Construction Of Biomimetic Collagen Model And Its Application On In Vitro Study Of Breast Cancer

Construction of disease models in vitro is the current development trend for drug screening and pathological study.Nowadays,breast cancer metastasis has become a major enemy threatening women's health.In the extracellular matrix?ECM?of breast tumors,collagen I is deposited in large quantities and the collagen fibers present in an aligned manner.ECM model can be used to explore the occurrence and metastasis mechanism of breast cancer and to provide means for drug screening and therapy method.In the study,based on the structural analysis of collagen I fibers around breast tumor ECM,biomimetic collagen models were prepared using shear stressing.The structure and biomechanics characteristics of biomimetic collagen models and its effect on breast cancer cell migration rule were analyzed,and preliminary study of the application of the biomimetic collagen models in drug screening was explored.Osteoid-like model was produced via cell biomineralization on aligned collagen template,where the simulation of bone metastasis relative to breast cancer was studied.The main results are listed as follows:1.Collagen I in breast tumor ECM is abundant and presents its orderly alignment distribution in the form of corrugated fold according in vivo analysis.Non-aligned and aligned biomimetic collagen models were prepared by shear stressing using rat tail collagen I.On the aligned collagen model,the collagen fibers were arranged in a dense corrugated fold structure,which was similar to the native collagen fibers in tumor ECM in vivo.In addition,the aligned collagen model showed more hydrophilicity,less roughness and larger rigid by atomic force microscopy?AFM?in the wet state,which similar to the structure and physicochemical properties of tumor ECM in vivo,basically meet the needs of breast cancer research in vitro.2.Biomimetic collagen models could promote the proliferation of breast cancer MDA-MB-231 cells,and aligned collagen fibers of collagen model were more conducive to cell proliferation and adhesion.Compared with those cells on the tissue culture plate?TCP?and non-aligned collagen model,the cancer cells on the aligned collagen model were in long spindle shape with few pseudopods,which grew along the alignment of collagen fibers.AFM analysis of a single cell showed that the elastic modulus and roughness of the cancer cell decreased on the aligned collagen model,so the cells on the aligned collagen showed stronger"compliance".Affected by the changes of cell's behavior and the contact guidance of the aligned collagen fibers,the breast cancer cells migrated along the alignment direction of collagen fibers,and its migration rate was the fastest?about 19.52±1.12?m/h?on the aligned collagen model when compared with the unaligned one.The results indicated that the aligned collagen model could significantly guide the migration of breast cancer cells and its composition,structure and biomechanical properties were similar to the ECM of breast cell in vivo,which could be used in the study of migration mechanism of breast cancer in vitro.3.The application of biomimetic collagen models in drug screening were explored.At the dosage of paclitaxel?PTX?at a concentration of 18.31?g/m L(semi-inhibitory concentration IC₅₀),there was no significant difference of the inhibitory effect on cell proliferation and adhesion between on the non-aligned and the aligned one,and the inhibitory effect was lower than those on TCP.The cell on the biomimetic collagen models arrested at G₂/M phase that was significantly higher than the control group,but the apoptosis was slightly lower than the TCP.Similar to the TCP,the cell morphologies in both biomimetic collagen models changed from irregular polygon to spherical shape,and the cell migration rates varied slightly,both about 2.7?m/h.The results show that,on different biomimetic collagen models,there was no significant difference of the behavior of breast cancer cells under the treatment of PTX.The morphology and migration of cells were not changed by matrix material composition and surface structure when PTX was taken.These results proved the prepared collagen matrix models meet the basic needs for drug screening for breast cancer in vitro.4.Constructed an osteoid-like bone matrix model which is highly similar to the composition and structure of bone tissue.The possible mechanisms of bone metastasis relative to breast cancer was investigated by co-culture of breast cancer MDA-MB-231 cells and pre-osteogenic MC-3T3-E1 cells on the osteoid-like model.The study found that the spreading state of cancer cell changed from parallel to the surface of model to vertical to the surface,and the cell shape was elongated into a long spindle-shaped erosive to the osteoid.The cancer cells proliferated rapidly and invaded from the looser stroma to the surrounding areas,which indicated its strong osteolysis.After coculture,collagen I and hydroxyapatite in the osteoid model decreased rapidly;collagen fibers were broken,and the layered and aligned structure were seriously eroded.These findings revealed the basic process of bone metastasis relative to breast cancer to some extent.An in vitro ECM model with similar composition,structure and biomechanical properties to breast tumor ECM was successfully prepared.In particular,the aligned collagen model also had been applied to the study of osteolytic bone metastasis relative to breast cancer.The study not only successfully developed biomimetic models for breast cancer study,but also provided the foundation for further exploring disease model in vitro.