Canine Bone Marrow Research Articles

Effects of fixation and demineralization on histomorphology and DNA amplification of canine bone marrow.

Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of T cell receptor gamma and immunoglobulin heavy chain genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article.

Abstract 1772: A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified

Abstract Despite being the most common subtype of human peripheral T-cell lymphoma (PTCL), PTCL-not otherwise specified (PTCL-NOS) remains a poorly understood diagnosis of exclusion with poor survival times and treatment responses. Canine PTCL clinically and immunophenotypically resembles human PTCL-NOS, and PTCL is more common in dogs than humans, leading to their interest as a naturally occurring preclinical model. Canine models of cancer offer several unique advantages: in contrast to traditional rodent models, these are spontaneous tumors in genetically diverse and immunocompetent animals living in a shared environment with humans, but relative to their human counterparts, they offer accelerated lifespans with shorter clinical courses of disease. Here, we analyzed the gene expression profile of 96 canine CD4+ PTCLs to determine similarities to human PTCL-NOS and investigate a possible cell of origin. Bulk RNA-seq was performed on lymph node aspirates of 96 cases of canine CD4+ PTCL diagnosed by flow cytometry and sorted CD4+ nodal lymphocytes and CD4+ thymocytes from healthy control dogs. Raw reads were aligned to the Ensembl canine reference genome (ROS_Cfam_1.0) and tabulated. Data normalization and differential expression analysis was conducted with DESeq2, and differentially expressed genes were compared to human PTCL-NOS and various stages of T-cell development via gene set enrichment analyses (GSEA). Global gene expression in canine CD4+ PTCL was analogous to that of human PTCL-NOS (NES = 2.3, p < 0.001). Additionally, canine CD4+ PTCL had increased expression of GATA3 (log2fc = 1.9, padj = 6.5e-07) and was enriched for gene signatures associated with downregulation of PTEN (NES = 1.7, p = 0.0001) and upregulation of PI3K/AKT/mTOR signaling (NES = 1.5, padj = 0.0007), resembling the more aggressive human GATA3-PTCL subtype. Canine CD4+ PTCL was also enriched for human and murine gene signatures associated with early thymocyte progenitor cells and had increased expression of genes of immaturity, including CD34 (log2fc = 7.8, padj = 2.6e-14), KIT (log2fc = 3.3, padj = 7.7e-05), and CCR9 (log2fc = 4.8, padj = 3.9e-11), although surface CD34 expression is not detected by flow cytometry. These findings were confirmed when we compared the canine PTCL gene expression programs to a canine single cell transcriptomic atlas of hematopoietic precursors and T cells across canine bone marrow, thymic, and lymph node tissues. Canine CD4+ PTCL cells were significantly (padj < 0.05) enriched for gene signatures associated with early thymic and bone marrow precursors (NES 1.3-2.1), and negatively enriched for gene signatures associated with naïve nodal CD4+ T cells (NES = -3.2, padj = 0.006). In conclusion, the gene expression profile of canine CD4+ PTCL resembles human PTCL-NOS, supporting its potential as a naturally occurring preclinical model, and a subset of these neoplasms may arise from a thymic precursor cell of origin. Citation Format: Eileen Owens, Adam Harris, Anne Avery. A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1772.

Silencing of transient receptor potential canonical channel 4 inhibits endothelial progenitor cell angiogenesis by suppressing VEGF and SDF-1.

Endothelial progenitor cells (EPCs) play a crucial role in acquired angiogenesis and endothelial injury repair. Transient receptor potential canonical channel 4 (TRPC4), a key component of store-operated calcium channels, is essential for EPC function. While the role of TRPCs has been clarified in vascular diseases, the relationship between TRPC4 and EPC function, along with the underlying molecular mechanisms, remains unclear and requires further elucidation. EPCs were isolated from canine bone marrow and identified by morphology and flow cytometry. TRPC4 was transfected into EPCs using lentivirus or negative control, and its expression was assessed using real-time polymerase chain reaction (RT-PCR). Proliferation, migration, and tube formation were evaluated using Cell Counting Kit-8 (CCK-8), Transwell, and Matrigel assays, respectively. Levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were measured using enzyme-linked immunosorbent assay (ELISA). TRPC4 mRNA expression was significantly reduced in TRPC4-short hairpin RNA (shRNA) transfected EPCs compared to the normal control (NC)-shRNA groups. Migration and tube formation were significantly decreased after TRPC4 silencing, while proliferation showed no difference. Additionally, levels of SDF-1 and VEGF in EPCs were markedly reduced following TRPC4 silencing. TRPC4 plays a crucial role in regulating angiogenesis in EPCs. Silencing of TRPC4 can lead to decreased angiogenesis by inhibiting VEGF and SDF-1 expression, suggesting that TRPC4 knockdown might be a novel therapeutic strategy for vascular diseases.

