Studies Of Repair And Mechanism On Canine MSC In The Skin And Joint Injury

Mesenchymal stem cells（MSCs）are adult stem cells present in the organism interstitium.Under suitable conditions in vivo or in vitro,MSCs have the ability to differentiate into various cell types,including osteoblasts,chondrocytes,muscle cells,adipocytes,and even islet cells and nerve cells.Skin trauma and arthritis are common diseases in dogs,which causes illness to dogs and affects their quality of life severely.Stem cell therapy,as an emerging treatment method,has shown a good effect on the tissue regeneration and the repair of damaged tissue,and MSCs are currently the most widely used adult stem cells.To study the repair effect of MSCs on canine skin trauma and the damaged joint,this experiment carried out the following studies:1.Biological characteristics of BM-MSCs and UC-MSCs.In this study,BM-MSCs were isolated by a method of Ficoll density gradient separation,and the UC-MSCs were isolated ftom umbilical cord Wharton’s jelly by 0.1%type collagenase I,then both of them were cultured in vitro and their biological characteristics were studied.The results showed that both of the BM-MSCs and UC-MSCs grew with adherence,showing a long fusiform shape,arranging in a clear directionality in spiral shaped and had a strong refraction.They maintained a good growth activity even when they passed to the 9^th generation.The differentiation ability of BM-MSCs decreased as the number of passages increased and their population doubling time increased with the number of passages increased.Results revealed that the isolated cells were MSCs,because they both were positive for CD44 and negative for CD34 surface markers,and the cells were differentiated into adipocytes and osteoblasts.2.Repair effects of BM-MSCs on canine skin wounds.To observe the effect of the canine bone marrow mesenchymal stem cells on the skin trauma,A 3 cm×3 cm square wound were made on the both side of the buttocks of the dog,the right side was the experimental group.After the wound was made,1ml allogeneic canine bone marrow mesenchymal stem cells（1×10⁵cells/ml）suspension was injected subcutaneously around the wound in the experimental group on the 1^st and the 3^th day.The control group was injected subcutaneously around the wound with mesenchymal stem cell culture medium on the first and third days after the wound made,and the wound healing was observed dynamically.The results showed that the pale yellow inflammatory substances reduced and the growth rate of the granulation tissue was faster in the experiment group which was treated by BM-MSCs in the first week.The wound area of the experimental group was slightly smaller than that of the control group at 8 and12 days after the treatment of the cells transplantation,and the healing speed of the experimental group was slightly faster,but the difference of them was not significant statistically.This might be related to factors,such as the number of cells injection,the number of the treatment times,the number of injection sites and injection points.The result indicated that the BM-MSCs had the possibility or trend to inhibit the inflammatory response of the wound and promote the healing of the skin trauma.3.In this study a blank control group,a complete medium control group,and a saline control group were set up and the method of transwell co-culture was used to explore the effect of bone marrow,fat and umbilical cord derived MSCs on the proliferation of articular chondrocyte（AC）.The results showed that these three sources of MSCs could significantly enhance the cellular activity of AC（P<0.01）,of which UC-MSC had the best enhancement effect.The effects of UC-MSC-derived exosomes（EXO）and their conditioned medium（CM）with no EXO on AC were further compared.It was found that the CM with no EXO of UC-MSCs promoted the proliferation of AC more significantly than the UC-MSCs exosomes（P<0.01）.4.Repair effect of UC-MSCs on injury of the canine knee joint.In this study,Eight dogs were evenly assigned to two groups.The canine model of knee osteoarthritis was established by surgical manipulation of knee articular cartilage on these eight dogs.The suspension of allogeneic UC-MSCs（1×10⁶）and equal amount of physiological saline was injected into the cavitas articularis in the treated and untreated control group,respectively on days 1 and 3 post-treatment.The structure of the canine knee joint was observed by magnetic resonance imaging（MRI）,B-mode ultrasonography and X-ray imaging at the 3rd,7th,14th and 28th days after treatment.Concurrently,the body temperature,the blood routine,the biochemical indicators,and the inflammatory factors in blood of the examined dogs were measured.Moreover,the recovery of cartilage and patella surface in treated group and untreated group was compared by scanning electron microscope（SEM）and histological methods after a 35-day treatment.Imaging technology showed that as treatment time increased,the high signal in the MRI T2-weighted images decreased,the echo-free space in B ultrasonography images disappeared basically and the continuous linear hypoechoic region at the trochlear sulcus thickened.On X-ray images,the serrate defect at ventral cortex of patella improved,and the low density gap of ventral patella and trochlear crest gradually increased in the treated group.On the contrary,the high signal in the MRI T2-weighted images and the echo-free space in B ultrasonography images still increased after a 14-day treatment in untreated control group,and the linear hypoechoic region was discontinuous.On the X-ray images,there was no improvement in the serrate defect of the ventral cortex of the patella.The result of SEM showed that the cartilage neogenesis in the treated group had visible neonatal tissue and more irregular arrangement of new tissue fibers than that of the untreated control group.Furthermore,more vacuoles but without collagen fibers were observed in the cartilage of the untreated control group,and the thickness of the neogenetic cartilage in the treated group and the untreated control group showed significant difference（P<0.01）.The body temperature of the untreated control group was higher than that of the treated group at the first 11 days of the experiment,and the temperature had significant difference on the 5^th,6^th and 10^th day（P<0.05）.On the 14th day after the treatment,the count of the white blood cell the neutrophils of the untreated control group was significantly higher than the treated group（P<0.05）,while the number of lymphocytes was significantly lower than that of the treated group（P<0.05）.During the 7th-14th day of the experiment,the levels of IL-6,IL-7 and TNF-αin the blood of the untreated control group were significant higher than that of the treated group（P<0.05）,and the level of IL-6 has significant difference after 14 days treatment（P<0.01）.There were no significant difference between the two groups in other biochemical indexes,except the level of ALP was higher than those of the treated group after 7 days treatment（P<0.05）.The above results indicated that canine UC-MSCs may enhance proliferation of AC to promote the repair of cartilage and patella injury in osteoarthritis and improve the healing of the surrounding tissues.UC-MSCs can reduce the inflammatory response,its mechanism might be to reduce the fever and inflammation of the body by inhibiting the production of white blood cells and neutrophils,and by inhibiting the production of inflammatory factors to reduce the inflammation of OA,while promoting the production of the lymphocytes and erythrocyte to enhance the ability of immune capacity and anti-infective,and by increasing the production of platelets to promote the wound healing.