendon tear is a common shoulder injury with a relatively high rate of recurrence after surgical repair. In order to reinforce the repair and reduce the risk of clinical complications, a patch scaffold is typically sutured over the tendon tear to provide post-surgical mechanical support. However, despite considerable research effort in this area, a patch scaffold that provides both superior initial mechanical properties and supports cell proliferation at the same time has not yet been achieved. In this study, we engineered a collagen/poly(lactic acid) (COL/PLA) hybrid yarn to leverage mechanical strength of PLA yarn and the bioactivity of collagen. The COL/PLA yarns were used to fabricate a tissue engineering scaffold using textile weaving technology. This hybrid scaffold had a tensile strength of 354.0 ± 36.0 N under dry conditions and 267.2 ± 15.9 N under wet conditions, which was satisfactory to maintain normal tendon function. By introducing COL yarns into the hybrid scaffold, the proliferation of tendon-derived cells was significantly improved on the scaffold. Cell coverage after 28-days of in vitro cell culture was noticeably higher on the COL yarns compared to the PLA yarns as a result of a larger number of cells and more spread cell morphology on collagen. Cells spread in multiple directions on COL yarns, which resembled a more natural cell attachment on extracellular matrix. On the contrary, the cells attached to the PLA filaments presented an elongated morphology along the fiber's axial direction. Combining the mechanical robustness of PLA and the biological activity of collagen, the woven COL/PLA hybrid scaffold has shown its potential to be a promising candidate for tendon repair applications., (© 2022 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Calcium (Ca 2+ ) signaling is the first messenger signal exhibited by osteocytes. The present study aimed to better understand the link between Ca 2+ concentration, and the levels of bone mineralization regulator proteins [phosphate‑regulating neutral endopeptidase on chromosome X (PHEX), matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein 1 (DMP1)] and the levels of oxidative stress in osteocytes. The viability of MLO‑Y4 cells was determined using the live/dead assay following treatment with various Ca 2+ concentrations (1.8, 6, 12, 18, 24 and 50 mM) for different durations (15 and 60 min, and 24 h). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and NADPH oxidase (NOX) enzymes were analyzed using a colorimetric method. Apoptosis was detected by caspase‑3 analysis. Furthermore, the protein expression levels of PHEX, MEPE and DMP1 were analyzed using immunoblotting, and oxidative stress was examined using the total antioxidant and total oxidant status (TOS) assay. Notably, after 15 min, there were more live cells than dead cells; however, after 60 min, the number of dead cells was increased following treatment with 24 and 50 mM Ca 2+ . After 24 h, there were more dead cells than live cells following treatment with 50 mM Ca 2+ . After 24 h of Ca 2+ treatment, the highest protein expression levels of PHEX, MEPE and DMP1 were measured in cells treated with 24 mM Ca 2+ . In addition, as Ca 2+ concentration increased, the TOS and the oxidative stress index values were also increased. In conclusion, these results suggested that 24 mM Ca 2+ may trigger bone mineralization proteins, such as PHEX, MEPE and DMP1, and could be considered an applicable dosage for the treatment of bone damage in the future.

Developing strategies to regulate the immune response poses significant challenges with respect to the clinical translation of tissue-engineered scaffolds. Prominent advancements have been made relating to macrophage-based therapies and biomaterials. Macrophages exhibit the potential to influence healing trajectory, and predominance of particular subtypes during early onset of healing influences repair outcomes. This study evaluated short- and long-term healing response and postoperative mechanical properties of genipin-cross-linked, electrochemically aligned collagen biotextiles with comparative administration of M0, M1, and M2 subtypes. Irrespective of macrophage subtype seeded, all the groups demonstrated existence of M2 macrophages at both time points as typified by arginase and Ym-1 expressions, and distinct absence of M1 macrophages, as indicated by lack of inducible nitric oxide synthase (iNOS) and interleukin-1β expression in all the groups for both time points. M2 macrophage-seeded collagen biotextiles revealed promising host tissue responses, such as reduced fibrous capsule thickness and minimal granulation tissue formation. Furthermore, the M2-seeded group displayed more abundant interstitial collagen deposition following degradation of the collagen threads. M2 macrophage supplementation improved structural and mechanical properties at the tissue and cellular level as indicated by increased modulus and stiffness. This study demonstrates improved biomechanical and histological outcomes following incorporation of M2 macrophages into genipin-cross-linked collagen biotextiles for tissue repair and offers future strategies focused on connective tissue regeneration. Impact statement Macrophages exhibit significant plasticity with complex phenotypes ranging from proinflammatory (M1) to proregenerative (M2). They release cytokines and chemokines governing immunological stability, inflammation resolution, and tissue healing and regeneration. However, utilization of macrophages as therapeutic tools for tissue engineering remains limited. In this study, genipin-cross-linked collagen biotextiles were employed to deliver M0, M1, and M2 macrophages and evaluate tissue responses and postsurgical mechanical properties in vivo . M2-seeded collagen biotextiles showed reduced fibrous capsule and favorable healing response. These outcomes shed new light on designing tissue-engineered constructs that offer a novel cell-based therapeutic approach for applications requiring structural augmentation.

