Impact of Canine Amniotic Mesenchymal Stem Cell Conditioned Media on the Wound Healing Process: In Vitro and In Vivo Study.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco's Modified Eagle's Medium-32.55%) and the positive control (Dulbecco's Modified Eagle's Medium supplemented with FGF2, N2, B27, and EGF-82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

A Chitosan-Based Biomaterial Combined with Mesenchymal Stem Cell-Conditioned Medium for Wound Healing and Skin Regeneration.

The aim of this study was to provide a beneficial treatment effect of novel chitosan bio-polymeric material enriched with mesenchymal stem cell products derived from the canine adipose tissue (AT-MSC) on the artificial skin defect in a rabbit model. For the objectivity of the regeneration evaluation, we used histological analysis and a scoring system created by us, taking into account all the attributes of regeneration, such as inflammatory reaction, necrosis, granulation, formation of individual skin layers and hair follicles. We observed an acceleration and improvement in the healing of an artificially created skin defect after eight and ten weeks in comparison with negative control (spontaneous healing without biomaterial). Moreover, we were able to described hair follicles and epidermis layer in histological skin samples treated with a chitosan-based biomaterial on the eighth week after grafting.

Tetracalcium Phosphate Biocement Hardened with a Mixture of Phytic Acid-Phytase in the Healing Process of Osteochondral Defects in Sheep.

Hyaline articular cartilage has unique physiological, biological, and biomechanical properties with very limited self-healing ability, which makes the process of cartilage regeneration extremely difficult. Therefore, research is currently focused on finding new and potentially better treatment options. The main objective of this in vivo study was to evaluate a novel biocement CX consisting of tetracalcium phosphate-monetit biocement hardened with a phytic acid-phytase mixture for the regeneration of osteochondral defects in sheep. The results were compared with tetracalcium phosphate-monetit biocement with classic fast-setting cement systems and untreated defects. After 6 months, the animals were sacrificed, and the samples were evaluated using macroscopic and histologic methods as well as X-ray, CT, and MR-imaging techniques. In contrast to the formation of fibrous or fibrocartilaginous tissue on the untreated side, treatment with biocements resulted in the formation of tissue with a dominant hyaline cartilage structure, although fine fibres were present (p < 0.001). There were no signs of pathomorphological changes or inflammation. Continuous formation of subchondral bone and hyaline cartilage layers was present even though residual biocement was observed in the trabecular bone. We consider biocement CX to be highly biocompatible and suitable for the treatment of osteochondral defects.

Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

Impact of mesenchymal stem cells derived conditioned media on neural progenitor cells.

Neurodegenerative diseases are common problem for companion animals. Due to the limited ability of injured axons to regenerate, innovative therapies combined with rehabilitation have been applied and evaluated. Among them, stem cells and their conditioned media implantation, which can ameliorate damaged tissue has been suggested as a promising treatment strategy. The main goal of our study was to characterize mesenchymal stem cells (MSC) derived from canine adipose tissue (AT-MSC) and umbilical cord (UC-MSC) and analyse effect of their conditioned media (CM) on neurite outgrowth of neural progenitor cells isolated from the brain cortex of neonatal rats. MSC from both sources showed high osteogenic and chondrogenic potential and expression of CD90 and CD29. Furthermore, both UC-MSCCM and AT-MSCCM stimulated neurite growth. Interestingly, this effect was more pronounced with UC-MSCCM when compared to AT-MSCCM in vitro, which may be related to the different content of neurotrophic factors included in the CM.

Spinal Cord Injury: Animal Models, Imaging Tools and the Treatment Strategies.

Spinal cord injury (SCI) often leads to irreversible neuro-degenerative changes with life-long consequences. While there is still no effective therapy available, the results of past research have led to improved quality of life for patients suffering from partial or permanent paralysis. In this review we focus on the need, importance and the scientific value of experimental animal models simulating SCI in humans. Furthermore, we highlight modern imaging tools determining the location and extent of spinal cord damage and their contribution to early diagnosis and selection of appropriate treatment. Finally, we focus on available cellular and acellular therapies and novel combinatory approaches with exosomes and active biomaterials. Here we discuss the efficacy and limitations of adult mesenchymal stem cells which can be derived from bone marrow, adipose tissue or umbilical cord blood and its Wharton's jelly. Special attention is paid to stem cell-derived exosomes and smart biomaterials due to their special properties as a delivery system for proteins, bioactive molecules or even genetic material.