Evaluation of safety and efficacy of the bone marrow mesenchymal stem cell and gelatin-nano-hydroxyapatite combination in canine femoral defect repair.

Femoral shaft fracture is a common bone trauma in dogs. The limitation of mesenchymal stem cells in bone defect applications is that the cell suspension cannot be fixed to the bone defect site. In the study, our objective was to substantiate the combined application of canine bone marrow mesenchymal stem cells (cBMSCs) and gelatin-nano-hydroxyapatite (Gel-nHAP) and evaluate its therapeutic effect on bone defect diseases in dogs. Experiments were performed to evaluate the following: (1) the porosity of Gel-nHAP; (2) the adhesion of cBMSCs to Gel-nHAP; and (3) the effect of Gel-nHAP on cBMSC proliferation. The efficacy and safety of the combination of cBMSC and Gel-nHAP in the repair of femoral shaft defects were evaluated in animal experiments. The results showed that Gel-nHAP supported the attachment of cBMSCs and exhibited good biocompatibility. In the animal bone defect repair experiment, significant cortical bone growth was observed in the Gel-nHAP group at week 8 (p < 0.05) and in the cBMSCs-Gel-nHAP group at week 4 (p < 0.01). We demonstrated that Gel-nHAP could promote the repair of bone defects, and the effect of cBMSC-Gel-nHAP on the repair of bone defects was profound.

Canine bone marrow peri-adipocyte cells could therapeutically benefit acute spinal cord injury through migration and secretion of hepatocyte growth factor to inflammatory milieu.

Spinal cord injury (SCI) is a common neurological disorder in dogs. A secondary injury that occurs in the acute phase causes expansion of inflammation, resulting in lesion extension and further loss of function. Mesenchymal stem cells (MSCs) have trophic effects and the ability to migrate toward injured tissues; therefore, MSC-based therapy is considered promising for the treatment of canine SCI. We recently reported that bone marrow peri-adipocyte cells (BM-PACs) can be obtained from canine bone marrow and have stem cell potential superior to that of conventional bone marrow MSCs (BMMSCs). However, their therapeutic potential for SCI have been still unknow. Here, we first evaluated the ability of BM-PACs to secrete hepatocyte growth factor (HGF) and their migration ability toward inflammatory milieu in vitro. BM-PACs can secrete HGF in response to pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-1β, and exhibit migration ability toward these cytokines. Next, BM-PACs were intravenously administered into nude mice with acute SCI to analyze the homing ability and therapeutic effects of HGF secreted by BM-PACs. BM-PACs homed to the injured spinal cord, where the HGF expression level increased 7 days after administration. Intravenous administration of BM-PACs induced functional recovery and pathological improvement, indicated by less demyelinating area, more preserved axons, and less glial scar formation compared with the mice only received vehicle. These findings suggest that the intravenous administration of BM-PACs can be a novel therapeutic intervention for acute canine SCI.

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

Hsp20 Promotes Endothelial Progenitor Cell Angiogenesis via Activation of PI3K/Akt Signaling Pathway under Hypoxia.

Mandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia. Mandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2) concentration. Hypoxic effects, caused by CoCl2, were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above. CD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1mM CoCl2, in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway. In summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

Local Administration of Metformin Improves Bone Microarchitecture and Biomechanical Properties During Ruptured Canine Achilles Tendon–Calcaneus Interface Healing

Background: Tendon-bone interface (TBI) healing is a clinical dilemma that is closely relevant to new bone formation and remodeling at the repair site. Previous studies showed that metformin is an osteogenic inducer for stem cells in vitro and capable of stimulating bone regeneration in vivo. Hypothesis: Metformin would be effective for promoting TBI healing by enhancing new bone formation and remodeling. Study Design: Controlled laboratory study. Methods: Canine bone marrow stem cells (BMSCs) were cultured with various concentrations of metformin (0, 10, 50, 100, 200 μM). The effect of metformin on the osteogenic differentiation of canine BMSCs was evaluated via alizarin red staining and osteogenic gene expression. Eighteen mature beagles were included in a bilateral Achilles tendon–calcaneus (ATC) interface injury model. The right interface was reattached via surgical repair only, while the left was surgically reattached after implanting a fibrin glue containing metformin. At postoperative week 4 or 8, the healing quality of the wounded ATC interfaces was evaluated. Results: In vitro experiments determined that metformin was an osteogenic inducer for canine BMSCs. In vivo experiments showed that the metformin-treated ATC interfaces were repaired with significantly greater failure load and stiffness than was the no-metformin control site (P < .05 for all). Micro–computed tomography analysis showed that the metformin-treated specimens presented significantly higher bone volume/total volume and trabecular thickness than did the no-metformin control specimens (P < .05 for all), as confirmed via hematoxylin and eosin staining. Immunohistochemical staining showed that significantly more osteocalcin-positive cells were located at the newly formed bones treated with metformin than at the no-metformin control site at week 4 (P < .05). Masson trichrome staining showed that significantly more oriented collagen fibers anchored into the newly formed bone of the metformin-treated site than the no-metformin control site (P < .05). Conclusion: Metformin induced the osteogenesis of canine BMSCs in vitro, and local administration of metformin provided an improvement of bone microarchitecture at the calcaneus as well as an increase in the tensile properties of the repaired ATC interfaces in canines. Clinical Relevance: Findings of the study indicate that local administration of metformin may be an effective strategy for TBI healing in clinic.

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow.

Hepcidin which is the crucial regulator of iron homeostasis, produced in the liver in response to anemia, hypoxia, or inflammation. Recent studies have suggested that hepcidin and iron metabolism are involved in osteoporosis by inhibiting osteoblast function and promoting osteoclastogenesis. Pentosan polysulfate (PPS) is a heparin analogue and promising novel therapeutic for osteoarthritis (OA). This study was undertaken to determine whether PPS inhibits hepcidin-facilitated osteoclast (OC) differentiation and iron overload. Canine (n = 3) bone marrow mononuclear cells were differentiated to OC by macrophage colony-stimulating factor and receptor-activator of nuclear factor kappaB ligand with the treatment of hepcidin1 (200, 400, 800, 1200 nmol/L) and PPS (1, 5, 10, 20, 40 μg/mL). Differentiation and function of OC were accessed using tartrate-resistant acid phosphate staining and bone resorption assay while monitoring ferroportin1 (FPN1) and iron concentration by immunocytochemistry. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9, nuclear factor of activated-T-cells cytoplasmic 1 and FPN1 was examined. Hepcidin1 showed significant enhancement of OC number at 800 nmol/L (p<0.01). PPS impeded hepcidin-facilitated OC at 1, 5 and 10 μg/mL and reduction of resorption pits at 5 and 10 μg/mL (p< 0.01). All OC specific genes were downregulated with PPS, specifically in significant manner with CTK at higher concentrations. However, heparin induced FPN1 internalization and degradation was inhibited at higher concentrations of PPS while restoring iron-releasing capability of OC. We demonstrate for the first time that PPS is a novel-inhibitor of hepcidin-facilitated OC formation/function which might be beneficial for treatment of OA and osteoporosis.

TGF-β1-overexpressing mesenchymal stem cells reciprocally regulate Th17/Treg cells by regulating the expression of IFN-γ.