Background: Successful management of massive rotator cuff (RC) tendon tears represents a treatment challenge because of the limited intrinsic healing capacity of native tendons and the risk of repair failure. Biologic augmentation of massive RC tears utilizing scaffolds-capable of regenerating bulk tendon tissue to achieve a mechanically functional repair-represents an area of increasing clinical interest., Purpose: To investigate the histological and biomechanical outcomes after the use of a novel biologic scaffold fabricated from woven electrochemically aligned collagen (ELAC) threads as a suture-holding, fully load-bearing, defect-bridging scaffold with or without mesenchymal stem cells (MSCs) compared with direct repair in the treatment of critically sized RC defects using a rabbit model., Study Design: Controlled laboratory study., Methods: A total of 34 New Zealand White rabbits underwent iatrogenic creation of a critically sized defect (6 mm) in the infraspinatus tendon of 1 shoulder, with the contralateral shoulder utilized as an intact control. Specimens were divided into 4 groups: (1) gap-negative control without repair; (2) direct repair of the infraspinatus tendon-operative control; (3) tendon repair using ELAC; and (4) tendon repair using ELAC + MSCs. Repair outcomes were assessed at 6 months using micro-computed tomography, biomechanical testing, histology, and immunohistochemistry., Results: Specimens treated with ELAC demonstrated significantly less tendon retraction when compared with the direct repair group specimens ( P = .014). ELAC + MSCs possessed comparable biomechanical strength (178 ± 50 N) to intact control shoulders (199 ± 35 N) ( P = .554). Histological analyses demonstrated abundant, well-aligned de novo collagen around ELAC threads in both the ELAC and the ELAC + MSC shoulders, with ELAC + MSC specimens demonstrating increased ELAC resorption (7% vs 37%, respectively; P = .002). The presence of extracellular matrix components, collagen type I, and tenomodulin, indicating tendon-like tissue formation, was appreciated in both the ELAC and the ELAC + MSC groups., Conclusion: The application of MSCs to ELAC scaffolds improved biomechanical and histological outcomes when compared with direct repair for the treatment of critically sized defects of the RC in a rabbit model., Clinical Relevance: This study demonstrates the feasibility of repairing segmental tendon defects with a load-bearing, collagen biotextile in an animal model, showing the potential applicability of RC repair supplementation using allogeneic stem cells.

