Search

Complex injury and open reconstructive surgeries of the knee often lead to joint dysfunction that may alter the normal biomechanics of the joint. Two major complications that often arise are excessive deposition of fibrotic tissue and acquired heterotopic endochondral ossification. Knee arthrofibrosis is a fibrotic joint disorder where aberrant buildup of scar tissue and adhesions develop around the joint. Heterotopic ossification is ectopic bone formation around the periarticular tissues. Even though arthrofibrosis and heterotopic ossification pose an immense clinical problem, limited studies focus on their cellular and molecular mechanisms. Effective cell-targeted therapeutics are needed, but the cellular origin of both knee disorders remains elusive. Moreover, all the current animal models of knee arthrofibrosis and stiffness are developed in rats and rabbits, limiting genetic experiments that would allow us to explore the contribution of specific cellular targets to these knee pathologies. Here, we present a novel mouse model where surgically induced injury and hyperextension of the knee lead to excessive deposition of disorganized collagen in the meniscus, synovium, and joint capsule in addition to formation of extra-skeletal bone in muscle and soft tissues within the joint capsule. As a functional outcome, arthrofibrosis and acquired heterotopic endochondral ossification coupled with a significant increase in total joint stiffness were observed. By employing this injury model and genetic lineage tracing, we also demonstrate that Gli1+ mesenchymal progenitors proliferate after joint injury and contribute to the pool of fibrotic cells in the synovium and ectopic osteoblasts within the joint capsule. These findings demonstrate that Gli1+ cells are a major cellular contributor to knee arthrofibrosis and acquired heterotopic ossification that manifest after knee injury. Our data demonstrate that genetic manipulation of Gli1+ cells in mice may offer a platform for identification of novel therapeutic targets to prevent knee joint dysfunction after chronic injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Magallanes, Liu, Zhang, Ouyang, Mkaratigwa, Bian, Van Handel, Skorka, Petrigliano and Evseenko.)

At the present time there are no consistently satisfactory treatment options for some challenging bone loss scenarios. We have previously reported on the properties of a novel 3D-printed hydroxyapatite-composite material in a pilot study, which demonstrated osteoconductive properties but was not tested in a rigorous, clinically relevant model. We therefore utilized a rat critical-sized femoral defect model with a scaffold designed to match the dimensions of the bone defect. The scaffolds were implanted in the bone defect after being loaded with cultured rat bone marrow cells (rBMC) transduced with a lentiviral vector carrying the cDNA for BMP-2. This experimental group was compared against 3 negative and positive control groups. The experimental group and positive control group loaded with rhBMP-2 demonstrated statistically equivalent radiographic and histologic healing of the defect site (p > 0.9), and significantly superior to all three negative control groups (p < 0.01). However, the healed defects remained biomechanically inferior to the unoperated, contralateral femurs (p < 0.01). When combined with osteoinductive signals, the scaffolds facilitate new bone formation in the defect. However, the scaffold alone was not sufficient to promote adequate healing, suggesting that it is not substantially osteoinductive as currently structured. The combination of gene therapy with 3D-printed scaffolds is quite promising, but additional work is required to optimize scaffold geometry, cell dosage and delivery., (© 2021 Wiley Periodicals LLC.)

