Minimally Invasive Intradiscal Delivery of BM-MSCs via Fibrous Microscaffold Carriers.

Current treatments of degenerated intervertebral discs often provide only temporary relief or address specific causes, necessitating the exploration of alternative therapies. Cell-based regenerative approaches showed promise in many clinical trials, but limitations such as cell death during injection and a harsh disk environment hinder their effectiveness. Injectable microscaffolds offer a solution by providing a supportive microenvironment for cell delivery and enhancing bioactivity. This study evaluated the safety and feasibility of electrospun nanofibrous microscaffolds modified with chitosan (CH) and chondroitin sulfate (CS) for treating degenerated NP tissue in a large animal model. The microscaffolds facilitated cell attachment and acted as an effective delivery system, preventing cell leakage under a high disc pressure. Combining microscaffolds with bone marrow-derived mesenchymal stromal cells demonstrated no cytotoxic effects and proliferation over the entire microscaffolds. The administration of cells attached to microscaffolds into the NP positively influenced the regeneration process of the intervertebral disc. Injectable poly(l-lactide-co-glycolide) and poly(l-lactide) microscaffolds enriched with CH or CS, having a fibrous structure, showed the potential to promote intervertebral disc regeneration. These features collectively address critical challenges in the fields of tissue engineering and regenerative medicine, particularly in the context of intervertebral disc degeneration.

Pulmonary artery embolism: comprehensive transcriptomic analysis in understanding the pathogenic mechanisms of the disease.

BACKGROUND: Pulmonary embolism (PE) is a severe disease that usually originates from deep vein thrombosis (DVT) of the lower extremities. This study set out to investigate the changes in the transcriptome of the pulmonary artery (PA) in the course of the PE in the porcine model. METHODS: The study was performed on 11 male pigs: a thrombus was formed in each right femoral vein in six animals, and then was released to induce PE, the remaining five animals served as a control group. In the experimental animals total RNA was isolated from the PA where the blood clot lodged, and in the control group, from the corresponding PA segments. High-throughput RNA sequencing was used to analyse the global changes in the transcriptome of PA with induced PE (PA-E). RESULTS: Applied multistep bioinformatics revealed 473 differentially expressed genes (DEGs): 198 upregulated and 275 downregulated. Functional Gene Ontology annotated 347 DEGs into 27 biological processes, 324 to the 11 cellular components and 346 to the 2 molecular functions categories. In the signaling pathway analysis, KEGG 'protein processing in endoplasmic reticulum' was identified for the mRNAs modulated during PE. The same KEGG pathway was also exposed by 8 differentially alternative splicing genes. Within single nucleotide variants, the 61 allele-specific expression variants were localised in the vicinity of the genes that belong to the cellular components of the 'endoplasmic reticulum'. The discovered allele-specific genes were also classified as signatures of the cardiovascular system. CONCLUSIONS: The findings of this research provide the first thorough investigation of the changes in the gene expression profile of PA affected by an embolus. Evidence from this study suggests that the disturbed homeostasis in the biosynthesis of proteins in the endoplasmic reticulum plays a major role in the pathogenesis of PE.

Ultrasonographic Imaging Protocol and Sonoanatomy of the Lumbar Spine in Healthy Dogs.

Ultrasound is an imaging technique commonly used in veterinary medicine. Ultrasound devices are widely available, their means of examination are relatively short and cheap, and they do not generate ionizing radiation. In addition, ultrasound generally does not need to be performed under general anesthesia. This study was performed on 23 canine cadavers with full clinical histories and with no confirmed pathological changes in the spine region. The imaging modalities were established in dogs in lateral recumbency, with the selected side being the uppermost angle, in a neutral position. All dogs were examined in the transverse and longitudinal planes. Sacral crest, intertransverse ligament, vertebral canal floor, vertebral body, and intervertebral discs were only visible in the longitudinal plane. Vertebral arch, supraspinal ligament, dorsal wall of the vertebral canal and muscles were visualized only in the transverse plane. This article provides a brief and relatively easy-to-perform protocol for ultrasound imaging of the lumbar spine of dogs. In addition, it presents a detailed description of the sonoanatomy of the area under investigation.

Two in One: Use of Divalent Manganese Ions as Both Cross-Linking and MRI Contrast Agent for Intrathecal Injection of Hydrogel-Embedded Stem Cells.

