Preparation Of 3D Printed Porous Hydroxyapatite Scaffold Coated With Collagen/rhBMP-2 Chitosan Microspheres And Ectopic Bone Formation Investigation In Vivo

PART I Preparation of vanilline cross-linked rhBMP-2/chitosan microspheres and its effect on hMSCsBackground:RhBMP-2 was an important bone inductive protein. It could effectively induce bone formation in vivo. However, previous researches had demonstrated that the release pattern of rhBMP-2 affect its osteoinductivity. Initial burst release combined with slow sustained release of rhBMP-2 would possibly induce more bone formation in vivo. Chitosan was nature biomaterials with good biocompatibility. It acted as carriers for many cytokines. Vanilline was a favorable cross-linking reagent with better biocompatibility compared with traditional cross-linking reagent such as glutaraldehyde. So rhBMP-2/chitosan microspheres cross-linked by vanilline would possibly have excellent biocompatibility and controlled release pattern.Objective:To prepare the rhBMP-2/chitosan microspheres(rhBMP-2 CMs) using the vanilline as cross-linking reagent, study the biocompatibility and drug release characteristic of the microspheres in vitro.Methods:Emulsion cross-linking method was utilized to prepare the rhBMP-2 CMs, Scanning electron microscope (SEM) was used to observe the microstructure of the microspheres. Leaching solution of the microspheres and the blank culture medium was set as experimental and control group respectively. They were cultured with the human Mesenchymal Stem Cells (hMSCs) to determine its cytotoxicity and its effect on the proliferation of hMSCs. Dynamic immersion method was used to examine the release characteristic of rhBMP-2 in vitro.ALP activity of hMSCs was determined to reveal the bioactivity of the released rhBMP-2.Results:The rhBMP-2 CMs were spherical in shape under SEM observation. After the leaching solution cultured the cell for 24 and 48 hours, there was no statistical difference of the OD values between the experimental and control group at both time points(24h:0.719±0.072 vs 0.726±0.051,p>0.05; 48h:1.192±0.108 vs 1.273± 0.058,p>0.05). When the cells were cultured with the leaching solution for 1,3 and 7 days, the number of the cells increased with time at both groups, and the OD values of each tested time point were not statistically different. Five milligram of rhBMP-2 CMs were soaking for 19 days, and rhBMP-2 was released gradually. The concentration of rhBMP-2 was still 216.14±20.00ng/mL at the 19th day. The ALP activity of hMSCs cultured at 3d- and 7d-collected medium was (0.504± 0.065),(0.684±0.060)μmol pNPP/min/mg protein, and they were higher than the blank culture medium group(0.135±0.011) (p<0.05).Conclusions:The rhBMP-2 CMs prepared using the vanilline as cross-linking reagent had good biocompatibility. It could be a carrier for the control release of rhBMP-2 and the encapsulated rhBMP-2 was still bioactive.PART Ⅱ Preparation of porous HA scaffold coated with collagen/rhBMP-2 and biocompatibility investigation in vitro.Background:Many materials could be used for fabricating individualized bone repaired scaffold with the advancement of rapid prototyping technique. It was a material increase manufacturing technique and could control the fine internal structure of materials. Recently, porous bone tissue engineering scaffold with excellent interconnectivity could be made via this technique, however sufficient osteogenesis of large scaffold could not simply relay on the modification of structure. An ideal bone tissue engineering scaffold should have the property of osteoinductivity. Porous composite scaffold combined with osteoinductive protein would be a good option for the repair of large bone defect.Purpose:Construct a novel 3D porous HA scaffold coated with collagen/rhBMP-2 chitosan microspheres (CMs), and then assess its effect on the adhesion and osteogenic differentiation of MSCs.Methods:The 3D model of the porous scaffold was created and then printed using the extrusion deposition system. Composite scaffold was coated with collagen (HC) and collagen/rhBMP-2 chitosan microspheres (HCC)。Scanning electron microscopy was used to observe the surface of HA, HC and HCC. Human Mesenchymal Stem Cells (hMSCs) was used to determine the Cytotoxicity of the scaffold and its impact on the cell proliferation. HMCSs were co-cultured with the scaffold to contrast the cell adhesion ability of different scaffold. Dynamic soaking method was used to determine the release characteristic of HCC in vitro. Extraction of HCC scaffold collected at day 1,3 and 7 was used to culture hMSCs, and then ALP activity was detected to reveal the bioactivity of the released rhBMP-2.Results:Scanning electron microscopy observation revealed that a layer of collagen was coated on the surface of HC. The rhBMP-2 CMs were adhered on the surface of HCC and agglomerates of the microspheres could be observed. Cytotoxicity of the HCC extraction was no statistical different with HA and HC after 24 and 48 hours of culturing. Cell viability of different group was no statistical different when co-culture with the composite scaffold, and the number of cells increase with time for all groups. Cell adhesion test revealed that HCC scaffold could facilitate the adhesion of hMSCs, and the number of cells grew on HCC was more than that of HA and HC visually. The release of rhBMP-2 in vitro could last for over 3 week, and the concentration of rhBMP-2 of each tested time pointed was beyond 112.8ng/ml. The extraction fluid of HCC scaffold could successfully enhanced the ALP activity of hMSCs.Conclusions:Collagen/rhBMP-2 chitosan microspheres coating was an ideal way to combine the osteoinductive protein to the 3D printed scaffold. The constructed composited scaffold had good biocompatibility and could released rhBMP-2 in control manner. It could support the proliferation and adhesion of hMSCs in vitro.PART Ⅲ Ectopic bone formation by collagen/rhBMP-2 chitosan microspheres coated porous HA scaffold in vivoBackground:Close integration of bone repair material with the host bone could reduce the risk of loosening, shifting or fatigue fracture of the implants. The pure porous materials could improve its osteoconductivity thought adjusting the porosity or surface modification to improve the cell adhesion rate. However, when large bone repair scaffold was made, the effect on improving bone growth to the inside materials through increase the osterconductivity of materials was limited. Thus, it was important to make osteoinductive materials which had the ability to induce tissue cells to the osteoblast differentiation and osteogenesis.Purpose:To compare the components of tissue grown inside the scaffold of HA, HC and HCC after they were implanted in the muscle of rabbits and observe the bone distribution within the collagen/rhBMP-2 chitosan microsphere coated HA scaffold.Methods:The general view of the sample at 8 weeks showed that the tissue was loose and easy to separate in HA and HC while it was tight and hard to detach in HCC. Micro-CT observation revealed that the bone volume fraction of HCC was 11.56% at 4 weeks, while it was 17.14% at 8 weeks. The trabecular thickness (4 weeks:0.074± 0.05mm; 8 weeks:0.083±0.05mm) inside the HCC increased with time. The bone volume at 8 weeks (7.12±1.56mm3) was more than that of 4 weeks (4.69± 1.09mm3).The hard tissue slicing of the HA and HC shown that only fibrous tissue and adipose tissue was filled inside the scaffold and no bone could be observed, while in HCC scaffold a layer a new bone was evenly grown on the wall of the pore. The trabecular thickness of 8 weeks was thicker than that of 4 weeks. New blood vessels could be found in the pores of all scaffolds.Conclusions:The collagen/rhBMP-2 chitosan microspheres coated HA scaffold could successfully induce ectopic bone formation within the scaffold. The collagen/rhBMP-2 chitosan microspheres coating method was an ideal way to combine the scaffold with osteogenetic protein and construct a composite scaffold that had both osteoinductivity and osteoconductivity.