Regional gene therapy using a lentiviral vector containing the BMP-2 complementary DNA (cDNA) has been shown to heal critical-sized bone defects in rodent models. An appropriate "cellular dose" needs to be defined for eventual translation into human trials. The purpose of this study was to evaluate bone defect healing potential and quality using three different doses of transduced human bone marrow cells (HBMCs). HBMCs were transduced with a lentiviral vector containing either BMP-2 or green fluorescent protein (GFP). All cells were loaded onto compression-resistant matrices and implanted in the bone defect of athymic rats. Treatment groups included femoral defects that were treated with a low-dose (1 × 10 6 cells), standard-dose (5 × 10 6 cells), and high-dose (1.5 × 10 7 cells) HBMCs transduced with lentiviral vector containing BMP-2 cDNA. The three control groups were bone defects treated with HBMCs that were either nontransduced or transduced with vector containing GFP. All animals were sacrificed at 12 weeks. The bone formed in each defect was evaluated with plain radiographs, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. Bone defects treated with higher doses of BMP-2-producing cells were more likely to have healed (6/14 of the low-dose group; 12/14 of the standard-dose group; 14/14 of the high-dose group; χ 2 (2) = 15.501, p  < 0.001). None of the bone defects in the control groups had healed. Bone defects treated with high dose and standard dose of BMP-2-producing cells consistently outperformed those treated with a low dose in terms of bone formation, as assessed by microCT and histomorphometry, and biomechanical parameters. However, statistical significance was only seen between defects treated with high dose and low dose. Larger doses of BMP-2-producing cells were associated with a higher likelihood of forming heterotopic ossification. Femurs treated with a standard- and high-dose BMP-2-producing cells demonstrated similar healing and biomechanical properties. Increased doses of BMP-2 delivered through higher cell doses have the potential to heal large bone defects. Adapting regional gene therapy for use in humans will require a balance between promoting bone repair and limiting heterotopic ossification. Impact statement Critical bone loss may result from complex traumatic bone injury (i.e., open fracture or blast injury), revision total joint arthroplasty, and spine pseudoarthrosis. This is a challenging clinical problem to treat and regional gene therapy is an innovative means of addressing it. This study provides information regarding the quantity of cells or "cell dose" of transduced cells needed to treat a critical-sized bone defect in a rat model. This information may be extrapolated for use in humans in future trials.