Cell therapy is a promising tool for treating central nervous system (CNS) disorders; though, the translational efforts are plagued by ineffective delivery methods. Due to the large contact surface with CNS and relatively easy access, the intrathecal route of administration is attractive in extensive or global diseases such as stroke or amyotrophic lateral sclerosis (ALS). However, the precision and efficacy of this approach are still a challenge. Hydrogels were introduced to minimize cell sedimentation and improve cell viability. At the same time, contrast agents were integrated to allow image-guided injection. Here, we report using manganese ions (Mn2+) as a dual agent for cross-linking alginate-based hydrogels and magnetic resonance imaging (MRI). We performed in vitro studies to test the Mn2+ alginate hydrogel formulations for biocompatibility, injectability, MRI signal retention time, and effect on cell viability. The selected formulation was injected intrathecally into pigs under MRI control. The biocompatibility test showed a lack of immune response, and cells suspended in the hydrogel showed greater viability than monolayer culture. Moreover, Mn2+-labeled hydrogel produced a strong T1 MRI signal, which enabled MRI-guided procedure. We confirmed the utility of Mn2+ alginate hydrogel as a carrier for cells in large animals and a contrast agent at the same time.

Usefulness of Imaging Techniques in the Diagnosis of Selected Injuries and Lesions of the Canine Tarsus. A Review.

Tarsus lesions are not common in dogs, but they can cause serious health problem. They can lead to permanent changes in the joint and, in dogs involved in canine sports, to exclusion from training. The most common diseases and injuries involving the tarsal joint are osteochondrosis, fractures and ruptures of the Achilles tendon. These conditions can be diagnosed primarily through accurate orthopedic examination, but even this may be insufficient for performing a proper diagnosis. Imaging modalities such as radiography, ultrasonography, magnetic resonance imaging or computed tomography can facilitate the detection and assessment of lesions in the canine tarsal joint. This review paper briefly presents some characteristics of the above-mentioned imaging techniques, offering a comparison of their utility in the diagnosis of lesions and injuries involving the canine tarsus.

Transcriptomic Profiling of Femoral Veins in Deep Vein Thrombosis in a Porcine Model.

Deep vein thrombosis (DVT) is a severe disease affecting the human venous system, accompanied by high morbidity and mortality rates caused by early and late complications. The study aimed at analyzing the changes in the transcriptome of the femoral vein caused by DVT in the porcine model based on the formation of the thrombus in vivo. The study was performed on 11 castrated male pigs: A thrombus was formed in each left femoral vein in six animals; the remaining five served as a control group. Total RNA was isolated from the left femoral veins of the experimental and control animals. High-throughput RNA sequencing was used to analyze the global changes in the transcriptome of veins with induced DVT. Applied multistep bioinformatics revealed 1474 differentially expressed genes (DEGs): 1019 upregulated and 455 downregulated. Functional Gene Ontology annotated 1220 of DEGs into 225 biological processes, 30 molecular functions and 40 cellular components categories. KEGG analysis disclosed TNF, NF-κB and apoptosis pathways' overexpression in DVT samples. A thorough analysis of the detected DEGs indicated that a dysregulated inflammatory response and disturbed balance between clotting and anti-clotting factors play a crucial role in the process of DVT.

The Effect of Horse Shoeing with Egg Bar Shoes and Shoes with Wedge Pads on the Results of Thermal Imaging of the Equine Distal Limb.

The presented manuscript provides reference for practitioners when measuring normal hoof temperature, as well as controlling the temperature after shoeing with particular shoes. The aim of this study was to determine the effect of horse shoeing with egg bar shoes and shoes with wedge pads on hoof temperature measured by thermography. This was a prospective study conducted on 16 horses. The horses were divided into two groups: horses from group 1 were shod with egg bar shoes, while horses from group 2 were shod with shoes with wedge pads. Thermographic examination was performed below the metacarpophalangeal joint before and one month after shoeing. After shoeing with egg bar shoes, there was a decrease in the median of the minimal temperature in the palmar view. After shoeing with wedge pads, thermography revealed decreased hoof temperature in the dorsal and palmar views. Horse shoes may have a negative impact on the blood circulation and metabolism within the distal part of the limb; however, our study found this only to a minor extent.