Stem Cell Conditioned Medium Treatment for Canine Spinal Cord Injury: Pilot Feasibility Study.

Spinal cord injury (SCI) involves nerve damage and often leads to motor, sensory and autonomic dysfunctions. In the present study, we have designed a clinical protocol to assess the feasibility of systemic delivery of allogenic canine bone marrow tissue-derived mesenchymal stem cell conditioned medium (BMMSC CM) to dogs with SCI. Four client-owned dogs with chronic SCI lasting more than six months underwent neurological and clinical evaluation, MRI imaging and blood tests before being enrolled in this study. All dogs received four intravenous infusions with canine allogenic BMMSC CM within one month. Between the infusions the dogs received comprehensive physiotherapy, which continued for three additional months. No adverse effects or complications were observed during the one, three and six months follow-up periods. Neither blood chemistry panel nor hematology profile showed any significant changes. All dogs were clinically improved as assessed using Olby locomotor scales after one, three and six months of BMMSC CM treatment. Furthermore, goniometric measurements revealed partial improvement in the range of joint motion. Bladder function improved in two disabled dogs. We conclude that multiple delivery of allogenic cell-derived conditioned medium to dogs with chronic SCI is feasible, and it might be clinically beneficial in combination with physiotherapy.

Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury.

It was recently shown that the conditioned medium (CM) of mesenchymal stem cells can enhance viability of neural and glial cell populations. In the present study, we have investigated a cell-free approach via CM from rat bone marrow stromal cells (MScCM) applied intrathecally (IT) for spinal cord injury (SCI) recovery in adult rats. Functional in vitro test on dorsal root ganglion (DRG) primary cultures confirmed biological properties of collected MScCM for production of neurosphere-like structures and axon outgrowth. Afterwards, rats underwent SCI and were treated with IT delivery of MScCM or vehicle at postsurgical Days 1, 5, 9, and 13, and left to survive 10 weeks. Rats that received MScCM showed significantly higher motor function recovery, increase in spared spinal cord tissue, enhanced GAP-43 expression and attenuated inflammation in comparison with vehicle-treated rats. Spared tissue around the lesion site was infiltrated with GAP-43-labeled axons at four weeks that gradually decreased at 10 weeks. Finally, a cytokine array performed on spinal cord extracts after MScCM treatment revealed decreased levels of IL-2, IL-6 and TNFα when compared to vehicle group. In conclusion, our results suggest that molecular cocktail found in MScCM is favorable for final neuroregeneration after SCI.

Two Amnion-Derived Mesenchymal Stem-Cells Injections to Osteoarthritic Elbows in Dogs-Pilot Study.

The aim of the study was to investigate the potential of cell-based regenerative therapy for elbow joints affected by osteoarthritis. Interest was focused on two intra-articular applications of amnion-derived mesenchymal stem cells (A-MSCs) to a group of different breeds of dogs with elbow osteoarthritis (13 joints). Two injections were performed 14 days apart. We evaluated synovial fluid biomarkers, such as IFN-γ, IL-6, IL-15, IL-10, MCP-1, TNF-α, and GM-CSF, by multiplex fluorescent micro-bead immunoassay in the treated group of elbows (n = 13) (day 0, day 14, and day 28) and in the control group of elbows (n = 9). Kinematic gait analysis determined the joint range of motion (ROM) before and after each A-MSCs application. Kinematic gait analysis was performed on day 0, day 14, and day 28. Kinematic gait analysis pointed out improvement in the average range of motion of elbow joints from day 0 (38.45 ± 5.74°), day 14 (41.7 ± 6.04°), and day 28 (44.78 ± 4.69°) with statistical significance (p < 0.05) in nine elbows. Correlation analyses proved statistical significance (p < 0.05) in associations between ROM (day 0, day 14, and day 28) and IFN-γ, IL-6, IL-15, MCP-1, TNF-α, and GM-CSF concentrations (day 0, day 14, and day 28). IFN-γ, IL-6, IL-15, MCP-1, GM-CSF, and TNF- α showed negative correlation with ROM at day 0, day 14, and day 28, while IL-10 demonstrated positive correlation with ROM. As a consequence of A-MSC application to the elbow joint, we detected a statistically significant (p < 0.05) decrease in concentration levels between day 0 and day 28 for IFN-γ, IL-6, and TNF-α and statistically significant increase for IL-10. Statistical significance (p < 0.05) was detected in TNF-α, IFN-γ, and GM-CSF concentrations between day 14 and the control group as well as at day 28 and the control group. IL-6 concentrations showed statistical significance (p < 0.05) between day 14 and the control group.