Traditionally, ex vivo gene therapy involves a two-step approach, with culture expansion of cells prior to transduction and implantation. We have tried to simplify this strategy and eliminate the time and cost associated with culture expansion, by introducing "next-day" regional gene therapy using human bone marrow cells. The purpose of this study was to determine whether a lentiviral vector (LV) carrying the cDNA for BMP-2 can transduce freshly isolated human BM cells, leading to abundant BMP production and bone formation in vivo, and evaluate the in vivo osteoinductive potential of "next-day" gene therapy and the standard "two-step" tissue culture expansion approach. To this end, human bone marrow cells (HBMC) from patients undergoing total hip arthroplasty were harvested, transduced with a BMP-2-expressing LV either overnight ("next day" gene therapy; ND) or after culture expansion (cultured "two-step" approach; C) and then implanted into a rat critical-sized femoral defect. The animals were randomly assigned to one of the following groups: I; ND-HBMC transduced with LV-TSTA BMP-2, II; ND-HBMC transduced with LV-TSTA GFP, III; non-transduced ND-HBMC; IV; C-HBMC transduced with LV-TSTA BMP-2, V; C-HBMC transduced with LV-TSTA-GFP, VI; non-transduced C-HBMC. Treatment with either "next-day" or cultured HBMC demonstrated a significant increase in new bone formation compared with all negative control groups as seen in plain radiographs, microCT and histologic/histomorphometric analysis. At 12 weeks post-op, complete defect union on plain X-rays occurred in 7/14 animals in the ND-HBMC/BMP-2 group and 12/14 in the C-HBMC/BMP-2 treated rats. The two-step approach was associated with more consistent results, a higher union rate, and superiority with regards to all of the studied bone healing parameters. In this study we demonstrate proof of concept that BMP-2-transduced human bone marrow cells can be used to enhance bone healing in segmental bone defects, and that regional gene therapy using lentiviral transduction has the osteoinductive potential to heal large bone defects in clinical settings., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Physical activity is emerging as an alternative nonpharmaceutical strategy to prevent and treat a variety of cardiovascular diseases due to its cardiac and skeletal muscle beneficial effects. Oxidative stress occurs in skeletal muscle of chronic heart failure (CHF) patients with possible impact on muscle function decline. We determined the effect of voluntary-free wheel running (VFWR) in preventing protein damage in Tgαq*44 transgenic mice (Tg) characterized by a delayed CHF progression. In the early (6 months) and transition (12 months) phase of CHF, VFWR increased the daily mean distance covered by Tg mice eliminating the difference between Tg and WT present before exercise at 12 months of age (WT Pre-EX 3.62 ± 1.66 vs. Tg Pre-EX 1.51 ± 1.09 km, P < 0.005; WT Post-EX 5.72 ± 3.42 vs. Tg Post-EX 4.17 ± 1.8 km, P > 0.005). This effect was concomitant with an improvement of in vivo cardiac performance [(Cardiac Index (mL/min/cm 2 ): 6 months, untrained-Tg 0.167 ± 0.005 vs. trained-Tg 0.21 ± 0.003, P < 0.005; 12 months, untrained-Tg 0.1 ± 0.009 vs. trained-Tg 0.133 ± 0.005, P < 0.005]. Such effects were associated with a skeletal muscle antioxidant response effective in preventing oxidative damage induced by CHF at the transition phase (untrained-Tg 0.438 ± 0.25 vs. trained-Tg 0.114 ± 0.010, P < 0.05) and with an increased expression of protein control markers (MuRF-1, untrained-Tg 1.12 ± 0.29 vs. trained-Tg 14.14 ± 3.04, P < 0.0001; Atrogin-1, untrained-Tg 0.9 ± 0.38 vs. trained-Tg 7.79 ± 2.03, P < 0.01; Cathepsin L, untrained-Tg 0.91 ± 0.27 vs. trained-Tg 2.14 ± 0.55, P < 0.01). At the end-stage of CHF (14 months), trained-Tg mice showed a worsening of physical performance (decrease in daily activity and weekly distance and time of activity) compared to trained age-matched WT in association with oxidative protein damage of a similar level to that of untrained-Tg mice (untrained-Tg 0.62 ± 0.24 vs. trained-Tg 0.64 ± 0.13, P > 0.05). Prolonged voluntary physical activity performed before the onset of CHF end-stage, appears to be a useful tool to increase cardiac function and to reduce skeletal muscle oxidative damage counteracting physical activity decline., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Chameleon species have recently been adopted as models for evo-devo and macroevolutionary processes. However, most anatomical and developmental studies of chameleons focus on the skeleton, and information about their soft tissues is scarce. Here, we provide a detailed morphological description based on contrast enhanced micro-CT scans and dissections of the adult phenotype of all the forelimb and hindlimb muscles of the Veiled Chameleon (Chamaeleo calyptratus) and compare these muscles with those of other chameleons and lizards. We found the appendicular muscle anatomy of chameleons to be surprisingly conservative considering the remarkable structural and functional modifications of the limb skeleton, particularly the distal limb regions. For instance, the zygodactyl autopodia of chameleons are unique among tetrapods, and the carpals and tarsals are highly modified in shape and number. However, most of the muscles usually present in the manus and pes of other lizards are present in the same configuration in chameleons. The most obvious muscular features related to the peculiar opposable autopodia of chameleons are: (1) presence of broad, V-shaped plantar and palmar aponeuroses, and absence of intermetacarpales and intermetatarsales, between the digits separated by the cleft in each autopod; (2) oblique orientation of the superficial short flexors originating from these aponeuroses, which may allow these muscles to act as powerful adductors of the "super-digits"; and (3) well-developed abductor digiti minimi muscles and abductor pollicis/hallucis brevis muscles, which may act as powerful abductors of the "super-digits.", (© 2017 Wiley Periodicals, Inc.)

Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo . Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR -/- ) in vivo , in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1-7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR -/- mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate ® FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T 1 ) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1-7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR -/- mice, Ach induced a vasoconstriction in BCA that amounted to -7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1-7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1-9) and Ang-(1-7) and a proportional decrease in Ang II and its active metabolites. Finally, MNA and perindopril treatment resulted in an increase in L-arginine/ADMA ratio by 107% (MNA) and 140% (perindopril), as compared to non-treated mice. Functional and biochemical endothelial profiling in ApoE/LDLR -/- mice in vivo revealed that 2-month treatment with MNA (100 mg/kg/day) displayed a similar profile of vasoprotective effect as 2-month treatment with perindopril (10 mg/kg/day): i.e., the improvement in endothelial function that was associated with the beneficial changes in ACE/Ang II-ACE2/Ang (1-7) balance and in L-arginine/ADMA ratio in plasma.