A New Experimental Porcine Model of Venous Thromboembolism.

Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a severe disease affecting the human venous system, accompanied by high morbidity and mortality rates. The aim of the study was to establish a new porcine VTE model based on the formation of the thrombus in vivo. The study was performed on 10 castrated male pigs: thrombus was formed in each closed femoral vein and then successfully released from the right femoral vein into the circulation of animals. In six pigs PE was confirmed via both computed tomography pulmonary angiography and an autopsy. Our research presents a novel experimental porcine model of VTE that involves inducing DVT and PE in the same animal in vivo, making it suitable for advanced clinical research and testing of future therapies.

Intra-arterial transplantation of stem cells in large animals as a minimally-invasive strategy for the treatment of disseminated neurodegeneration.

Stem cell transplantation proved promising in animal models of neurological diseases; however, in conditions with disseminated pathology such as ALS, delivery of cells and their broad distribution is challenging. To address this problem, we explored intra-arterial (IA) delivery route, of stem cells. The goal of this study was to investigate the feasibility and safety of MRI-guided transplantation of glial restricted precursors (GRPs) and mesenchymal stem cells (MSCs) in dogs suffering from ALS-like disease, degenerative myelopathy (DM). Canine GRP transplantation in dogs resulted in rather poor retention in the brain, so MSCs were used in subsequent experiments. To evaluate the safety of MSC intraarterial transplantation, naïve pigs (n = 3) were used as a pre-treatment control before transplantation in dogs. Cells were labeled with iron oxide nanoparticles. For IA transplantation a 1.2-French microcatheter was advanced into the middle cerebral artery under roadmap guidance. Then, the cells were transplanted under real-time MRI with the acquisition of dynamic T2*-weighted images. The procedure in pigs has proven to be safe and histopathology has demonstrated the successful and predictable placement of transplanted porcine MSCs. Transplantation of canine MSCs in DM dogs resulted in their accumulation in the brain. Interventional and follow-up MRI proved the procedure was feasible and safe. Analysis of gene expression after transplantation revealed a reduction of inflammatory factors, which may indicate a promising therapeutic strategy in the treatment of neurodegenerative diseases.

The Influence of Haemostatic Dressing Prototypes for the Emergency Services on the Histopathological Parameters of Porcine Muscle.

BACKGROUND/AIM: Haemostatic dressings for the uniformed and rescue services are an integral part of life-saving equipment for controlling post-traumatic haemorrhage. The aim of this study was to assess the influence of active constituent substances and materials of haemostatic dressings on muscle tissue and muscle regeneration after traumatic injury. MATERIALS AND METHODS: Three hemostatic dressing prototypes were analysed: OBR/G/S sponge: dressing material sponge made of Na-Ca chitosan/algal composite microfibers and nanofibers; OBR/MBT/S: tactic gauze modified with a polymer mixture of Na-Ca chitosan/algal composite microfibers and nanofibers, impregnated with a moderate amount of procoagulants (22.9 g/m2); and OBR/MS/S: seton gauze modified with a polymer mixture of Na-Ca chitosan/algal composite microfibers and nanofibers, impregnated with a moderate amount of procoagulants (18.0 g/m2), with chitosan (ChitoClearhqg 95) and sodium alginate (Protanal LF10/60 FT) as the coagulants. The experiment was conducted on 20 pigs which were euthanised 24 h, 7 or 14 days after wound dressing. Samples of porcine muscle tissue were subjected to qualitative histopathological analysis. RESULTS: Histopathological analysis of muscle tissues from the experimental pigs revealed that the application of modified seton (OBR/MS/S) produced the most satisfactory results. The observed changes were similar on all dates that samples were collected and in all experimental groups, and minor differences in their extent were observed between groups. Regenerative processes were most advanced, and retrograde changes were least apparent in animals treated with OBR/MS/S. CONCLUSION: Modified seton (OBR/MS/S) induced the least tissue reaction and was most effective in promoting tissue regeneration after injury.

The Effect of Haemostatic Dressing Prototypes for the Emergency Services in the Porcine Haemostatic System.

