The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells (PSCs) into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group (P<0.05). Quantitative real-time PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group (P<0.01). ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group's PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P<0.01). The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering.
Objective: To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor β3 vector (hTGF-β3) and transfecting it into rat's precartilaginous stem cells (PSCs).Methods: Gene engineering technique was introduced to construct eukaryotic expression plasmid pcDNA3.1 ( + )-hTGF-β3. PSCs of rats were isolated and purified with method of immunomagnetic microbeads. Then PSCs were cotransfected with plasmid hTGF-β3 and pcDNA3.1 ( + ) -enhanced green fluorescence protein (EGFP) by liner polyethyleneimine (PEI). And 48 hours later the transient expression of EGFP was observed under a fluorescence microscope, and the expression of hTGF-β3 was detected with reverse transcription-polymerase chain reaction (RTPCR) and enzyme linked immunosorbent assay (ELISA).Results: The sequences of the recombinants were consistent with that from Genebank. Cotransfection of EGFP provided fast visual confirmation of successful transduction. The hTGF-β3 mRNA and protein expression could be detected by RT-PCR and ELISA.Conclusions: The recombinant plasmid is correctly constructed and successfully transfected into rat's PSCs,which is an important step to treat epiphyseal injury or other osteo-cartilage diseases with transgenic therapy.