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1β levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1β induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Background: Structural cortical bone allografts are a reasonable treatment option for patients with large cortical bone defects caused by trauma, tumors, or complications of arthroplasty. Although structural cortical bone allografts provide the benefit of an osteoconductive material, they are susceptible to fatigue failure (fracture) and carry a risk of disease transmission. Radiation-sterilization at the recommended dose of 25 kGy decreases the risk of disease transmission. However, previous studies demonstrated that radiation sterilization at this dose can negatively impact the high cycle-fatigue life of cortical bone. Although the effects of higher doses of radiation on cortical bone allografts are well described, the effects of lower doses of radiation on a high-cycle fatigue life of cortical bone are poorly understood., Questions/purposes: (1) Does the cycle-fatigue life of human cortical allograft bone vary with gamma radiation dose levels of 0 (control), 10 kGy, 17.5 kGy, and 25 kGy? (2) What differences in Raman spectral biomarkers are observed following varying doses of gamma radiation exposure?, Methods: The high-cycle fatigue behavior of human cortical bone specimens was examined at different radiation sterilization doses under physiologic stress levels (35 MPa) and in a 37° C phosphate-buffered saline bath using a custom-designed rotating-bending fatigue device. Six human femora from three donors were obtained for this study (two male, 63 and 61 years old, respectively, and one female, 48 years old). Test specimens were allocated among four treatment groups (0 kGy [control], 10 kGy, 17.5 kGy, and 25 kGy) based on donor and anatomic location of harvest site (both length and cross-sectional quadrant of femoral diaphysis) to ensure equal variation (n = 13 per group). Specimens underwent high-cycle fatigue testing to failure. The number of cycles to failure was recorded. Raman spectroscopy (a noninvasive vibrational spectroscopy used to qualitatively assess bone quality) was used to detect whether any changes in Raman spectral biomarkers occurred after varying doses of gamma radiation exposure., Results: There was a decrease in the log-transformed mean high-cycle fatigue life in specimens irradiated at 25 kGy (5.39 ± 0.32) compared with all other groups (0 kGy: 6.20 ± 0.50; 10k Gy: 6.35 ± 0.79; 17.5 kGy: 6.01 ± 0.53; p = 0.001). Specimens irradiated at 25 kGy were also more likely to exhibit a more brittle fracture surface pattern than specimens with more ductile fracture surface patterns irradiated at 0 kGy, 10 kGy, and 17.5 kGy (p = 0.04). The Raman biomarker for the ratio of the relative amount of disordered collagen to ordered collagen showed a decrease at the 10 kGy radiation level from 1.522 ± 0.025 preirradiation to 1.489 ± 0.024 postirradiation (p = 0.01); no other detectable changes in Raman biomarkers were observed., Conclusion: The high-cycle fatigue life of cortical bone undergoes a nonlinear, dose-dependent decrease with an increase in gamma radiation sterilization in a clinically relevant dose range (0-25 kGy). Importantly, a notable drop-off in the high-cycle fatigue life of cortical bone appeared to occur between 17.5 kGy and 25 kGy, correlating to a sixfold decrease in mean cycles to failure. We speculate that the decrease in the Raman biomarker for disordered collagen at 10 kGy with no loss in high-cycle fatigue life may be caused by an increased amount of nonenzymatic crosslinking of the collagen backbone relative to collagen chain-scission (whereas the benefits of crosslinking may be outweighed by excess scission of the collagen backbone at higher radiation doses), but future studies will need to ascertain whether this in fact is the case., Clinical Relevance: Radiation sterilization at the industry standard of 25 kGy has a substantial negative impact on the high-cycle fatigue life of cortical bone. Given these findings, it is possible to provide a meaningful increase in the high-cycle fatigue life and improve the overall functional lifetime of cortical bone allografts by lowering the radiation-sterilization dose below 25 kGy. Future work on radiation-sterilization methods at these clinically relevant doses is warranted to aid in preserving the high cycle fatigue life of cortical bone allografts while maintaining sterility., Competing Interests: All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2022 by the Association of Bone and Joint Surgeons.)

Bacterial infections routinely cause inflammation and thereby impair osseointegration of orthopedic implants. Acinetobacter spp., which cause osteomyelitis following trauma, on or off the battlefield, were, however, reported to cause neither osteomyelitis nor osteolysis in rodents. We therefore compared the effects of Acinetobacter strain M2 to those of Staphylococcus aureus in a murine implant infection model. Sterile implants and implants with adherent bacteria were inserted in the femur of mice. Bacterial burden, levels of proinflammatory cytokines, and osseointegration were measured. All infections were localized to the implant site. Infection with either S. aureus or Acinetobacter strain M2 increased the levels of proinflammatory cytokines and the chemokine CCL2 in the surrounding femurs, inhibited bone formation around the implant, and caused loss of the surrounding cortical bone, leading to decreases in both histomorphometric and biomechanical measures of osseointegration. Genetic deletion of TLR2 and TLR4 from the mice partially reduced the effects of Acinetobacter strain M2 on osseointegration but did not alter the effects of S. aureus. This is the first report that Acinetobacter spp. impair osseointegration of orthopedic implants in mice, and the murine model developed for this study will be useful for future efforts to clarify the mechanism of implant failure due to Acinetobacter spp. and to assess novel diagnostic tools or therapeutic agents.

We investigated for the first time zonal-dependent water distribution in articular cartilage by Raman spectroscopy (RS). We further investigated the association of histopathologic score with RS- and magnetic resonance imaging (MRI)-based water measurements. Cadaveric human cartilage plugs (N = 16) with different osteoarthritis (OA) severity were used. Water content distribution in cartilage zones was probed using RS- and MRI-based techniques. Histopathologic scoring was performed by two independent observers blindly. Moderate associations existed between RS- and MRI-based water measurements across all cartilage zones. RS-based analysis of different water compartments helped assign the origin of the T2 signal collected from the various cartilage zones. RS-based water parameters significantly correlated with OA-severity score, whereas MRI-based water measurements did not. RS can probe different water compartments in cartilage zones and predict up to 66% of the variation observed in the histopathologic score. RS-based water measurement could be developed further to assess cartilage quality in the clinic., (© 2021 Wiley-VCH GmbH.)

