The Design And Research Of Lysine-cyclic RGD Peptide Applied To Selective Cell Retention Technology

Background and objectives: Suitable bone repair materials are desired by bone surgery for curing serious trauma, infection, tumor abscission and joint fusion, etc. But there are many obstacles needed to be removed, even the most hopeful bone tissue engineering also meets various questions on its three elements including seed cells, scaffold materials and construction strategy that restrict its development. The Selective Cell Retention technology(SCR) has provided a hopeful method which is an improved avenue of construction strategy of traditional bone tissue engineering. SCR employs autogenous cells of bone marrow as seed cells, which could solve many intractable issues of traditional bone tissue engineering including long culture period, potential oncogenicity, immunogenicity and ethical problems. Thus, the performance of scaffold material is crucial to SCR that requires scaffold material possesses satisfactory biocompatibility, osteoinduction and rapid cell adhesion ability. Based on above-mentioned background, we design the Lysine-cyclic RGD peptide(Lc RGD) to coordinate non-specificity cell adhesion ability of lysine sequence and specificity cell adhesion ability as well as osteoinduction of RGD sequence, aimed to enhance cell adhesion ability and osteoinduction ability of scaffold material engaged in SCR.Method:(1)Referred to bioinformatics methods, we designed the structure of Lc RGD peptide and using solid state synthesis method and HPLC to manufacture, purify and identify the Lc RGD peptide. We apply LcRGD peptide to decorate DBM and test the availability and stability of the modification with a laser scanning confocal microscope, ESEM and energy spectrum analysis.(2) Centrifugal cell adhesion assay, CCK-8 method, Wstern Blot, qPCR and ALP activity analysis kit to determine the cell adhesion ability and osteoinduction of Lc RGD peptide in vitro.(3)In vivo osteoinduction performance and adhesion ability for bone marrow cells of DBM/LRGD scaffold applied in SCR was determined by flow cytometry, athymic mice, radiological methods and pathology methods.Results:(1)We successfully designed and achieved highly purified LcRGD peptide. After being modified with Lc RGD peptide, the roughness of DBM surfaces was increased detected by ESEM, the energy spectrum analysis also confirmed the availability of modification. FITC-labeled Lc RGD peptide was modified to DBM and scanned by LSM, then the DBM/LcRGD-FITC scaffolds were subjected to a mimetic SCR procedure using PBS and observed again using the LSM, the results showed the stability of modification.(2) Centrifugal cell adhesion assay shown Lc RGD peptide hold preferable cell adhesion ability than cRGD peptide; CCK-8 method confirmed after mimetic SCR procedure using MSCs suspension, DBM/Lc RGD scaffolds detained more MSCs than DBM/cRGD and DBM scaffolds, and the number of MSCs within DBM/LcRGD scaffolds peaked earlier. After mimetic SCR procedure using MSCs suspension, DBM/Lc RGD, DBM/cRGD and DBM scaffolds were maintained in osteogenic medium then subjected to detect the Runx2, ALP, OPN, OCN m RNA and protein expression level by Western Blot, q PCR and ALP activity analysis kit, results shown DBM/LcRGD group has higher osteoinduction.(3)After SCR procedure using bone marrow, the cells detained on scaffolds were detected by flow cytometry. Results shown DBM/LcRGD detained higher concentration of karyocytes, monocytes, CD90+/CD105+ cells and CD34+ cells compared to DBM/cRGD and DBM alone, which implied DBM/Lc RGD had a preferable cell adhesion ability and specificity cell adhesion ability to a certain extent. Finally, in vivo experiments in athymic mice confirmed the enhanced osteogenic capability of the DBM/LcRGD scaffolds subjected to SCR procedure using bone marrow.Conclusion: The LcRGD peptide we designed for Selective Cell Retention technology displayed excellent adherent ability and osteoinduction. As a short nanoscle biomaterial, the LcRGD peptide has preferable biocompatibility, stability and accessibility that can be used to modify medical materials and is particularly suitable for selective cell retention technology to rapidly construct tissue engineering bone in the clinic.