Transforming growth factor (TGF)-β1 and mesenchymal stromal cells (MSCs) are two effective immunosuppressive agents for organ transplantation technology. This study aims to explore the molecular mechanism of TGF-β1-overexpressed MSCs on T cell immunosuppression. To achieve that, BM-MSCs were isolated from canine bone marrow, and their osteogenic differentiation and surface markers were detected. The TGF-β1 gene was transferred into lentivirus and modified MSCs (TGF-β1/MSCs) by lentivirus transfection. Furthermore, TGF-β1/MSCs were co-cultured with T cells to investigate their effect on differentiation and immune regulation. Results showed that TGF-β1/MSCs significantly downregulated the proportion of CD4+ CD8+ T cells in lymphocytes and significantly upregulated the proportion of CD4+ CD25+ T cells. Moreover, TGF-β1/MSCs significantly upregulated the expression of IL-10 in CD4+ T cells and downregulated the expression of IL-17A, IL-21, and IL-22. Meanwhile, interferon-γ (IFN-γ) neutralizing antibody blocked the effects of TGF-β1/MSCs on the differentiation inhibition of Th17. Overall, our results confirm the strong immunosuppressive effect of TGF-β1/MSCs in vitro and demonstrate that IFN-γ mediates the immunosuppressive effect of TGF-β1/MSC.

Curcumin Alleviates the Senescence of Canine Bone Marrow Mesenchymal Stem Cells during In Vitro Expansion by Activating the Autophagy Pathway.

Senescence in mesenchymal stem cells (MSCs) not only hinders the application of MSCs in regenerative medicine but is also closely correlated with biological aging and the development of degenerative diseases. In this study, we investigated the anti-aging effects of curcumin (Cur) on canine bone marrow-derived MSCs (cBMSCs), and further elucidated the potential mechanism of action based on the modulation of autophagy. cBMSCs were expanded in vitro with standard procedures to construct a cell model of premature senescence. Our evidence indicates that compared with the third passage of cBMSCs, many typical senescence-associated phenotypes were observed in the sixth passage of cBMSCs. Cur treatment can improve cBMSC survival and retard cBMSC senescence according to observations that Cur (1 μM) treatment can improve the colony-forming unit-fibroblasts (CFU-Fs) efficiency and upregulated the mRNA expression of pluripotent transcription factors (SOX-2 and Nanog), as well as inhibiting the senescence-associated beta-galactosidase (SA-β-gal) activities and mRNA expression of the senescence-related markers (p16 and p21) and pro-inflammatory molecules (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)). Furthermore, Cur (0.1 μM~10 μM) was observed to increase autophagic activity, as identified by upregulation of microtubule-associated protein 1 light chain 3 (LC3), unc51-like autophagy-activating kinase-1 (ULK1), autophagy-related gene (Atg) 7 and Atg12, and the generation of type II of light chain 3 (LC3-II), thereby increasing autophagic vacuoles and acidic vesicular organelles, as well as causing a significant decrease in the p62 protein level. Moreover, the autophagy activator rapamycin (RAP) and Cur were found to partially ameliorate the senescent features of cBMSCs, while the autophagy inhibitor 3-methyladenine (3-MA) was shown to aggravate cBMSCs senescence and Cur treatment was able to restore the suppressed autophagy and counteract 3-MA-induced cBMSC senescence. Hence, our study highlights the important role of Cur-induced autophagy and its effects for ameliorating cBMSC senescence and provides new insight for delaying senescence and improving the therapeutic potential of MSCs.

Transdifferentiation of canine mesenchymal stem cells into neuron-like cells by induction with β-mercaptoethanol

The objective of this study was to check whether β- mercaptoethanol in a culture medium can induce the neuronal differentiation of canine MSCs. The canine bonemarrow derived MSCs were first pre-inducted with 1 mM BME for 24 hrs followed by induction in a serum-free medium supplemented with 4 mM BME without FBS for another 6 days. Morphological changes in MSCs from spindle-shaped to neuron-like branching from the edges of the cells were noticed at the end of induction. These neuronlike cells were found positive for the immunophenotypic expression of different neural cell markers β-tubulin III, MAP-2 and Nestin. In RT-PCR analysis, it was also evident that the relative expressions of these representative genes were significantly higher in the differentiated cells. On the basis of our observations, it can be summarized that the BME induction of canine MSCs resulted in morphological changes that resembled neuron-like cells which were found to express the representative neuronal markers. Therefore, inducing canine MSCs with BME resulted in the generation of neuron-like cells that might be utilized for the prospective therapeutic applications in veterinary medicine.