Objective: To assess alterations in perfusion and liver function in the concanavalin A (ConA)-induced mouse model of acute liver failure (ALF) using two magnetic resonance imaging (MRI)-based methods: dynamic contrast-enhanced MRI (DCE-MRI) with Gd-EOB-DTPA contrast agent and arterial spin labelling (ASL)., Materials and Methods: BALB/c mice were studied using a 9.4 T MRI system. The IntraGateFLASH TM and FAIR-EPI pulse sequences were used for optimum mouse abdomen imaging., Results: The average perfusion values for the liver of the control and ConA group were equal to 245 ± 20 and 200 ± 32 ml/min/100 g (p = 0.008, respectively). DCE-MRI showed that the time to the peak of the image enhancement was 6.14 ± 1.07 min and 9.72 ± 1.69 min in the control and ConA group (p < 0.001, respectively), while the rate of the contrast wash-out in the control and ConA group was 0.037 ± 0.008 and 0.021 ± 0.008 min -1 (p = 0.004, respectively). These results were consistent with hepatocyte injury in the ConA-treated mice as confirmed by histopathological staining., Conclusions: Both the ASL and DCE-MRI techniques represent a reliable methodology to assess alterations in liver perfusion and hepatocyte integrity in murine hepatitis., Competing Interests: The authors declare that they have no conflict of interest. Welfare of animals All applicable national and institutional guidelines for the care and use of animals were followed. All procedures were approved by the local Ethics Committee on Animal Testing.

ApoE/LDLR(-/-) mice represent a reliable model of atherosclerosis. However, it is not clear whether cardiac performance is impaired in this murine model of atherosclerosis. Here, we used MRI to characterize cardiac performance in vivo in apoE/LDLR(-/-) mice with advanced atherosclerosis. Six-month-old apoE/LDLR(-/-) mice and age-matched C57BL/6J mice (control) were examined using highly time-resolved cine-MRI [whole-chamber left ventricle (LV) imaging] and MR tagging (three slices: basal, mid-cavity and apical). Global and regional measures of cardiac function included LV volumes, kinetics, time-dependent parameters, strains and rotations. Histological analysis was performed using OMSB (orceine with Martius, Scarlet and Blue) and ORO (oil red-O) staining to demonstrate the presence of advanced coronary atherosclerosis. MR-tagging-based strain analysis in apoE/LDLR(-/-) mice revealed an increased frequency of radial and circumferential systolic stretch (25% and 50% of segments, respectively, p ≤ 0.012), increased radial post-systolic strain index (45% of segments, p = 0.009) and decreased LV untwisting rate (-30.3° (11.6°)/cycle, p = 0.004) when compared with control mice. Maximal strains and LV twist were unchanged. Most of the cine-MRI-based LV functional and anatomical parameters also remained unchanged in apoE/LDLR(-/-) mice, with only a lower filling rate, longer filling time, shorter isovolumetric contraction time and slower heart rate observed in comparison with control mice. The coronary arteries displayed severe atherosclerosis, as evidenced by histological analysis. Using comprehensive MRI methods, we have demonstrated that, despite severe coronary atherosclerosis in six-month-old apoE/LDLR(-/-) mice, cardiac performance including global parameters, twist and strains, was well preserved. Only subtle diastolic alterations, possibly of ischemic background, were uncovered. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

