A Novel Strategy For In Situ Bone Tissue Engineering By CD271 Antibody Mediated Bone Marrow Mesenchymal Stem Cells Recruiting Chitosan Microspheres

Objective: The objective of this study is to fabricate a kind of functional microsphere that consists of chitosan(CS) microsphere/CD271 antibody/polydopamine(PDA) coating, and then test its ability of capturing human bone marrow mesenchymal stem cells(hBM-MSCs) and facilitating the attachment and proliferation of cells.Methods:1. The chitosan microspheres were prepared by differentiating chitosan solution into droplets under high-voltage electrostatic field which were followed by being hardened with sodium hydroxide. During fabricating the CD271/PDA/CS microspheres, chitosan microspheres were firstly modified with PDA coating by reacting with dopamine solution in a weak alkaline buffer solution. After inducing biotin to the surface of microspheres, biotinylated CD271 antibody was further connected through the streptavidin-biotin pair. CD271/CS microspheres and blank CS microspheres were also produced as control groups. 2. Testing the CD271 expression of hBM-MSCs through immunofluorescent staining and flow cytometry. 3. Cells were previously stained by DiI. Microspheres and h BM-MSCs were co-cultured for 5 hours and then uncaptured cells were filtered by 40 μm strainer. The result of capture was evaluated by fluorescence microscope and MTS analysis. 4. Microspheres with captured cells were cultured for 21 days. Cell expansion of each group was assessed by DAPI/Palloidine staining and MTS analysis.Results: 1. Submillimeter-sized chitosan microspheres were well fabricated. After modified with PDA, the color of microspheres changed from white to black. Results of fluorescence staining revealed that CD271 antibody could be successfully connected to both chitosan microsphere and PDA modified chitosan microsphere. 2. CD271 was highly expressed in hBM-MSCs. The ratio of expression was 97.9%. 3. Both CD271/PDA/CS group and CD271/CS group could capture a lot of hBM-MSCs without significantly statistic difference between the two groups(P > 0.05), which was much more than blank chitosan microspheres group(P < 0.05). 4. Obvious cell proliferation was observed in CD271/PDA/CS group after cultured for 21 days, while in CD271/CS group most cells were dropped from microspheres to the surface of petri dish during the first week which led to the significantly decreased amount of cells on the microspheres.Conclusion: Chitosan microsphere modified with PDA coating and CD271 antibody has been successfully produced. Results provide first evidence for the ability of CD271 antibody modified in microspheres to capture hBM-MSCs from cell suspension, and demonstrate that DPA coating can facilitate cell attachment and proliferation on the surface of microspheres.