ECM components include a number of osteoinductive and osteoconductive factors, which are involved in bone fracture healing. In this study, a combination of adipose derived mesenchymal stem cells (Ad-MSCs), cancellous bone graft (CBG), and chitosan hydrogel (CHI) was applied to the non-union bone fracture and healing effects were evaluated for the first time. After creation of animal models with non-union fracture in rats, they were randomly classified into seven groups. Radiography at 0, 2, 4, and 8 weeks after surgery, indicated the positive effects of Ad-MSCs + CBG + CHI and Ad-MSCs + CBG in treatment of bone fractures as early as 2 weeks after the surgery. These data were confirmed with both biomechanical and histological studies. Gene expression analyses of Vegf and Bmp2 showed a positive effect of Ad-MSCs on vascularization and osteogenic differentiation in all groups receiving Ad-MSCs, as shown by real-time PCR. Immunofluorescence analysis and RT-PCR results indicated existence of human Ad-MSCs in the fractured region 8 weeks post-surgery. In conclusion, we suggest that application of Ad-MSCs, CBG, and CHI, could be a suitable combination for osteoinduction and osteoconduction to improve non-union bone fracture healing. Further investigations are required to determine the exact mechanisms involved in this process before moving to clinical studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2018.

BackgroundApplication of competent cells such as mesenchymal stem cells (MSCs) for treatment of musculoskeletal disorders in equine athletes is increasingly needed. Moreover, similarities of horse and human in size, load and types of joint injuries, make horse as a good model for MSCs therapy studies. This study was designed to isolate and characterize stemness signature of equine bone marrow-derived mesenchymal stem cells (BM-MSCs).MethodsBM of three mares was aspirated and the mononuclear cells (MNCs) were isolated using density gradient. The primary MNCs were cultured and analyzed after tree passages (P3) for growth characteristics, differentiation potentials, and the expression of genes including CD29, CD34, CD44, CD90, CD105, MHC-I, MHC-II and pluripotency related genes (Nanog, Oct-4, Sox-2, SSEA-1, -3, -4) using RT-PCR or immunocytochemistry techniques.ResultsThe isolated cells in P3 were adherent and fibroblast-like in shape with doubling times of 78.15 h. Their clonogenic capacity was 8.67±4% and they were able to differentiate to osteogenic, adipogenic and chondrogenic lineages. Cells showed expression of CD29, CD44, CD90, MHC-I and Sox-2 while no expression for CD34, MHC-II, CD105, and pluripotency stemness markers was detected.ConclusionsIn conclusion, data showed that isolated cells have the basic and minimal criteria for MSCs, however, expressing only one pluripotency gene (sox-2).

The effects of selenium-methionine (Se-Met) and chromium-methionine (Cr-Met) supplementation on performance and response to transportation stress were studied on 24 Baluchi ewe lambs (18-20 weeks of age) for 9 weeks. The lambs were randomly assigned to four dietary treatments: (1) control; (2) 1.5 mg supplemental Se-Met/kg dry matter (DM) of diet; (3) 0.8 mg supplemental Cr-Met/kg DM of diet; and (4) 1.5 mg Se-Met plus 0.8 mg Cr-Met/kg DM of diet (Se-Cr-Met). At the commencement of week 8, a road transportation stress (TS) was carried out for 30 min. Lambs fed Cr-Met and Se-Cr-Met diets had higher feed intake than the control and Se-Met animals (P < 0.0001). Lambs on Cr-Met diet showed higher average daily gain (ADG) compared to the control group (P = 0.007). Se-Met and Cr-Met supplementation alone or in combination significantly (P < 0.05) reduced feed conversion ratio (FCR). The animals that received Se-Met (P = 0.014), Cr-Met (P = 0.005), and Se-Cr-Met (P = 0.003) supplemented diets had lower glucose concentration than the control. Lambs on Cr-Met had higher blood T3 concentration than control animals (P = 0.040), while Cr-Met (P = 0.039) and Se-Cr-Met (P = 0.032) supplementation increased triiodothyronine (T3) to thyroxin (T4) ratio. Animals fed Se-Met and/or Cr-Met supplements had lower blood malondialdehyde (MDA) in week 9 of the experiment (P < 0.05). Blood ferric-reducing antioxidant power (FRAP) tended to be higher in the Se-Met- and Se-Cr-Met-supplemented groups (P < 0.1).TS reduced feed intake in lambs fed the control diet in week 8 of the experiment (P = 0.003). The lambs given with supplemental Cr-Met exhibited lower glucose concentration before transportation (BT) (P = 0.029) and after transportation (AT) (P = 0.016) compared to the control. Lambs fed Se-Cr-Met had the lowest cortisol concentration BT (P < 0.05). It was concluded that feeding Se-Met and/or Cr-Met supplements could improve growth performance and be beneficial in attenuating the adverse effects of transportation stress in Baluchi ewe lambs.