While a healthy endothelium serves to maintain vascular haemostasis, a malfunctioning endothelium leads to various cardiovascular diseases, including atherothrombosis. Endothelial dysfunction is characterized by increased vascular permeability, impaired endothelium-dependent responses and various pro-inflammatory and pro-thrombotic changes in endothelial phenotype, all of which could provide the basis for an in vivo diagnosis of endothelial dysfunction. In the present review, we briefly summarize the magnetic resonance imaging (MRI)-based methods available for assessing endothelial function in animal models, especially in mice. These methods are aimed to assess biochemical phenotype using molecular imaging, endothelium-dependent responses or changes in endothelial permeability. All these approaches provide a complementary insight into the endothelial dysfunction in vivo and may offer a unique opportunity to study endothelium-based mechanisms of diseases and endothelial response to treatment., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Purpose: To assess the cardiac response to low (0.15-0.5 mg/kg i.p.) and high (1.5-20 mg/kg i.p.) doses of dobutamine in Tgαq*44 mice with dilated cardiomyopathy at the stage of advanced heart failure., Materials and Methods: Inotropic, lusitropic, and chronotropic response to β(1) -adrenergic stimulation was assessed by the cine magnetic resonance imaging (MRI) protocol based on the electrocardiogram (ECG)-triggered bright-blood images of one midventricular short-axis slice., Results: In wildtype mice increasing doses of dobutamine resulted in subsequent increase in the left ventricular function and heart rate acceleration, but significant inotropic, lusitropic, and chronotropic cardiac response was observed only after high doses of dobutamine, what is typical. In the Tgαq*44 mice low doses of dobutamine significantly increased inotropic and lusitropic cardiac performance without chronotropic changes. An increased heart rate was observed only after high doses of dobutamine, but then inotropic and lusitropic cardiac functional reserve was lost., Conclusion: We described MRI stress test protocol based on a low and high dose of dobutamine induced response that proves useful in revealing alternation in cardiac function in mice with heart failure., (Copyright © 2012 Wiley Periodicals, Inc.)

The compendial method of evaluation of orodispersible tablets (ODT) is the same disintegration test as for conventional tablets. Since it does not reflect the disintegration process in the oral cavity, alternative methods are proposed that are more related to in vivo conditions, e.g. modified dissolution paddle apparatus, texture analyzer, rotating shaft apparatus, CCD camera application, or wetting time and water absorption ratio measurement. In this study, three different co-processed excipients for direct compression of orally disintegrating tablets were compared (Ludiflash, Pharmaburst, F-Melt). The properties of the prepared tablets such as tensile strength, friability, wetting time and water absorption ratio were evaluated. Disintegration time was measured using the pharmacopoeial method and the novel apparatus constructed by the authors. The apparatus was based on the idea of Narazaki et al., however it has been modified. Magnetic resonance imaging (MRI) was applied for the analysis of the disintegration mechanism of prepared tablets. The research has shown the significant effect of excipients, compression force, temperature, volume and kind of medium on the disintegration process. The novel apparatus features better correlation of disintegration time with in vivo results (R(2) = 0.9999) than the compendial method (R(2) = 0.5788), and presents additional information on the disintegration process, e.g. swelling properties.

Objective: The aim of the present work was to analyze coronary endothelial function in the transgenic mouse model of dilated cardiomyopathy (Tgalphaq*44 mice)., Methods: Coronary vasodilatation, both NO-dependent (induced by bradykinin) and PGI(2)-dependent (induced by acetylcholine), was assessed in the isolated hearts of Tgalphaq*44 and FVB mice. Cardiac function was analyzed in vivo (MRI)., Results: In Tgalphaq*44 mice at the age of 2-4 months cardiac function was preserved and there were no alterations in endothelial function. By contrast, in Tgalphaq*44 mice at the age of 14-16 months cardiac function was significantly impaired and NO, but not PGI(2)-dependent coronary function was altered. Interestingly, the basal level of PGI(2) in coronary circulation increased fourfold as compared to FVB mice. Cardiac O(2) (-) production increased 1.5-fold and 3-fold in Tgalphaq*44 vs. FVB mice at the age of 2-6 and 14-16 months, respectively, and was inhibited by apocynin. Interestingly, inhibition of NADPH oxidase or NOS-3 normalized augmented PGI(2) production in Tgalphaq*44 mice. There was also an increased expression of gp91phox in Tgalphaq*44 vs. FVB hearts, without evident alterations in the expression of COX-1, COX-2, NOS-3 and PGI(2)-synthase., Conclusions: In the mouse model of dilated cardiomyopathy, endothelial dysfunction in coronary circulation is present in the late but not the early stage of heart failure pathology and is characterized by a decrease in NO bioavailability and a compensatory increase in PGI(2). Both the decrease in NO activity and the increase in PGI(2) activity may result from excessive O(2) (-) production by cardiac NADPH oxidase in Tgalphaq*44 hearts.

Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.