The role of primed and non-primed MSC-derived conditioned media in neuroregeneration.

Introduction: With growing significance in nervous system repair, mesenchymal stem cell-derived conditioned media (MSCCM) have been used in cell-free therapies in regenerative medicine. However, the immunomodulatory and neuroregenerative effects of MSCCM and the influence of priming on these effects are still poorly understood. Methods: In this study, by various methods focused on cell viability, proliferation, neuron-like differentiation, neurite outgrowth, cell migration and regrowth, we demonstrated that MSCCM derived from adipose tissue (AT-MSCCM) and amniotic membrane (AM-MSCCM) had different effects on SH-SY5Y cells. Results and discussion: AT-MSCCM was found to have a higher proliferative capacity and the ability to impact neurite outgrowth during differentiation, while AM-MSCCM showed more pronounced immunomodulatory activity, migration, and re-growth of SH-SY5Y cells in the scratch model. Furthermore, priming of MSC with pro-inflammatory cytokine (IFN-γ) resulted in different proteomic profiles of conditioned media from both sources, which had the highest effect on SH-SY5Y proliferation and neurite outgrowth in terms of the length of neurites (pAT-MSCCM) compared to the control group (DMEM). Altogether, our results highlight the potential of primed and non-primed MSCCM as a therapeutic tool for neurodegenerative diseases, although some differences must be considered.

Non-Exosomal and Exosome-Derived miRNAs as Promising Biomarkers in Canine Mammary Cancer.

Canine mammary cancer (CMC), similar to human breast cancer (HBC) in many aspects, is the most common neoplasm associated with significant mortality in female dogs. Due to the limited therapy options, biomarkers are highly desirable for early clinical diagnosis or cancer progression monitoring. Since the discovery of microRNAs (miRNAs or miRs) as post-transcriptional gene regulators, they have become attractive biomarkers in oncological research. Except for intracellular miRNAs and cell-free miRNAs, exosome-derived miRNAs (exomiRs) have drawn much attention in recent years as biomarkers for cancer detection. Analysis of exosomes represents a non-invasive, pain-free, time- and money-saving alternative to conventional tissue biopsy. The purpose of this review is to provide a summary of miRNAs that come from non-exosomal sources (canine mammary tumor, mammary tumor cell lines or canine blood serum) and from exosomes as promising biomarkers of CMC based on the current literature. As is discussed, some of the miRNAs postulated as diagnostic or prognostic biomarkers in CMC were also altered in HBC (such as miR-21, miR-29b, miR-141, miR-429, miR-200c, miR-497, miR-210, miR-96, miR-18a, miR19b, miR-20b, miR-93, miR-101, miR-105a, miR-130a, miR-200c, miR-340, miR-486), which may be considered as potential disease-specific biomarkers in both CMC and HBC.

A Comparative Study of Canine Mesenchymal Stem Cells Isolated from Different Sources.

