title: Combining mesenchymal stem cell sheets with platelet-rich plasma gel/calcium phosphate particles: a novel strategy to promote bone regeneration.
abstract: BACKGROUND
  Promotion of bone regeneration is important for successful repair of bony defects. This study aimed to investigate whether combining bone marrow-derived mesenchymal stem cell (BMSC) sheets with platelet-rich plasma (PRP) gel/calcium phosphate particles could promote bone formation in the femoral bone defects of rats.
  
  
  METHODS
  The proliferation and differentiation of BMSCs or BMSC sheets cultured with calcium phosphate particles and/or PRP were investigated in in vitro. In vivo, 36 2.5 × 5 mm bone defects were randomly divided into groups and treated with either BMSCs/PRP gel, calcium phosphate particles, PRP gel/calcium phosphate particles, a BMSC sheet/calcium phosphate particles, a BMSC sheet/PRP gel/calcium phosphate particles, or were left untreated (n = 6/group). A further 15 bone defects were treated with chloromethyl-benzamidodialkylcarbocyanine (CM-Dil)-labelled BMSC sheet/PRP gel/calcium phosphate particles and observed using a small animal in vivo fluorescence imaging system to trace the implanted BMSCs at 1 day, 3 days, 7 days, 2 weeks, and 4 weeks after surgery.
  
  
  RESULTS
  The expression of collagen type I and osteocalcin genes of BMSCs or BMSC sheets treated with PRP and calcium phosphate particles was significantly higher than that of BMSCs or BMSC sheets treated with calcium phosphate particles or the controls (P <0.05). PRP can promote gene expression of collagen III and tenomodulin by BMSCs and in BMSC sheets. The VEGF, collagen I and osteocalcin gene expression levels were higher in the BMSC sheet than in cultured BMSCs (P <0.05). Moreover, alizarin red staining quantification, ALP quantification and calcein blue fluorescence showed the osteogenic potential of BMSCs treated with PRP and calcium phosphate particles The implanted BMSCs were detectable at 1 day, 3 days, 7 days, 2 weeks and 4 weeks after surgery by a small animal in vivo fluorescence imaging system and were visualized in the defect zones by confocal microscopy. At 4 weeks after implantation, the defects treated with the BMSC sheet/PRP gel/calcium phosphate particles showed significantly more bone formation than the other five groups.
  
  
  CONCLUSIONS
  Incorporation of an BMSC sheet into the PRP gel/calcium phosphate particles greatly promoted bone regeneration. These BMSC sheet and tissue engineering strategies offer therapeutic opportunities for promoting bone defect repair clinically.
authors.affiliation: Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. yiyingqi@163.com.
authors.forename: Yiying
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Department of Orthopedic Surgery, People's Hospital of Dongping County, Shandong, China. allgoodstuff@163.com.
authors.forename: Lie
authors.initials: L
authors.lastname: Niu
authors.affiliation: Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. zhaotengfei12@gmail.com.
authors.forename: Tengfei
authors.initials: T
authors.lastname: Zhao
authors.affiliation: Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. unumah@gmail.com.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Shi
authors.affiliation: Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. mikewqyan@gmail.com.
authors.forename: Tuoyu
authors.initials: T
authors.lastname: Di
authors.affiliation: Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. abfarthood@163.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Feng
authors.affiliation: Department of Orthopedic Surgery, Hangzhou TCM Hospital, Hangzhou, China. dushaoh39@gmail.com.
authors.forename: Junhua
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Orthopedic Surgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310009, China. doctorhzm@gmail.com.
authors.forename: Zhongming
authors.initials: Z
authors.lastname: Huang
mesh_terms: D000818:Animals; D001672:Biocompatible Materials; D001861:Bone Regeneration; D002130:Calcium Phosphates / Q000008:administration & dosage; D002454:Cell Differentiation; D049109:Cell Proliferation; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D005269:Femur / Q000293:injuries / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression; D008297:Male; D008422:Materials Testing; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D053657:Platelet-Rich Plasma; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D001672:Biocompatible Materials; D002130:Calcium Phosphates; D015675:Osteocalcin; D003094:Collagen; C020243:calcium phosphate