Preparation Of Micro Topography And Research On Its Application In Bone And Cartilage Repairing

The microenvironment in which cell reside can affect the cells proliferation, differentiation et.al. With the development of molecular biology, regenerative medicine, etc, people realized that the character of material surface such as hydrophilic-hydrophobic property, chemical component, topography etc can greatly affect cell fate. Micro-nano topography is one of the important factors which can influence the cell behaviors in vivo and vitro. It is significant to study its effect on cells’ hehaviros and the mechanism for embryonic development, tissue repairing, tissue engineering and regenerative medicine. It benefits from the development of microfabrication that humans can research the effect of micro-nano topography of material surface on cells and get much knowledge in recent years. It is more clear that the interaction between cells and their microenvironment where they stay. Besides, more basic theroy can be used to guide the fabrication of artificial tissue and surface treatment of implant.Nano topography can interact with molecule on cell surface and influence cell fate. Submicro topography can interact with organelle, influence cell morphology and fate. Micro topography can impact the organization, migration of collective cells, further more affect the morphogenesis and tissue healing. Most researches focus on the effect of submicro and nano topography on cells. Few researchers pay attention to the effect of micro topography on cells. Nano, submicro and micro are three different level structure of extracellular matrix and scaffold. Micro structure is very important for the macrosturcture formation of bone and cartilage. Besides, the structure of compact bone and cartilage are multiscale and well-organized and are closely related to their function. The researches on bone and cartilage repair focus on the gene expression and protein secretion, but pay little attention to the matrix organization and structrue. In this study, we hope build a magic micro topography surface which is composed a few kinds of shapes. The magic surface can guide cell migration and recruitment, then promote fracture healing and make integration of bone and cartilage repair to be possible. Besides, the different shape micro topography on the magic surface can promote cell osteogenic differentiaton or chondrogenic differentiation in different sites. The micro topography can guide the organization of collective cells at micro meter and guide the deposition of extracellular matrix. Their structure maybe will similar to the nature structure of bone and cartilage. This will be better for tissue repair and function reestablishment.There are so many methods to fabricate topography. Soft-lithography is used to prepare micro topography on biomaterials surface. Thick photoresist of Fururrex company is choose in this study. The photolithography technique of thick photoresist is very different from traditional photolithography technique because its thick is to a few tens to hundreds micrometers. Combining the character of thick photoresist and the condition of our laboratory, we optimized the parameters of photolithography. Based the photolithography of thick photoresist, using a few methods of soft-lithography, different shape micro topography with various dimensions were made on the surface of polycaprolactone(PCL), polymethyl methacrylate(PMMA), chitosan(CS), bioglass(BG). BG/PCL compound surface also be made.Mesenchymal stem cells attracted much interesting in researching of bone and cartilage repairing. In this study, hMSCs exhibited complex behavior in addition to “contact guidance” on micro-grooved substrates. A unique growth pattern was demonstrated that hMSCs spanned across grooves with 100μm widths between adjacent plateaus. It is benefit to tissue repair or not? So this phenomenon was systemly and deeply study by us. It was discovered that hMSCs on the bottom of groove spanned across grooves by climbing the side walls of grooves. Besides, only when the ratio of groove width to depth was less than two, cells able to be spanned across a groove. Furthermore, disorganized parallel actin stressfibers and enhanced “actin edge-bundles” were obviously revealed by characterization of F-action. However, according to results from AFM, the stiffness of bridging hMSCs was not different from that of normal spreading hMSCs. The result of osteocalcin immuofluorescent staining, alizarin red S and alkaline phosphate staining indicate that the bridging hMSCs were not osteogenic differentiation. A part of bridging hMSCs expressed desmin especially the strentched hMSCs according to desmin immuofluorescent staining. In addition to hMSCs, ATDC5, MG-63 and C2C12 also can span across the micro groove, and their behaviors are different with each other.In order to reseach the effect of topography on fracture healing and its ability to recruit cell to injury site, we used a wound healing to study the effect of microgroove on cells directed growth in vitro. Microgroove could promote the MG-63 growth along the direction parallel to the microgroove, but prevented MG-63 growth along the direction perpendicular to the microgroove. Moreover, the groove widths did not show obvious influence on the speed of MG-63 growth along the direction parallel to the microgroove whereas the smaller groove depths tended to favor the MG-63 growth along the direction parallel to the microgroove. The microgrooved substrates accelerated / prevented the MG-63 growth along the direction parallel / perpendicular to the microgroove by facilitating / hindering the collective cell migration into the wound gaps but not by promoting / inhibuting the cell proliferation. Cells on the edge of groove were first polarized and experienced “contact guidance”. As cellular proliferation progresses, the cells experiencing “contact guidance” by the groove edge could transmit the signal from microgroove to other cells, then the collective cells were polarized and migrated along the direction parallel to the microgroove. Addition to MG-63, D25W200 microgrooves were also found to promote the growth of human mesenchymal stem cells(hMSCs) and C2C12 along the direction parallel to the microgroove, but not ATDC5.Finally, we study the influence of microtopography on osteogenic differentiation and chondrogenic differentiation of cells. The result of osteogenic differentiation of hMSCs on D25W200 PCL microgroove surface indicated that osteogenic differentiation of hMSCs was not improved on the microgrooved substrate. Collagen I and minerals deposited by hMSCs were in an aligned pattern. Such alignment was similar to the organization of the same components in natural bone. BG/PCL compound surface could influence the spreading, morphology and distribution of hMSCs, but did not promote its osteogenic differentiation. The micro pits on PMMA surface could accelerate ATDC5 aggregation into the pits, and promoted collagenΙ and glycosaminoglycan gene expression, secretion of cartilage matrix.These findings suggest the necessity of using combined topography of microgroove and micropit for enhancing fracture healing, guidancing stem cells to migrate to cartilage injury site, organization the cell matrix and realization of integration of bone/cartilage repair.