Protective effect of MitoQ on oxidative stress-mediated senescence of canine bone marrow mesenchymal stem cells via activation of the Nrf2/ARE pathway.

The destruction of biological activity such as senescence and apoptosis caused by oxidative stress could play a pivotal role in the poor therapeutic efficiency of bone marrow mesenchymal stem cells (BMSCs) transplantation. Mitoquinone (MitoQ) has a highly effective mitochondrial antioxidant effect, and has been widely used in many oxidative damage models. This study aimed to investigate the protective effect of MitoQ on the oxidative stress-mediated senescence of canine BMSCs and the underlying mechanism. The senescence of BMSCs was determined by senescence-associated β-galactosidase staining and quantitative real-time PCR. The expression of p-Nrf2 protein was detected by Western blotting. The results demonstrated that, as BMSCs were expanded in vitro, the senescent phenotype appeared. And the senescence of BMSCs may be caused by oxidative stress, manifested by increasing the level of ROS and decreasing the activity of antioxidant enzymes. Treatment of MitoQ down-regulated the mRNA levels of senescence-related and apoptosis-related genes, but up-regulated the mRNA levels of proliferation-related genes. Meanwhile, ROS generation and senescent activity were reduced in MitoQ-treated BMSCs. Further mechanism studies showed that MitoQ obviously promoted Nrf2 phosphorylation, and also facilitated the translocation of Nrf2 into the nucleus. Moreover, treatment of MitoQ increased the mRNA levels of downstream antioxidant genes and enhanced the activities of superoxide dismutase, catalase, and glutathione peroxidase. Thus, our study revealed that MitoQ, via the Nrf2/ARE signaling pathway, exerts an antioxidant effect as well as potentially delays OS-mediated senescence during BMSCs that were expanded in vitro, which may serve as a novel strategy to optimize the clinical application of BMSCs.

Osteogenic and Angiogenic Potency of VEGF165-Transfected Canine Bone Marrow Mesenchymal Cells Combined with Coral Hydroxyapatite in Vitro.

To explore the osteogenic and angiogenic potential of human vascular endothelial growth factor 165 (hVEGF165) gene-transfected canine bone marrow mesenchymal stem cells (BMSCs) combined with coral hydroxyapatite (CHA) scaffold. We constructed a lentiviral vector and transfected canine BMSCs with the best multiplicity of infection. Osteogenesis was induced in the transfected groups (GFP-BMSCs group and hVEGF-BMSCs group) and non-transfected group (BMSCs group), followed by the evaluation of alkaline phosphatase (ALP) activity and alizarin red S staining. Cells from the three groups were co-cultured with CHA granules, respectively to obtain the tissue-engineered bone. MTT assay and fluorescence microscopy were employed to assess cell proliferation and adhesion. The expression of osteogenic and angiogenic related genes and proteins were evaluated at 7, 14, 21, and 28days post osteoinduction in cell culture alone and cell co-culture with CHA, respectively using RT-PCR and ELISA. The hVEGF165 gene was transfected into BMSCs successfully. Higher ALP activity and more calcified nodules were found in the hVEGF-BMSCs group than in the control groups (p < 0.001). Cells attached and proliferated in CHA particles. Both cells cultured alone and cells co-culture with CHA expressed more osteogenic and angiogenic related genes and proteins in the hVEGF-BMSCs group compared to the GFP-BMSCs and BMSCs groups (p < 0.05). High expression of hVEGF165 in BMSCs potentially promote the osteogenic potential of BMSCs, and synergically drive the expression of other osteogenic and angiogenic factors. hVEGF-BMSCs co-cultured with CHA expressed more osteogenic and angiogenic related factors, creating a favorable microenvironment for osteogenesis and angiogenesis. Also, the findings have allowed for the construction of a CHA-hVEGF-BMSCs tissue-engineered bone.

