The Preliminary Research On The Immortalized Mouse Cranial Suture Stem Cells Differentiation Induced By BMP9

Background:The bony defects are very common which are caused by the traumatic, tumorous, degenerative or congenital reasons. Although orthopedic fixation and surgical techniques have greatly improved, it is still a great challenge in bone reconstruction. The main reason is the restrictions on the amount and autologous bone donor site. With the development of regenerative medicine and bone tissue engineering, it provides a new choice for bone reconstruction.Seed cells, biomaterials, bioactive factors are the three elements of bone tissue engineering. The seed cells are the most basic element in the bone tissue engineer. As the seed cells provide the main source of the active ingredient in the regenerative medicine and tissue engineering, so it is the core part. The stem cells have pluripotent differentiated ability, therefore, are the most widely used as seed cells currently.Cranial suture tissue has a strong proliferative ability, it plays a crucial role in the formation of the skull in its first three decades of life. Not as the long bone, the osteogenesis of crania mainly through intramembranous ossification. The mesenchymal stem cells in cranial suture accumulate at the ossification sites which overlapping each other when under certain condition. Studies have shown that cranial sutures stem cells have the differentiation ability. So through research on the cranial suture stem cells, not only we could further explore the mechanism and character of intramembranous ossification, explore a good seed cells in regenerative medicine and tissue engineering, but also make sense in some cranial sutures diseases, such as Craniosynostosis.In normal circumstances, the growth of normal tissue cells often have the growth arrest after cultured for some time in vitro. That is called M1 (aging period) stage. When the cells continue to proliferate, it will come into M2 (the crisis period) stage. And then the cells gradually degraded, and death will happen. So it brings many restrictions to regenerative medicine and tissue engineering, especially those rare or hard to obtained. The cell immortalization can provide the cells have a continuous proliferative ability, and could provide enough seed cells to regenerative medicine and tissue engineering. So it benefits the conducive to experimental research.BMPs belong to the TGF-β super-family which has at least 20 members, and BMPs are expressed in most species. BMPs play a wider role in the body, in addition to widely known of ability of osteo-inducetion, it participates in a variety of tissues and organs formation and repair. Many BMP had been reported that had the osteogenesis induce ability, and BMP2, BMP4, BMP6, BMP7, BMP9 were most reported. Our previous experiments showed, BMP9 had the strongest effect of osteogenic differentiation ability in MSCs, as well as adipogenic and chondrogenic ability under certain conditions.Biomaterials are very important part of regenerative medicine and tissue engineering, and the regenerative medicine and tissue engineering development is a great extent due to biomaterials development. Nowdays, it applied in more and more scientific experiments and clinical applications. The basic properties of biomaterial are to provide a three-dimensional structure for cells migration, proliferation and differentiation. In some weight-bearing areas, it can also play a temporary mechanical structure support. An ideal scaffold material required to meet the following characteristics:① the three-dimensional and porous structure of biomaterial is conducive to cell growth, nutrients and metabolites transport.② surface properties is conducive to cell attachment, migration, proliferation and differentiation.③it has a good biocompatible feature, not cause an immune response and has a controllable biodegradability rates in the growth of cells/tissues.