Biological Characteristics Of Bovine Adipose-derived Mesenchymal Stem Cells And The Construction Of The Model For Treatment In Achilles Tendon Injury Of Mice

Objective:Tendon injuries are common in athletes with suddenly repetitive running or jumping movements in their major special project.Due to the problems of poor recovery and easy recurrence,tendon injuries have become the key factors restricting the personal development,training and competition of athletes.At present,the regenerative therapy based on adipose-derived mesenchymal stem cells(ADSCs)is expected to have broad clinical application prospects.To investigate the therapeutic effect of ADSCs on tendon injury and its possible mechanism to involve in the repair of damaged tendon,we established a culture system of bovine ADSCs in vitro and collagenase-induced tendon injury in mice model.Methods:An enzyme digestion method was used to obtain the ADSCs in fetal bovine pericardial adipose tissue;the growth curve of bovine ADSCs was made and colony-forming ability was detected;bovine ADSCs surface markers were detected by RT-PCR,immunofluorescence and flow cytometry;bovine ADSCs were induced into osteoblasts,adipocytes and chondrocytes in vitro;The collagenase-induced Achilles tendon injury model in mice was established,then we respectively observed the histological appearance,cell-labeled and differentiation in vivo of tendon tissue in control group(C),injury control group(I),injury + placebo injection group(IS)and injury + ADSCs transplantation group(IC)on 1d,3d,5d,7d,14 d,21d,28 d and 35 d.Results:1.The enzyme digestion method was used to successfully separate and obtain the fetal bovine ADSCs.After the isolation,ADSCs had good self-proliferation ability,and could be subcultured in vitro to passage 50.ADSCs recovered after freezing are still highly active.2.We detected ADSCs surface markers by RT-PCR,immunofluorescence and flow cytometry,the results showed these ADSCs were positive for CD29,CD44,CD73,CD90,CD105,OCT4,SOX2,NANOG and MHC-I,but were negative in CD45 and MHC-II.3.The cultured ADSCs could be differentiated into osteoblasts,adipocytes and chondrocytes in vitro,the expression of BGLAP,bALP and Runx2 were detected after 14 days of osteogenic induction by RT-PCR,with Alizarin red S staining positive;LPL and PPAR-γ were expressed in 14 days after induction,which was confirmed by Oil Red-O staining.And chondrocyte related genes such as COL2A1 and ACAN were detected post 21 days of induction,with Alcian blue staining positive.4.H&E staining showed that 24 h after injury,the structure of normal tendon tissue was destructive,and there were still un-healing structures in I group and IS group after 35 d.The tendon tissues of IC group had a large number of inflammatory cells at 24 h,3d and 5d after injury,and the structure was basically recovered to normal after 35 d.The cell immunofluorescence showed that the CM-Dil labeled ADSCs could migrate to the site of injured tendon and express the tenocyte-related protein TNMD in vivo.Conclusions:This study successfully established an isolation,culture and amplification system of fetal bovine ADSCs in vitro.These bovine ADSCs had a strong self-renewal and expansion potential in vitro and could be subcultured to passage 50,without their stemness and low immunogenicity changed.The transplanted bovine ADSCs could migrate rapidly into collagenase type Ⅰ-induced tendon injury of mice,and expressed tenogenic lineage marker,which promoted the regeneration and repair in native tendon tissue.