Osteoarthritic degeneration of cartilage is a major social health problem. Tissue engineering of cartilage using combinations of scaffold and mesenchymal stem cells (MSCs) is emerging as an alternative to existing treatment options such as microfracture, mosaicplasty, allograft, autologous chondrocyte implantation, or total joint replacement. Induction of chondrogenesis in high-density pellets of MSCs is generally attained by soluble exogenous TGF-β3 in culture media, which requires lengthy in vitro culture period during which pellets gain mechanical robustness. On the other hand, a growth factor delivering and a mechanically robust scaffold material that can accommodate chondroid pellets would enable rapid deployment of pellets after seeding. Delivery of the growth factor from the scaffold locally would drive the induction of chondrogenic differentiation in the postimplantation period. Therefore, we sought to develop a biomaterial formulation that will induce chondrogenesis in situ, and compared its performance to soluble delivery in vitro . In this vein, a heparin-conjugated mechanically robust collagen fabric was developed for sustained delivery of TGF-β3. The amount of conjugated heparin was varied to enhance the amount of TGF-β3 uptake and release from the scaffold. The results showed that the scaffold delivered TGF-β3 for up to 8 days of culture, which resulted in 15-fold increase in GAG production, and six-fold increase in collagen synthesis with respect to the No TGF-β3 group. The resulting matrix was cartilage like, in that type II collagen and aggrecan were positive in the spheroids. Enhanced chondrogenesis under in situ TGF-β3 administration resulted in a Young's modulus of ∼600 kPa. In most metrics, there were no significant differences between the soluble delivery group and in situ heparin-mediated delivery group. In conclusion, heparin-conjugated collagen scaffold developed in this study guides chondrogenic differentiation of hMSCs in a mechanically competent tissue construct, which showed potential to be used for cartilage tissue regeneration. Impact statement The most significant finding of this study was that sustained release of TGF-β3 from heparinized collagen scaffold had chondroinductive effect on pelleted human mesenchymal stem cells (hMSCs). The effect was comparable to that observed in hMSC pellets that were cultured in chondrogenic media supplemented with TGF-β3. The stiffness of scaffolds at the baseline was about 50% that of native cartilage and over 28 days the combined stiffness of pellet/scaffold complex converged to the stiffness of native cartilage. These data indicate that the scaffold system can generate a load-bearing cartilage-like tissue by using hMSCs pellets in a mechanically competent framework.

M2 macrophages are associated with deposition of interstitial collagen and other extracellular matrix proteins during the course wound healing and also inflammatory response to biomaterials. Developing advanced biomaterials to promote the M2 subtype may be an effective way to improve tissue reinforcement surgery outcomes. In this study, the effect of genipin, a naturally derived crosslinking agent, on M0 → M2-polarization was investigated. Genipin was introduced either indirectly by seeding cells on aligned collagen biotextiles that are crosslinked by the agent or in soluble form by direct addition to the culture medium. Cellular elongation effects on macrophage polarization induced by the collagen biotextile were also investigated as a potential inducer of macrophage polarization. M0 and M2 macrophages demonstrated significant elongation on the surface of aligned collagen threads, while cells of the M1 subtype-maintained a round phenotype. M0 → M2 polarization, as reflected by arginase and Ym-1 production, was observed on collagen threads only when the threads were crosslinked by genipin, implicating genipin as a more potent inducer of the regenerative phenotype compared to cytoskeletal elongation. The addition of genipin to the culture medium directly also drove the emergence of pro-regenerative phenotype as measured by the markers (arginase and Ym-1) and through the activation of the pSTAT6-PPAR-gamma pathway. This study indicates that genipin-crosslinked collagen biotextiles can be used as a delivery platform to promote regenerative response after biomaterial implantation. STATEMENT OF SIGNIFICANCE: The immune response is one of the key determinants of tissue repair and regeneration rate, and outcome. The M2 macrophage subtype is known to resolve the inflammatory response and support tissue repair by producing pro-regenerative factors. Therefore, a biomaterial that promotes M2 sub-type can be a viable strategy to enhance tissue regeneration. In this study, we investigated genipin-crosslinked electrochemically aligned collagen biotextiles for their capacity to induce pro-regenerative polarization of M0 macrophages. The results demonstrated that genipin, rather than matrix-induced cellular elongation, was responsible for M0 → M2 polarization in the absence of other bioinductive factors and maintaining the M2 polarized status of macrophages. Furthermore, we identified that genipin polarizes the M2 macrophage phenotype via activation of the pSTAT6-PPAR-gamma pathway., Competing Interests: Declaration of Competing Interest Drs. Akkus and Hijaz disclose employment and equity ownership in association with CollaMedix Inc. Dr. Akkus is an inventor on a patent that is associated with electrochemical compaction of collagen as threads. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. All rights reserved.)