BACKGROUND/AIM: Coagulopathy can develop when hemostatic dressings are used to stop massive bleeding, even in patients without prior history of clotting disorders. The selection of procoagulants, which effectively control bleeding and prevent disseminated intravascular coagulation (DIC) and thrombosis, is a significant challenge. The aim of this study was to evaluate the effect of two prototypes of haemostatic dressing in the porcine haemostatic system. MATERIALS AND METHODS: The total number of animals used in our experiments was 24. Group I: pigs were treated with the developed prototype of sponge dressing, made of Na-Ca chitosan/algal composite of microfibers and nanofibers. Group II: animals were treated with a seton gauze modified with a polymer mixture of Na-Ca chitosan/algal composite of microfibers and nanofibers. Group III: animals were treated with non-hemostatic dressing and this group was the control. Blood was sampled five times to determine changes in the coagulation and fibrinolytic profiles: before injury: i) at 1 h, ii) at 24 h, iii) at 7, and iv) at 14 days following injury. RESULTS: Significant changes were observed in the coagulation parameters, in the total numbers of white blood cells and platelets in groups I and II, compared to controls. CONCLUSION: The modified haemostatic dressings used in this study produced a strong procoagulant effect in pigs. This, together with high fibrinogen concentrations, which can cause DIC, require further studying.

Evaluation of regenerative processes in the pig model of intervertebral disc degeneration after transplantation of bone marrow-derived mesenchymal stem cells.

INTRODUCTION: The pathophysiology of degenerative disc disease (DDD) is complex and not fully understood. While surgical treatment and appropriate rehabilitation offer relief of acute symptoms, there is a need to find tissue engineering strategies for intervertebral disc repair to restore healthy higher and histological structure. The purpose of this study was to estimate the survival rate of transplanted cells and their post-delivery integration level at the damage site. MATERIAL AND METHODS: We used an in vivo porcine model to investigate autogenic bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation for intervertebral disc repair. In our experiment we used a large animal model of DDD induced by percutaneous laser light deliveries. The percutaneous approach has also been used for delivery of BM-MSCs into the intervertebral disc space. RESULTS: After MSC transplantation, we observed a deceleration of the degenerative process in the intervertebral disc, relative to degenerative discs without MSC transplantation. CONCLUSIONS: By using a large animal model that mimicked the development of intervertebral degenerative disc disease, the present results are indicative of the clinical feasibility of this procedure.

MRI-guided intrathecal transplantation of hydrogel-embedded glial progenitors in large animals.

Disseminated diseases of the central nervous system such as amyotrophic lateral sclerosis (ALS) require that therapeutic agents are delivered and distributed broadly. Intrathecal route is attractive in that respect, but to date there was no methodology available allowing for optimization of this technique to assure safety and efficacy in a clinically relevant setting. Here, we report on interventional, MRI-guided approach for delivery of hydrogel-embedded glial progenitor cells facilitating cell placement over extended surface of the spinal cord in pigs and in naturally occurring ALS-like disease in dogs. Glial progenitors used as therapeutic agent were embedded in injectable hyaluronic acid-based hydrogel to support their survival and prevent sedimentation or removal. Intrathecal space was reached through lumbar puncture and the catheter was advanced under X-ray guidance to the cervical part of the spine. Animals were then transferred to MRI suite for MRI-guided injection. Interventional and follow-up MRI as well as histopathology demonstrated successful and predictable placement of embedded cells and safety of the procedure.

MRI-guided intracerebral convection-enhanced injection of gliotoxins to induce focal demyelination in swine.

Demyelinating disorders such as multiple sclerosis (MS) or transverse myelitis are devastating neurological conditions with no effective cure. Prevention of myelin loss or restoration of myelin are key for successful therapy. To investigate the disease and develop cures animal models with good clinical relevance are essential. The goal of the current study was to establish a model of focal demyelination in the brain of domestic pig using MRI-guided gliotoxin delivery. The rationale for developing a new myelin disease model in the domestic pig was based on the fact that the brain in pigs is anatomically and histologically much more similar to that of humans compared to the rodent brain. For MRI-assisted gliotoxin injection, eight 30 kg pigs were subjected to treatment with lysolecithin (20, 30 mg/ml); or with ethidium bromide (0.0125, 0.05, 0.2 mg/ml). Animals were placed in an MRI scanner for intraparenchymal targeting of gliotoxin into the corona radiata (250 µl over 1h), with real-time monitoring of toxin distribution on T1 scans and monitoring of lesion evolution over seven days using both T1 and T2 scans. After the last MRI, animals were transcardially perfused and brains were processed for histological and immunofluorescent analysis. Gadolinium-enhanced T1 MRI during injection demonstrated biodistribution of the contrast (as a surrogate marker for toxin distribution) and its diffusion through the brain parenchyma. Lesion induction was confirmed on T2-weighted MRI and histopathology, thus enabling the establishment of optimal doses of gliotoxins. To conclude, MRI-guided focal demyelination in swine is accurate and provides real-time confirmation of gliotoxin, thus facilitating placement of focal lesions with high precision. This new model of focal demyelination can be used for further investigation and development of novel therapeutic approaches.