Histopathological Parameters of Canine Bone Marrow in Cell-Block Preparations

Studies using the cell-block technique for bone marrow (BM) analysis are limited in the veterinary literature. This work assessed whether the histopathological analysis of canine BM was feasible using cell-block cytoinclusions prepared from fine-needle sternal aspirate samples. Eight clinically healthy young-to-middle-aged dogs underwent both fine-needle sternal aspiration for BM cell-block (BM-Cb) processing and iliac-crest BM core biopsy (BM-B). Histopathologic parameters were compared between the 2 methods. There were no statistically significant histopathological differences between hematopoietic tissue areas (P = .6294) in the BM-Cb and BM-B sections, and they had similar microscopic characteristics and microarchitecture. Cellularity and reticulin-fiber staining were equivalent in the BM-Cb and BM-B preparations in 87.5% (7/8) and 100% (8/8) of the sections, respectively. However, the quantitative results of the megakaryocytic series differed between BM-Cb and BM-B in 37.5% (3/8) of the sections, and the myeloid:erythroid (M:E) ratios differed between the 2 methods in 25% (2/8). These preliminary data indicate that cell-block preparations made from sternal fine-needle aspiration samples warrant continued evaluation in a larger number of animals, including those with various diseases affecting the bone marrow.

Comparison of iron staining and scoring methods on canine bone marrow aspirates.

Insufficient iron for erythropoiesis can occur in multiple conditions, including absolute iron deficiency, which is often caused by chronic external hemorrhage in dogs. Distinguishing this from other causes of iron-restricted erythropoiesis allows appropriate intervention. Decreased marrow iron assessed by Prussian blue staining is a method to diagnose absolute iron deficiency, but scoring systems for marrow iron are not validated in dogs. Our objectives were to (a) evaluate the technical performance of two bone marrow iron scoring systems used in human medicine and (b) examine the effects of destaining and restaining on iron stores after Wright-stained marrow slides are destained and restained with a Prussian blue stain. Two marrow aspirate slides were included from each of 12 ill dogs in which marrow was collected during clinical evaluation. One slide was directly stained with Prussian blue. The other was first stained with Wright stain, then destained and restained with Prussian blue. Three blinded observers scored the presence of iron in each of the 24 randomized slides using the Gale (scale 0-6) and sideroblast methods (percentage score). Slides were then re-randomized and rescored. For the Gale method, interobserver agreement was fair, and intraobserver agreement was substantial to perfect. There was less agreement using the sideroblast method, with a significant observer effect. Iron scores were significantly lower in destained slides compared with those stained directly. Interobserver and intraobserver agreements were acceptable for the Gale method, but the sideroblast method should be used cautiously. A destaining procedure before Prussian blue staining could decrease marrow iron scores.

Pancytopenia in a Dog Induced by Concomitant Use of Myelotoxic Drugs

Background: The indiscriminate use of drugs is an issue in Veterinary Medicine, as it has serious consequences for the animals. Many drugs are myelotoxic and cause a decrease in the production of blood cells, which may be irreversible in some cases. The present work reports a case of pancytopenia induced by the concomitant use of myelotoxic drugs (estrogen, metamizole and phenobarbital) in a dog and describes findings on myelotoxicity, hematological alterations and treatment success.Case: A 7-year-old Lhasa Apso bitch was referred to the Veterinary Hospital of Federal University of Paraná, Curitiba campus, with hematuria and a history of treatment with phenobarbital [2 mg/kg twice a day (bis in die, BID)], metamizole [25 mg/kg three times a day (ter in die, TID)], and use of estrogen hormone (estradiol cypionate). At physical examination, the animal was normohydrated and exhibited normal palpable lymph nodes, pale mucous membranes, galactorrhea, and a body temperature of 36°C. A complete blood count including reticulocyte count and a total plasma protein (TPP) exam were requested. The results revealed pancytopenia (18% hematocrit, 1,400 total leucocytes/µL, and 22,000 reticulocytes/µL). An abdominal ultrasound exam did not detect any relevant alterations. In view of the results obtained, medullary aplasia was suspected. A bone marrow aspiration was performed. A myelogram revealed a decrease in cellularity (erythrocytic and granulocytic hypoplasia), with presence of rare erythroid and granulocytic precursors. The diagnosis was medullary aplasia. The animal was treated, and the evolution of the hematological alterations was monitored. The treatment consisted of administration of erythropoietin (100UI/kg subcutaneously every 48 h), prednisone (2 mg/kg BID), Leucogen (3 mg/kg BID), interferon (0.2 IU/kg BID) and Eritrós Dog Tabs [1 tablet once a day (semel in die, SID)]. After five days of treatment, the patient’s clinical picture improved (30% hematocrit, 5,300 total leukocytes/µL, 84,000 platelets/µL, and 195,000 reticulocytes/µL), and the hematological alterations were resolved after 25 days of treatment (43% hematocrit, 5,100 total leukocytes/µL, and 333,000 platelets/µL). The bitch was discharged after 89 days of treatment.Discussion: The hematological alterations found in the patient were consistent with pancytopenia, and the myelogram allowed the establishment of a diagnosis of medullary aplasia. There are various causes of pancytopenia in dogs; in this case, it was caused by medications, as the drugs administered to the patient (estrogens, metamizole, and phenobarbital) are myelotoxic. Canine bone marrow is susceptible to suppression by estrogens, which can induce medullary aplasia even with a single dose. No reports on hematological alterations caused by dipyrone (metamizole) in dogs were found; however, in humans, development of aplastic anemia, agranulocytosis, nephrotoxicity, and allergic reactions have been attributed to the use of this drug. Phenobarbital can cause adverse reactions that lead to anemia, leukopenia, and thrombocytopenia. Evaluating the bone marrow of animals with pancytopenia is important because this procedure allows the establishment of a diagnosis that may prompt treatment while hematopoietic precursors are still present in the bone marrow. In this case, a treatment using hematopoietic stimulants was employed owing to the presence of erythrocytic and myelocytic precursors in the patient’s bone marrow. The treatment instituted was efficacious, as only five days of therapy already improved the hematological condition of the patient, who was discharged after 89 days of treatment.