Background: Critical shortages of personal protective equipment, especially N95 respirators, during the coronavirus disease 2019 (COVID-19) pandemic continues to be a source of concern. Novel methods of N95 filtering face-piece respirator decontamination that can be scaled-up for in-hospital use can help address this concern and keep healthcare workers (HCWs) safe., Methods: A multidisciplinary pragmatic study was conducted to evaluate the use of an ultrasonic room high-level disinfection system (HLDS) that generates aerosolized peracetic acid (PAA) and hydrogen peroxide for decontamination of large numbers of N95 respirators. A cycle duration that consistently achieved disinfection of N95 respirators (defined as ≥6 log10 reductions in bacteriophage MS2 and Geobacillus stearothermophilus spores inoculated onto respirators) was identified. The treated masks were assessed for changes to their hydrophobicity, material structure, strap elasticity, and filtration efficiency. PAA and hydrogen peroxide off-gassing from treated masks were also assessed., Results: The PAA room HLDS was effective for disinfection of bacteriophage MS2 and G. stearothermophilus spores on respirators in a 2,447 cubic-foot (69.6 cubic-meter) room with an aerosol deployment time of 16 minutes and a dwell time of 32 minutes. The total cycle time was 1 hour and 16 minutes. After 5 treatment cycles, no adverse effects were detected on filtration efficiency, structural integrity, or strap elasticity. There was no detectable off-gassing of PAA and hydrogen peroxide from the treated masks at 20 and 60 minutes after the disinfection cycle, respectively., Conclusion: The PAA room disinfection system provides a rapidly scalable solution for in-hospital decontamination of large numbers of N95 respirators during the COVID-19 pandemic.

Collagen is the major component of the extracellular matrix in human tissues and widely used in the fabrication of tissue engineered scaffolds for medical applications. However, these forms of collagen gels and films have limitations due to their inferior strength and mechanical performance and their relatively fast rate of degradation. A new form of continuous collagen yarn has recently been developed for potential usage in fabricating textile tissue engineering scaffolds. In this study, we prepared the continuous electrochemical aligned collagen yarns from acid-soluble collagen that was extracted from rat tail tendons (RTTs) using 0.25 M acetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Fourier transform infrared spectroscopy confirmed that the major component of the extracted collagen contained alpha 1 and alpha 2 chains and the triple helix structure of Type 1 collagen. The collagen solution was processed to monofilament yarns in continuous lengths by using a rotating electrode electrochemical compaction device. Exposing the non-crosslinked collagen yarns and the collagen yarns crosslinked with 1-ethyl-3-(-3-dimethyl-aminopropyl) carbodiimide hydrochloride to normal physiological hydrolytic degradation conditions showed that both yarns were able to maintain their tensile strength during the first 6 weeks of the study. Cardiosphere-derived cells showed significantly enhanced attachment and proliferation on the collagen yarns compared to synthetic polylactic acid filaments. Moreover, the cells were fully spread and covered the surface of the collagen yarns, which confirmed the superiority of collagen in terms of promoting cellular adhesion. The results of this work indicated that the aligned RTT collagen yarns are favorable for fabricating biotextile scaffolds and are encouraging for further studies of various textile structure for different tissue engineering applications.

