Bao-yi Liu1, De-wei Zhao. 1. Department of Orthopedics, Affiliated Zhongshan Hospital of Dalian University, Dalian 116001, China.
Abstract
OBJECTIVE: To understand the biological characteristics and osteogenic potential of hVEGF-165 gene modified marrow stromal stem cells and investigate the effect and value of treatment for osteonecrosis of femoral head by hVEGF-165 gene modified marrow stromal stem cells under arthroscope. METHODS: rAAV-2-hVEGF-165 plasmids were extracted and transfected into rabbit marrow stromal stem cells. hVEGF-165 mediated by adeno-associated virus (AAV) was used to transfect rMSCs. The transfection efficiency was detected with enhanced green fluorescent protein under fluorescence microscope. hVEGF-165 mediated by adeno-associated virus (AAV) was used to transfect rMSCs. Virus transfection stayed overnight after 90% cell converged. MOI was 105. The transcription and expression of hVEGF-165 protein expression were detected by RT-PCR and Western blot. The necrotic bone was emptied and then MSCs were implanted under arthroscope. The histology of femoral head was inspected at postoperative 2 - 8 weeks. RESULTS: The expression of hVEGF-165 gene could be found distinctly in the transfected rabbit MSCs and hVEGF-165 protein in the supernatants of transfected cell cultures. The transfection efficiency of adeno-associated virus (AAV) transfected rMSCs was 70%. And rAAV-2-hVEGF-165 transfected rMSCs achieved an effective expression by RT-PCR and Western blot. hVEGF-165 could be found after a 48-hour transfection and peaked at Day 10. Immunohistochemical detection showed that the implanted rMSCs was positive at Week 2 and strong positive at Week 8. The compressive strength of the hVEGF-165 gene group approached that of normal control. CONCLUSION: hVEGF-165 gene transfected rabbit MSCs can express hVEGF-165 with highly biological activity. It provides provided a basis for employing hVEGF-165 gene and MSCs based gene therapy for ONFH repairing and regeneration. rAAV-2-hVEGF-165/MSCs may be implanted accurately under arthroscope. Implantation of human BMP-2 gene transfected BMSCs can repair early-stage experimental femoral head necrosis.
OBJECTIVE: To understand the biological characteristics and osteogenic potential of hVEGF-165 gene modified marrow stromal stem cells and investigate the effect and value of treatment for osteonecrosis of femoral head by hVEGF-165 gene modified marrow stromal stem cells under arthroscope. METHODS: rAAV-2-hVEGF-165 plasmids were extracted and transfected into rabbit marrow stromal stem cells. hVEGF-165 mediated by adeno-associated virus (AAV) was used to transfect rMSCs. The transfection efficiency was detected with enhanced green fluorescent protein under fluorescence microscope. hVEGF-165 mediated by adeno-associated virus (AAV) was used to transfect rMSCs. Virus transfection stayed overnight after 90% cell converged. MOI was 105. The transcription and expression of hVEGF-165 protein expression were detected by RT-PCR and Western blot. The necrotic bone was emptied and then MSCs were implanted under arthroscope. The histology of femoral head was inspected at postoperative 2 - 8 weeks. RESULTS: The expression of hVEGF-165 gene could be found distinctly in the transfected rabbit MSCs and hVEGF-165 protein in the supernatants of transfected cell cultures. The transfection efficiency of adeno-associated virus (AAV) transfected rMSCs was 70%. And rAAV-2-hVEGF-165 transfected rMSCs achieved an effective expression by RT-PCR and Western blot. hVEGF-165 could be found after a 48-hour transfection and peaked at Day 10. Immunohistochemical detection showed that the implanted rMSCs was positive at Week 2 and strong positive at Week 8. The compressive strength of the hVEGF-165 gene group approached that of normal control. CONCLUSION: hVEGF-165 gene transfected rabbit MSCs can express hVEGF-165 with highly biological activity. It provides provided a basis for employing hVEGF-165 gene and MSCs based gene therapy for ONFH repairing and regeneration. rAAV-2-hVEGF-165/MSCs may be implanted accurately under arthroscope. Implantation of humanBMP-2 gene transfected BMSCs can repair early-stage experimental femoral head necrosis.