In this study, we provide comprehensive analyses of mesenchymal stem cells (MSCs) isolated from three types of canine tissues: bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and amniotic tissue (AM-MSCs). We compare their morphology, phenotype, multilineage potential and proliferation activity. The BM-MSCs and AM-MSCs showed fibroblast-like shapes against the spindle shape of the AT-MSCs. All populations showed strong osteogenic and chondrogenic potential. However, we observed phenotypic differences. The BM-MSCs and AT-MSCs revealed high expression of CD29, CD44, CD90 and CD105 positivity compared to the AM-MSCs, which showed reduced expression of all the analysed CD markers. Similarly, the isolation yield and proliferation varied depending on the source. The highest isolation yield and proliferation were detected in the population of AT-MSCs, while the AM-MSCs showed a high yield of cells, but the lowest proliferation activity, in contrast to the BM-MSCs which had the lowest isolation yield. Thus, the present data provide assumptions for obtaining a homogeneous MSC derived from all three canine tissues for possible applications in veterinary regenerative medicine, while the origin of isolated MSCs must always be taken into account.

Canine Bone Marrow Mesenchymal Stem Cell Conditioned Media Affect Bacterial Growth, Biofilm-Associated Staphylococcus aureus and AHL-Dependent Quorum Sensing.

The present study investigated the in vitro antibacterial, antibiofilm and anti-Quorum Sensing (anti-QS) activities of canine bone marrow mesenchymal stem cell-conditioned media (cBM MSC CM) containing all secreted factors <30 K, using a disc diffusion test (DDT), spectrophotometric Crystal Violet Assay (SCVA) and Bioluminescence Assay (BA) with QS-reporter Escherichia coli JM109 pSB1142. The results show a sample-specific bacterial growth inhibition (zones varied between 7-30 mm), statistically significant modulation of biofilm-associated Staphylococcus aureus and Escherichia coli bioluminescence (0.391 ± 0.062 in the positive control to the lowest 0.150 ± 0.096 in the experimental group, cf. 11,714 ± 1362 to 7753 ± 700, given as average values of absorbance A550 ± SD versus average values of relative light units to growth RLU/A550 ± SD). The proteomic analysis performed in our previous experiment revealed the presence of several substances with documented antibacterial, antibiofilm and immunomodulatory properties (namely, apolipoprotein B and D; amyloid-ß peptide; cathepsin B; protein S100-A4, galectin 3, CLEC3A, granulin, transferrin). This study highlights that cBM MSC CM may represent an important new approach to managing biofilm-associated and QS signal molecule-dependent bacterial infections. To the best of our knowledge, there is no previous documentation of canine BM MSC CM associated with in vitro antibiofilm and anti-QS activity.

Novel Diagnostic Tools for Identifying Cognitive Impairment in Dogs: Behavior, Biomarkers, and Pathology.

Canine cognitive dysfunction syndrome (CCDS) is a progressive neurodegenerative disorder in senior dogs that is mainly associated with decreased ability to learn and respond to stimuli. It is commonly under-diagnosed because behavioral changes are often attributed to the natural process of aging. In the present study, we used for the first time a comprehensive approach enabling early diagnosis of canine patients with mild cognitive disorders (MiCI). We included CAnine DEmentia Scale (CADES) questionnaires, biochemical parameters, and biomarkers in blood serum, and correlated them with post-mortem histopathological changes. The CADES questionnaires enabled us to identify MiCI dogs developing changes mainly in domains corresponding to social interaction and spatial orientation, which seems to be crucial for delineating early cognitive disorders. Biochemical analyses in these dogs showed slightly elevated liver enzyme parameters (AST and ALT) and significantly decreased sodium and chloride levels in blood serum. Furthermore, we describe for the first time a significant increase of neurofilament light chain (NFL) in blood serum of MiCI dogs, compared to normal aging seniors and young controls, but no changes in TAU protein and amyloid-ß (Aß42) peptide levels. In canine brains with cognitive impairment, amyloid plaques of mainly diffuse and dense types were detected. Furthermore, activated microglia with amoeboid body and dystrophic processes occurred, in some cases with spheroidal and bulbous swellings. On the other hand, no TAU pathology or neurofibrillary tangles were detected. These results suggest that a combination of CADES questionnaire mainly with CNS injury biomarker (NFL) and with biochemical parameters (ALT, AST, Na, and Cl) in blood serum may predict CCDS in senior dogs.