Background: Fatty infiltration of the rotator cuff occurs after injury to the tendon and results in a buildup of adipose in the muscle. Fatty infiltration may be a biomarker for predicting future injuries and mechanical properties after tendon repair. As such, quantifying fatty infiltration accurately could be a relevant metric for determining the success of tendon repairs. Currently, fatty infiltration is quantified by an experienced observer using the Goutallier or Fuchs staging system, but because such score-based quantification systems rely on subjective assessments, newer techniques using semiautomated analyses in CT and MRI were developed and have met with varying degrees of success. However, semiautomated analyses of CT and MRI results remain limited in cases where only a few two-dimensional slices of tissue are examined and applied to the three-dimensional (3-D) tissue structure. We propose that it is feasible to assess fatty infiltration within the 3-D volume of muscle and tendon in a semiautomated fashion by selecting anatomic features and examining descriptive metrics of intensity histograms collected from a cylinder placed within the central volume of the muscle and tendon of interest., Questions/purposes: (1) Do descriptive metrics (mean and SD) of intensity histograms from microCT images correlate with the percentage of fat present in muscle after rotator cuff repair? (2) Do descriptive metrics of intensity histograms correlate with the maximum load during mechanical testing of rotator cuff repairs?, Methods: We developed a custom semiautomated program to generate intensity histograms based on user-selected anatomic features. MicroCT images were obtained from 12 adult female New Zealand White rabbits (age 8 to 12 months, weight 3.7 kg ± 5 kg) that were randomized to surgical repair or sham repair of an induced infraspinatus defect. Intensity histograms were generated from images of the operative and contralateral intact shoulder in these rabbits which were presented to the user in a random order without identifying information to minimize sources of bias. The mean and SD of the intensity histograms were calculated and compared with the total percentage of the volume threshold as fat. Patterns of fat identified were qualitatively compared with histologic samples to confirm that thresholding was detecting fat. We conducted monotonic tensile strength-to-failure tests of the humeral-infraspinatus bone-tendon-muscle complex, and evaluated associations between histogram mean and SDs and maximum load., Results: The total percentage of fat was negatively correlated with the intensity histogram mean (Pearson correlation coefficient -0.92; p < 0.001) and positively with intensity histogram SD (Pearson correlation coefficient 0.88; p < 0.001), suggesting that the increase in fat leads to a reduction and wider variability in volumetric tissue density. The percentage of fat content was also negatively correlated with the maximum load during mechanical testing (Pearson correlation coefficient -78; p = 0.001), indicating that as the percentage of fat in the volume increases, the mechanical strength of the repair decreases. Furthermore, the intensity histogram mean was positively correlated with maximum load (Pearson correlation coefficient 0.77; p = 0.001) and histogram SD was negatively correlated with maximum load (Pearson correlation coefficient -0.72; p = 0.004). These correlations were strengthened by normalizing maximum load to account for animal size (Pearson correlation coefficient 0.86 and -0.9, respectively), indicating that as histogram mean decreases, the maximum load of the repair decreases and as histogram spread increases, the maximum load decreases., Conclusion: In this ex vivo rabbit model, a semiautomated approach to quantifying fat on microCT images was a noninvasive way of quantifying fatty infiltration associated with the strength of tendon healing., Clinical Relevance: Histogram-derived variables may be useful as surrogate measures of repair strength after rotator cuff repair. The preclinical results presented here provide a foundation for future studies to translate this technique to patient studies and additional imaging modalities. This semiautomated method provides an accessible approach to quantification of fatty infiltration by users of varying experience and can be easily adapted to any intensity-based imaging approach. To translate this approach to clinical practice, this technique should be calibrated for MRI or conventional CT imaging and applied to patient scans. Further investigations are needed to assess the correlation of volumetric intensity histogram descriptive metrics to clinical mechanical outcomes., Competing Interests: All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2020 by the Association of Bone and Joint Surgeons.)

Aim: We evaluated cardiovascular (CV) risk stratification for nonfunctioning adrenal incidentalomas (NFAIs) via the coronary-artery-calcium (CAC) score., Materials and Methods: The participants were patients with NFAI (n = 55). They were compared to patients with chest pain, a low-intermediate Framingham-risk score, and a non-diagnostic treadmill- exercise test, which served as the control group (n = 49). Subsequently, the NFAI group was subdivided according to a CAC score of <100 Agatston units - mild coronary-artery calcification (n = 40) - and ≥100 Agatston units - moderate-to-severe calcification (n = 15)., Results: Similar rates of traditional risk factors were observed between the NFAI and control groups, and lower low-density lipoprotein cholesterol rates were observed in the NFAI group. The CAC score was significantly higher for the NFAI group than the control group. Glucose, potassium, adrenocorticotropic-hormone, and basal-cortisol levels were higher in those with a CAC score of ≥100. High-density-lipoprotein cholesterol estimated glomerular filtration rate and ejection fraction (EF) were higher in those with a CAC score of <100. Adenoma size and location were similar between the groups. Age, EF, and glucose were the most significant variables related to CAC score in patients with NFAI, at ≥100 Agatston units., Discussion: Patients with a low-intermediate CV risk profile and NFAI have a higher risk of atherosclerosis when compared to patients with a low-intermediate CV risk profile, but no NFAI., Conclusion: In patients with NFAI, CAC score evaluation may be used to predict increased atherosclerosis, especially in patients of an older age with higher glucose and decreased EF., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Tissue-engineered small caliber vascular grafts have attracted much research attention as a viable alternative to traditional vascular grafts with their biocompatibility and potential to achieve complete healing. However, the major challenge is to fabricate a scaffold with both satisfactory mechanical properties and fast endothelialization. In this study, a hybrid tubular vascular tissue engineered scaffold has been circular-knitted using novel electrochemically aligned collagen (ELAC) filaments plied together with traditional poly(lactic acid) (PLA) yarn. The collagen component was able to promote the recruitment and proliferation of endothelial cells by increasing the initial cell adhesion 10-fold and the eventual cell population 3.2 times higher than the PLA scaffold alone. At the same time, the PLA yarn was able to provide sufficient mechanical strength and structural stability, as well as facilitate scaffold fabrication on high speed textile production equipment. The tubular hybrid scaffold exhibited excellent bursting strength (1.89 ± 0.43 MPa) and suture retention strength (10.86 ± 0.49 N), and had comparable compliance (3.98 ± 1.94%/100 mmHg) to that of the coronary artery (3.8 ± 0.3%/100 mmHg) under normotensive pressure. With its excellent mechanical and biological performance, this prototype hybrid scaffold is a promising candidate for the construction of a clinically successful and easily translatable tissue-engineered small caliber vascular graft., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Implantable medical devices and hardware are prolific in medicine, but hardware associated infections remain a major issue., Objective: To develop and evaluate a novel, biologic antimicrobial coating for medical implants., Methods: Electrochemically compacted collagen sheets with and without crosslinked heparin were synthesized per a protocol developed by our group. Sheets were incubated in antibiotic solution (gentamicin or moxifloxacin) overnight, and in vitro activity was assessed with five-day diffusion assays against Pseudomonas aeruginosa. Antibiotic release over time from gentamicin-infused sheets was determined using in vitro elution and high performance liquid chromatography (HPLC)., Results: Collagen-heparin-antibiotic sheets demonstrated larger growth inhibition zones against P. aeruginosa compared to collagen-antibiotic alone sheets. This activity persisted for five days and was not impacted by rinsing sheets prior to evaluation. Rinsed collagen-antibiotic sheets did not produce any inhibition zones. Elution of gentamicin from collagen-heparin-gentamicin sheets was gradual and remained above the minimal inhibitory concentration for gentamicin-sensitive organisms for 29 days. Conversely, collagen-gentamicin sheets eluted their antibiotic load within 24 hours. Overall, heparin-associated sheets demonstrated larger inhibition zones against P. aeruginosa and prolonged elution profile via HPLC., Conclusion: We developed a novel, local antibiotic delivery system that could be used to coat medical implants/hardware in the future and reduce post-operative infections.

