Construction and application for tissue-engineered bone with BMSCs transfected with genes in combining manner

A tissue-engineered bone and gene transfection technology, used in tissue engineering and biomanufacturing, tissue-engineered bone tissue construction and its application in the repair of jaw defects, and biomedical materials, which can solve the expression of angiogenic factors and osteogenic factors. Low dose, poor treatment effect, and difficulty in meeting the needs of bone tissue repair in the defect area

CN104984397AActive Publication Date: 2015-10-21THE FIRST TEACHING HOSPITAL OF XINJIANG MEDICAL UNIVERCITY
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2015-10-21

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Abstract

The invention discloses construction and application for tissue-engineered bone with BMSCs transfected with genes in a combining manner. According to the invention, recombinant lentiviral vectors carrying human bFGF, BMP-2 and GFP genes are constructed, BMSCs are transfected with the genes, the osteogenesis function of BMSCs can be enhanced through human bFGF and BMP2 gene combined-transfection, the fact that bFGF / BMP2 can adjust OPN, collagen-I and other osteogenesis nonspecific gene expression mechanisms is proved, and a GFP-marked tissue-engineered bone is constructed by gene transfected BMSCs composite heterologous bone scaffold material, and a jaw defect animal model is replanted. According to the invention, defects of single gene transfection are overcome, blood vessel forming and osteogenesis functions for solving difficulties in tissue-engineered bone tissue construction and application thereof to jaw defect repairing are obtained, a bone grafting material which has a wide application prospect is provided for mouth rehabilitation and bone transplantation treatment, and a high practical value and practical significance are realized.
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Description

technical field

[0001] The invention belongs to the field of biomedical materials. Specifically, the present invention relates to a technique belonging to the technical field of tissue engineering and biomanufacturing. To be precise, the present invention relates to the technical field of tissue engineering bone tissue construction and its application in the repair of jaw defects. Background technique

[0002] Large-area bone defects are more complicated clinically, and require a large amount of bone regeneration, such as bone reconstruction of large-scale bone defects caused by trauma and infection, tumor resection and bone deformity, or under what circumstances, the regeneration process is destroyed, Including avascular necrosis of the femoral head, atrophic fracture and osteoporosis. Currently, the commonly used clinical treatment measures for large-area bone defects are autologous bone transplantation, allogeneic bone transplantation (allogeneic bone and xenograft bone...

Examples

Embodiment 1

[0089] Embodiment one: the construction of the tissue engineered bone of gene transfection BMSCs

[0090] The present invention provides a kind of construction of the tissue engineered bone of gene transfection BMSCs, obtains by following technical steps:

[0091] (1) Construction of human bFGF, BMP2, and GFP recombinant lentiviral vectors: construct recombinant lentiviral vectors carrying human bFGF, BMP2, and GFP genes respectively, and establish gene transfection technology. The research results confirm that human bFGF / BMP2 gene co-transfection can enhance BMSCs The osteogenic function of bFGF / BMP2 was preliminarily proved to regulate the expression mechanism of osteogenic non-specific genes such as OPN and Collagen-Ⅰ.

[0092] (2) BMSCs were transfected with human bFGF and BMP-2 genes, and BMSCs were transfected with GFP genes: Add the culture medium carrying human bFGF and BMP-2 recombinant lentivirus to the BMSCs induced in the direction of osteogenesis, and place in a C...

Embodiment 2

[0095] Embodiment two: the construction of the tissue engineered bone of gene transfection BMSCs

[0096] The present invention specifically provides a method for constructing tissue-engineered bone with gene-transfected BMSCs, which is specifically prepared by the following method:

[0097](1) BMSCs osteogenic induction culture and identification: extract sheep bone marrow, conduct primary culture of bone marrow mesenchymal stem and conduct osteogenic induction, RT-PCR detection of BMSCs non-specific osteogenic gene mRNA level expression, BMSCs osteogenic differentiation During the process, osteopontin, osteocalcin and type Ⅰ collagen were expressed in stages at the mRNA level, and BMSCs successfully differentiated into osteoblasts.

[0098] (2) Construction of three recombinant lentiviral vectors, gene transfection and identification:

[0099] Construct human bFGF, BMP2, GFP-plenti6 / V5-D-TOPO vector expression plasmids, and no frameshift mutations were confirmed by gene seq...

Embodiment 3

[0104] Embodiment three: the construction of the tissue engineered bone of gene transfection BMSCs

[0105] The present invention provides a tissue engineered bone with gene transfection BMSCs in detail, and the tissue engineered bone construction is specifically obtained by the following method:

[0106] (1) BMSCs osteogenic induction culture and identification: extract sheep bone marrow, conduct primary culture of bone marrow mesenchymal stem and conduct osteogenic induction, RT-PCR detection of BMSCs non-specific osteogenic gene mRNA level expression, BMSCs osteogenic differentiation During the process, osteopontin, osteocalcin and type Ⅰ collagen were expressed in stages at the mRNA level, and BMSCs successfully differentiated into osteoblasts.

[0107] (2) Construction of three recombinant lentiviral vectors, gene transfection and identification:

[0108] Construct human bFGF, BMP2, GFP-plenti6 / V5-D-TOPO vector expression plasmids, and no frameshift mutations were confir...