Background: The primary cell seeding density of bone marrow-derived mononuclear cells (BM-MNCs) affects several cellular behaviors, including attachment to the culture dish, proliferation, and differentiation. Methods: The aim of this study was to determine the best density of equine BM-MNCs in primary culture (P0) for obtaining the maximum bone marrow-derived mesenchymal stem cell (BM-MSC) yields at the end of P0. Bone marrow samples of two healthy mares were aspirated. The MNCs were isolated and cultured at different densities (1×10 5 , 2×10 5 , 4×10 5 , 8×10 5 , and 1×10 6 cells/cm 2). Within the 7 th and 14 th days after seeding, the colonies containing more than 15 cells were counted and the percentage of confluency and the number of cells were calculated on day 21. Results: The lowest density of MNCs was associated with the least number of colonies, number of adherent cells, and confluency percentage, whereas the highest density was associated with the maximum number of colonies and confluency percentage (P<0.05). However, the maximum number of cells at the end of P0 was associated with the intermediate (4×10 5 cells/cm 2) and the highest concentration (P<0.05). Conclusions: The maximum number of MSCs at the end of P0 was obtained at the densities of 1×10 6 and, especially, at 4×10 5 cells/cm 2 .

Spermatogonial stem cells (SSCs) are expected to participate in male infertility therapy, endangered species preservation, and transgenic animal technology by their unique unipotency to differentiate into spermatozoa. The main challenges, however, remain to be addressed including the appropriate conditions to reach good number of these cells and how to derive, culture, and maintain them in vitro. In the present study, the testicular tissues were isolated from 1-d-old male chickens to establish primary cell cultures. This culture led to development of distinguished colonies which were further characterized by alkaline phosphatase (AP) activity assay and gene expression analysis. They were shown to be positive for AP activity and expressed two main transcription factors of OCT4 and STRA8 as indicated by reverse transcription-polymerase chain reaction. These were indications of carrying characteristics of SSCs by these colonies. The cultures were also exposed to different concentrations of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) growth factors to seek optimum colony-forming conditions. Colony-forming activity assay indicated that they were able to propagate in vitro with an increased self-renewal property when cultured in the presence of 15 ng/mL of GDNF, 20 ng/mL of bFGF, and 15 ng/mL of LIF. The present work provides an easy and practical method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells and introduces appropriate cell culture conditions to improve and maintain their self-renewal property based on supplying the necessary growth factors.

An 11-year-old male dog was presented with a 1-week history of inappetence, weight loss and hind limb paralysis. Physical examination revealed weakness, depression, incoordination of the posterior limbs, peripheral lymphadenopathy and pale mucous membranes. Laboratory analysis of blood samples revealed anaemia, thrombocytopenia and low serum albumin concentration. The diagnosis was confirmed microscopically, by demonstrating the presence of Hepatozoon canis gametocytes within neutrophils in Giemsa-stained peripheral blood smears and bone marrow smear. Also, schizonts of H. canis were seen in tissue sections of muscles, lymph nodes, spleen and liver. To the best of authors' knowledge, this is the first description of H. canis infection in a dog in Iran.

Background: Sometimes it is necessary to use sedatives or even general anaesthetics to examine animals with spinal cord injuries. These drugs may affect spinal reflexes, alter the outcome of neurological examinations, and make it difficult to diagnose location of the lesion. Objectives:The aim of this study was to evaluate the effects of five pre-anaesthetic and anaesthetic agents commonly used in clinics on spinal reflexes in dogs.Methods: Ten native adult dogs were participated in three groups. In all groups, the dogs were premedicated with medetomidine and midazolam; then, in the first group, ketamine, in the second group, propofol and in the third group, isoflurane were used for induction of anaesthesia. The spinal reflexes were evaluated before injection, 15 min after medetomidine, 20 min after midazolam, and at 15, 30, 45 and 60 min after induction of anaesthesia. Results:Medetomidine did not reduce monosynaptic reflexes (patellar and cranial tibial reflexes) but increased them while it had no effect on the polysynaptic limb withdrawal reflexes. Midazolam had no effect on the spinal reflexes; Ketamine did not affect the patellar, cranial tibial and extensor carpi radialis reflexes, but reduced polysynaptic pain-related reflexes; and propofol and isoflurane abolished the all spinal reflexes. Conclusions: Medetomidine, midazolam and ketamine have no effect on reducing monosynaptic reflexes (patellar and cranial tibial reflexes) and may be used for neurological examination of restless animals in the clinic. Propofol and isoflurane eliminated all spinal reflex responses and are not suitable for neurological examinations.

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