Objective: We aimed to compare functional exercise capacity, respiratory and peripheral muscle strength, pulmonary function and quality of life between patients with stable angina and healthy controls., Methods: We compared 33 patients with stable angina (55.21 ± 6.12 years old, Canada Class II-III, left ventricular ejection fraction: 61.92 ± 7.55) and 30 healthy controls (52.70 ± 4.22 years old). Functional capacity (6-minute walk test (6-MWT)), respiratory muscle strength (mouth pressure device), peripheral muscle strength (dynamometer), pulmonary function (spirometer) and quality of life (Short Form 36 (SF-36)) were evaluated., Results: 6-MWT distance (499.20 ± 51.91 m versus 633.05 ± 57.62 m), maximal inspiratory pressure (85.42 ± 20.52 cmH 2 O versus 110.44 ± 32.95 cmH 2 O), maximal expiratory pressure (83.33 ± 19.05 cmH 2 O versus 147.96 ± 54.80 cmH 2 O) and peripheral muscle strength, pulmonary function and SF-36 sub-scores were lower in the angina group versus the healthy controls, respectively., Conclusion: Impaired peripheral and respiratory muscle strength, reduction in exercise capacity and quality of life are obvious in patients with stable angina. Therefore, these parameters should be considered in stable angina physiotherapy programmes to improve impairments.

Raman spectroscopy is a powerful non-invasive tool for detection and classification of chemical composition of materials including biological tissues. In this work, we report an in vitro Raman study on animal skin samples with a focus on high-frequency vibrations such as symmetric CH 3 stretching mode at 2934 cm -1 , and the symmetric CH 2 vibration mode at 2854 cm -1 , OH stretching modes near 3412 cm -1 , and bounded OH mode near 3284 cm -1 . Raman data was acquired with a customized InGaAs based Raman spectrometer that consolidates the NIR (866 nm) light and the InGaAs detector and is particularly suitable for probing high-frequency vibrations. The Raman spectra of fat, tendon, and muscle tissues are also analyzed to determine the spectroscopic identities of CH and OH groups in skin. Our results suggest that the protein is beneficial for the maintenance of skin hydration, as it has higher water capacity and greater capability to retain water than lipids. This conclusion is consistent with the additional discovery that water exists in fat mainly as unbound type, while part of water exists as bound type in muscle., Competing Interests: Disclosures The authors declare that there are no conflicts of interest related to this article.