④ mechanical properties should be fair with regional tissues⑤ it should have the plasticity. PCCN is a new scaffold that has the above features, and also has the temperature-sensitive and controllable re-shaping features. To validate the role of the novel scaffold materials in bone and cartilage repair, we conduct PCCN composite the immortalized cranial suture cells induced by BMP9 in vivo, and to verify the role of novel scaffold in tissue engineering.Objectives:1 To construct the immortalized mouse cranial suture cells by piggyBac system, and test if they have the stem cells marker.2 Compare the proliferation rate differences between the primary mouse cranial suture cells and the immortalized mouse cranial suture cells.3 To verify whether the immortalization of iSCCSs could be revised and monitor the whether they have tumorigenic tendency.4 To identify the potential differentiation ability of iSCCSs induced by BMP9 in vitro.5 To identify the potential differentiation ability of iSCCSs induced by BMP9 in vivo and the effect of PCCN in bone or cartilage formation.Methods1 Chose the one-week old sterile CD1 mice, and then sacrificed by cervical dislocation after onset of anesthesia. Aseptic expose the cranial suture, and obtained the primary cranial suture cells from the cranial suture by using collagenase type I dissolution method. Then transfected the immortalized gene SV40T antigen into the primary cell that conducted by piggyBac system to get the immortalized cranial suture cells.2 Test whether the iSCCSs have the mesenchymal stem cells marker through immunofluorescent staining.3 Compare the proliferation differences between primary SCCSs and iSCCSs by observations, crystal violet staining, WST-1 in vitro.4 Using the Flip/FRT recombination system to reverse the immortalization, and test the whether they could be reversed through observations, crystal violet staining, WST-1 and Touch-down qPCR methods in vitro. Identify whether the iSCCSs have tumorigenicity risk by imaging tests in vivo.5 Induced the iSCCSs with Ad-BMP2, Ad-BMP4, Ad-BMP6 Ad-BMP7, Ad-BMP9 through adenovirus method, and compare the osteogenic inductive ability of the BMPs by ALP staining, ALP activity reading, Alizarin red staining and Touch-down qPCR method.6 Induced the iSCCSs and other three stem cells (iCALs, iSCAPs, iMEFs) which had already been verified with a strong osteogenic ability by BMP9, and verify the osteogenic ability of the iSCCSs.7 Detect the time and dose-related correlation of iSCCSs induced by BMP9 through ALP staining and ALP activity readings. Test the OCN (Late osteogenesis index) expression induced by BMP9 through immunohistochemical staining method. Test the chondrogenic and adipogenics ability of iSCCSs induced by BMP9 through Alician Blue staining and Oil red O staining, respectively. And application Touch-down qPCR to identify relevant osteogenic, adipogenic, chondrogenic gene expression.8 Inject the iSCCSs induced by BMP9 with or without the new biomaterial PCCN into the subcutaneous and intramuscular area. And detect the osteogenic inductive ability of BMP9 and the effect of PCCN in bone formation. Conduct the HE staining, Alician Blue stain, Trichrome staining to test the osteogenic, adipogenic, chondrogenic ability of BMP9 to iSCCSs by the paraffin section of osteogenesis mass, and also check the effect of PCCN in bone and cartilage formation.Results:1 Successfully got the primary cranial suture stem cells, successfully constructed the immortalized mouse cranial suture cells by piggyBac system.2 About the mesenchymal stem cell marker, CD29 (intergin β1), CD73, CD113 (proml), CD40, CD90 (Thyl), CD117 (c-kit), CD166 (ALCAM), CD105 (Endglin) and BMPRII were expressed through immunofluorescence staining, Confirmed iSCCSs maintained mesenchymal stem cells properties.3 Through observations, crystal violet staining, WST-1 test. The iSCCSs had a greater proliferative capacity to the primary cells, the difference in the proliferation of primary and immortalized cells exist at the first day, and there are obvious differences in the third, five, seven days.4 After using the Flip/FRT recombination system to reverse the immortalization, through observation, crystal violet staining, WST-1 test. In the Ad-Flip group, the cells proliferation rate was significantly slower in the seven consecutive days, while the control group (Ad-GFP group) cell proliferation remains strong. Touch-down qPCR results showed SV40T antigen gene expression in the experimental group was significantly lower than the control group. It can be confirmed after SV40T antigen was removed the immortalization can