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-β 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.

The effect of fibrin formulation on cell migration in an in vitro tendon repair model

BackgroundThe purpose of this study was to determine the effect of fibrinogen concentration on cell viability and migration in a tissue culture tendon healing model. MethodsForty-eight canine flexor digitorum profundus tendons were randomly divided into three groups. In each group the tendons were lacerated and repaired augmented with a canine bone marrow stromal cell seeded fibrin interposition patch using either 5 mg/ml fibrinogen and 25 U/ml thrombin (physiological as a control), 40 mg/ml fibrinogen and 250 U/ml thrombin (low adhesive), or 80 mg/ml fibrinogen and 250 U/ml thrombin (high adhesive). The sutured tendons were cultured for two or four weeks. ResultsFailure load was not significantly different among the groups. Cell-labeling staining showed that the stromal cells migrated across the gap in the control and low adhesive groups, but there was no cell migration in the high adhesive group at two weeks. Conclusion: A high fibrinogen concentration in a fibrin patch or glue may impede early cell migration. Level of evidenceNot applicable because this study was a laboratory study.

Genome-Wide Analysis Reveals Changes in Long Noncoding RNAs in the Differentiation of Canine BMSCs into Insulin-Producing Cells.

Long noncoding RNAs (lncRNAs) have been extensively explored over the past decade, including mice and humans. However, their impact on the transdifferentiation of canine bone marrow mesenchymal stem cells (cBMSCs) into insulin-producing cells (IPCs) is largely unknown. In this study, we used a three-step induction procedure to induce cBMSCs into IPCs, and samples (two biological replicates each) were obtained after each step; the samples consisted of “BMSCs” (B), “stage 1” (S1), “stage 2” (S2), “stage 3” (S3), and “islets” (I). After sequencing, 15,091 lncRNAs were identified, and we screened 110, 41, 23, and 686 differentially expressed lncRNAs (padjusted < 0.05) in B vs. S1, S1 vs. S2, S2 vs. S3, and I vs. S3 pairwise comparisons, respectively. In lncRNA target prediction, there were 166,623 colocalized targets and 2,976,362 correlated targets. Gene Ontology (GO) analysis showed that binding represented the main molecular functions of both the cis- and trans-modes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that the insulin signaling pathway, Rap1 signaling pathway, tight junctions, MAPK signaling pathway, and cell cycle were enriched for these relative genes. The expression of lncRNAs was verified using qRT-PCR. This study provides a lncRNA catalog for future research concerning the mechanism of the transdifferentiation of cBMSCs into IPCs.