Introduction: Matrix damage sustained by bone tissue is repaired by the concerted action of bone cells. Previous studies have reported extracellular calcium ([Ca 2+ ] E ) efflux to originate from regions of bone undergoing diffuse microdamage termed as "diffuse microdamage-induced calcium efflux" (DMICE). DMICE has also been shown to activate and increase intracellular calcium ([Ca 2+ ] I ) signaling in osteoblasts via the involvement of voltage-gated calcium channels (VGCC). Past studies have assessed early stage (< 1 h) responses of osteoblasts to DMICE. The current study tested the hypothesis that DMICE has longer-term sustained effect such that it induces anabolic response of osteoblasts., Materials and Methods: Osteoblasts derived from mouse calvariae were seeded on devitalized bovine bone wafers. Localized diffuse damage was induced in the vicinity of cells by bending. The response of osteoblasts to DMICE was evaluated by testing gene expression, protein synthesis and mineralized nodule formation., Results: Cells on damaged bone wafers showed a significant increase in RUNX2 and Osterix expression compared to non-loaded control. Also, RUNX2 and Osterix expression were suppressed significantly when the cells were treated with bepridil, a non-selective VGCC inhibitor, prior to loading. Significantly higher amounts of osteocalcin and mineralized nodules were synthesized by osteoblasts on diffuse damaged bone wafers, while bepridil treatment resulted in a significant decrease in osteocalcin production and mineralized nodule formation., Conclusion: In conclusion, this study demonstrated that DMICE activates anabolic responses of osteoblasts through activation of VGCC. Future studies of osteoblast response to DMICE in vivo will help to clarify how bone cells repair diffuse microdamage.

Purpose: The classic fracture model, described by Bonnarens and Einhorn in 1984, enlists a blunt guillotine to generate a closed fracture in a pre-stabilized rodent femur. However, in less experienced hands, this technique yields considerable variability in fracture pattern and requires highly-specialized equipment. This study describes a reproducible and low-cost model of mouse fracture healing using an open femoral osteotomy., Methods: Femur fractures were produced in skeletally mature male and female mice using an open femoral osteotomy after intramedullary stabilization. Mice were recovered for up to 28 days prior to analysis with microradiographs, histomorphometry, a novel μCT methodology, and biomechanical torsion testing at weekly intervals., Results: Eight mice were excluded due to complications (8/193, 4.1%), including unacceptable fracture pattern (2/193, 1.0%). Microradiographs showed progression of the fracture site to mineralized callus by 14 days and remodelling 28 days after surgery. Histomorphometry from 14 to 28 days revealed decreased cartilage area and maintained bone area. μCT analysis demonstrated a reduction in mineral surface from 14 to 28 days, stable mineral volume, decreased strut number, and increased strut thickness. Torsion testing at 21 days showed that fractured femurs had 61% of the ultimate torque, 63% of the stiffness, and similar twist to failure when compared to unfractured contralateral femurs., Conclusions: The fracture model described herein, an open femoral osteotomy, demonstrated healing comparable to that reported using closed techniques. This simple model could be used in future research with improved reliability and reduced costs compared to the current options., (© 2020 The Authors.)

Background: Structural bone allografts are an established treatment method for long-bone structural defects resulting from such conditions as traumatic injury and sarcoma. The functional lifetime of structural allografts depends on resistance to cyclic loading (cyclic fatigue life), which can lead to fracture at stress levels well below the yield strength. Raman spectroscopy biomarkers can be used to non-destructively assess the 3 primary components of bone (collagen, mineral, and water), and may aid in optimizing allograft selection to decrease fatigue fracture risk. We studied the association of Raman biomarkers with the cyclic fatigue life of human allograft cortical bone., Methods: Twenty-one cortical bone specimens were machined from the femoral diaphyses of 4 human donors (a 63-year old man, a 61-year-old man, a 51-year-old woman, and a 48-year-old woman) obtained from the Musculoskeletal Transplant Foundation. Six Raman biomarkers were analyzed: collagen disorganization, mineral maturation, matrix mineralization, and 3 water compartments. The specimens underwent cyclic fatigue testing under fully reversed conditions (35 and 45 MPa), during which they were tested to fracture or to 30 million cycles ("runout"), simulating 15 years of moderate activity. A tobit censored linear regression model for cyclic fatigue life was created., Results: The multivariate model explained 60% of the variance in the cyclic fatigue life (R = 0.604, p < 0.001). Increases in Raman biomarkers for disordered collagen (coefficient: -2.74×10, p < 0.001) and for loosely collagen-bound water compartments (coefficient: -2.11×10, p < 0.001) were associated with a decreased cyclic fatigue life. Increases in Raman biomarkers for mineral maturation (coefficient: 3.50×10, p < 0.001), matrix mineralization (coefficient: 2.32×10, p < 0.001), tightly collagen-bound water (coefficient: 1.19×10, p < 0.001), and mineral-bound water (coefficient: 3.27×10, p < 0.001) were associated with an increased cyclic fatigue life. Collagen disorder accounted for 44% of the variance in the cyclic fatigue life, mineral maturation accounted for 6%, and all bound water compartments accounted for 3%., Conclusions: Increasing baseline collagen disorder was associated with a decreased cyclic fatigue life and had the strongest correlation with the cyclic fatigue life of human cortical donor bone. This model should be prospectively validated., Clinical Relevance: Raman analysis is a promising tool for the non-destructive evaluation of structural bone allograft quality for load-bearing applications.