be reversed.5 Successfully constructed a fluorescent luciferase (Fluc) labeled iSCCS cells (iSCCSs-Fluc) through piggyBac system. After the iSCCSs-Fluc subcutaneously injected, the imaging result showed at five-day the signal is strong, signal attenuation occurred at 10-day, at 15 days the signal had disappeared. No tumor sample was observed at the injection site. Indicates iSCCSs has strong proliferation without tumorigenicity.6 Early osteogenesis index (ALP staining, ALP readings) and late osteogenesis index (alizarin red staining) confirmed BMP2, BMP4, BMP6, BMP7, BMP9 had osteo-inductive ability in iSCCSs. But BMP2 and BMP9 had the stronger osteo-inductive ability, and BMP9 is the strongest one. The results of qPCR of osteogenic gene ALP, OPN, OSX, RUNX2 also supported the conclusion.7 The ALP staining and ALP readings showed the osteogenic capability of iSCCSs induced by BMP9 is strongest when compare to iCALs、iSCAPs、iMEFs in vitro, which had already a strong osteogenic ability induce by BMP9.8 To some extent, there were time and dose-related correlation of BMP9 that influence the osteogenic ability of iSCCSs.9 Immunofluorescence staining showed that the late Osteogenesis index OCN confirmed a high expression induced by BMP9.10 Oil Red O staining showing the cells induced by BMP9 showed vacuolar, red-staine. And the PPAR y 2 gene was unregulated. So the iSCCSs had the potential of adipogenesis.11 The cells mass of BMP9 groups were blue dye, and the SOX9 was unregulated. So the iSCCSs had the potential of chondrogenesis.12 Micro-CT showed:Either the cells only, or composite scaffolds, either subcutaneous or intramuscular injection, a visible high-density bone formation could be observed in BMP9 groups. The cortical bone, trabecula and other structure were visible. The GFP group showed no bone formation at a higher relative threshold. When increased the threshold to 950, the bone formation in the BMP9 group was still in a state of high density. It may be concerned with the region rich blood supply that conducted the scaffold a promotion of osteogenesis.13 HE staining showed:both of subcutaneous and BMP9 group could be observed the mesh mineralized structures, and the structures were interconnection. That suggested BMP9 could induce the iSCCSs into osteoblast in vivo. In the mass, the surrounding area had a better cell differentiation, and some of the adipocytes could be observed in this area. Compared with the BMP9 cells only group, the mesh structure of bone formation is connected more closely, extensive and more layers in the composite PCCN scaffolds group. PCCN was conducive to the formation of mature bone. Compared with the subcutaneous group with intramuscular group that with PCCN. The trabecula formed a higher maturity structure in the intramuscular group, which was similar with Micro-CT results. So a rich blood supply was conducive to bone formation.14 Alcian Blue staining showed a blue dye reticular structure was observed. Suggested BMP9 could induce iSCCSs into chondrocytes.15 Trichrome staining showed:a large number of reticular dark blue tissue formation. That suggested a large amount of collagen fibers and cartilage matrix formed. Further evidence BMP9 can effective differentiate the iSCCSs into osteoblasts and chondrocytes in vivo.Conclusion:1 The iSCCSs could be cultured pass multi-generation for a long time with the strong proliferative ability. And the iSCCSs retain the stem cells marker, suggesting that they may have MSC-like phenotypes.2 The iSCCSs’proliferation rate was significantly higher than that of primary cells. Flip recombinase can reverse the iSCCSs’proliferative activity. The iSCCSs have no tumorigenicity.3 Compare with other BMPs, BMP9 had the strongest effect of osteogenic differentiation ability to iSCCSs. In some extent, it has the time and dose-related correlation4 iSCCSs have a strong osteogenic ability induce by BMP9 in vitro.5 iSCCSs are capable of differentiating into multiple lineages (osteogenic, chondrogenic, adipogenic) upon BMP9 stimulation in vitro and in vivo.6 PCCN has the capabilities of cell adhesion, proliferation, differentiation and could provide better three-dimensional layered structure. And it is conductive to differentiate into chondrocytes and osteoblasts upon BMP9 stimulation.