The effect of hemostatic dressing prototypes for the uniformed services on selected blood coagulation parameters in pigs.

BACKGROUND: Serious injuries accompanied by severe bleeding are life-threatening. Post-traumatic hemorrhage involves the risk of developing coagulopathy. Hemostatic dressings are widely used to minimize bleeding. The application of procoagulants in control of hemorrhage may lead to thrombosis or disseminated intravascular coagulation. The aim of this study was to evaluate the effect of hemostatic dressing prototypes on the porcine coagulation system. RESULTS: Fibrinogen and D-dimer concentrations were significantly higher in the experimental groups where hemostatic dressings were used in comparison with the control group. Considerable differences in antithrombin III activity and thrombin-antithrombin complex concentrations were also observed between groups. CONCLUSIONS: The hemostatic dressing comprising modified seton impregnated with 18.0 g/m2 of procoagulant was most effective in preserving the physiological equilibrium between fibrinogenesis and fibrinolysis.

Real-time MRI for precise and predictable intra-arterial stem cell delivery to the central nervous system.

Stem cell therapy for neurological disorders reached a pivotal point when the efficacy of several cell types was demonstrated in small animal models. Translation of stem cell therapy is contingent upon overcoming the challenge of effective cell delivery to the human brain, which has a volume ∼1000 times larger than that of the mouse. Intra-arterial injection can achieve a broad, global, but also on-demand spatially targeted biodistribution; however, its utility has been limited by unpredictable cell destination and homing as dictated by the vascular territory, as well as by safety concerns. We show here that high-speed MRI can be used to visualize the intravascular distribution of a superparamagnetic iron oxide contrast agent and can thus be used to accurately predict the distribution of intra-arterial administered stem cells. Moreover, high-speed MRI enables the real-time visualization of cell homing, providing the opportunity for immediate intervention in the case of undesired biodistribution.

MR monitoring of minimally invasive delivery of mesenchymal stem cells into the porcine intervertebral disc.

PURPOSE: Bone marrow stem cell therapy is a new, attractive therapeutic approach for treatment of intervertebral disc (IVD) degeneration; however, leakage and backflow of transplanted cells into the structures surrounding the disc may lead to the formation of undesirable osteophytes. The purpose of this study was to develop a technique for minimally invasive and accurate delivery of stem cells. METHODS: Porcine mesenchymal stem cells (MSCs) were labeled with superparamagnetic iron oxide nanoparticles (SPIO, Molday ION rhodamine) and first injected into the explanted swine lumbar IVD, followed by ex vivo 3T MRI. After having determined sufficient sensitivity, IVD degeneration was then induced in swine (n=3) by laser-evaporation. 3 x 10(6) SPIO-labeled cells embedded within hydrogel were injected in 2 doses using a transcutaneous cannula and an epidural anesthesia catheter. T2-weighted MR images were obtained at 3T before and immediately after cell infusion. Two weeks after injection, histological examination was performed for detection of transplanted cells. RESULTS: MSCs were efficiently labeled with Molday ION rhodamine. Cells could be readily detected in the injected vertebral tissue explants as distinct hypointensities with sufficient sensitivity. MR monitoring indicated that the MSCs were successfully delivered into the IVD in vivo, which was confirmed by iron-positive Prussian Blue staining of the tissue within the IVD. CONCLUSION: We have developed a technique for non-invasive monitoring of minimally invasive stem delivery into the IVD at 3T. By using a large animal model mimicking the anatomy of IVD in humans, the present results indicate that this procedure may be clinically feasible.