title: Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair.
abstract: OBJECTIVE
  The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs.
  
  
  METHODS
  The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining.
  
  
  RESULTS
  An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs.
  
  
  CONCLUSIONS
  According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Dongyang
authors.initials: D
authors.lastname: Qian
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Bo
authors.initials: B
authors.lastname: Bai
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Guangbin
authors.initials: G
authors.lastname: Yan
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Shujiang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: a Department of Orthopaedics , the First Affiliated Hospital, Guangzhou Medical University , Guangzhou , P. R. China.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Tan
authors.affiliation: b Biomechanics & Medical Information Institute, Beijing University of Technology , Beijing , P. R. China.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Zeng
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000464:Alginates / Q000008:administration & dosage / Q000737:chemistry; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics* / Q000378:metabolism; D001861:Bone Regeneration / Q000187:drug effects*; D002214:Capsules / Q000008:administration & dosage / Q000138:chemical synthesis; D004318:Doxycycline / Q000494:pharmacology*; D005269:Femur / Q000187:drug effects / Q000293:injuries; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D011107:Polylysine / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000737:chemistry; D061251:Primary Cell Culture; D051381:Rats; D017207:Rats, Sprague-Dawley; D023822:Tissue Engineering; D014162:Transfection; D019076:Transgenes
chemical_list: D000464:Alginates; C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D002214:Capsules; C051546:alginate-polylysine-alginate; D011107:Polylysine; D004318:Doxycycline
keywords: APA microcapsule; Tet-On expression system; adenovirus; bone morphogenetic protein-2