| BackgroundCraniofacial cartilage defect is a common and difficult disease to treat in the field of plastic surgery.Common craniofacial cartilage defects include congenital nasal deformity,microtia,and ear and nasal cartilage defects caused by trauma.Due to the lack of nerves,blood vessels and lymphoid tissue in cartilage tissue,the regeneration capacity is limited,making it difficult to repair once damaged.At present,the methods for the treatment of cartilage defects mainly include autologous cartilage transplantation and allogeneic cartilage transplantation.However,such clinical treatment methods also have many shortcomings,such as large injury to patients,many postoperative complications,limited cartilage sampling,absorption after autologous transplantation,and immune rejection after allogeneic transplantation.Therefore,many scholars have been committed to seeking new treatment options.In recent years,the vigorous development of cartilage tissue engineering has provided new ideas and methods to solve this problem.Cartilage tissue engineering is an emerging discipline in which seed cells are planted on biocompatible materials and form complexes,and then the complexes are implanted into cartilage defects to repair damaged cartilage.The construction of tissue-engineered cartilage requires three key elements:（1）a suitable scaffold,（2）a sufficient number of seed cells with normal function,and（3）cytokines that promote cell proliferation and maintain cell phenotypic characteristics.As one of the three elements,the scaffold plays a crucial role in the successful construction of cartilage.An ideal cartilage tissue engineering scaffold should have the following characteristics:（1）sufficient mechanical strength,（2）suitable biodegradability,and（3）excellent biocompatibility.The scaffold should not only support cell survival,but also promote cartilage formation.It is difficult for a single biological material to satisfy all the above characteristics.Therefore,composite scaffolds come into being and are widely used in cartilage tissue engineering.Because the cartilage defect area of the craniofacial is small and the shape is irregular,the requirements for the scaffolds are more complicated.Larger scaffolds not only consume a lot of chondrocytes,but also have a difficult shape to meet the needs of implantation.Therefore,the construction of craniofacial cartilage scaffolds not only needs to meet all the needs of tissue engineering scaffolds,but also has the advantages of small size and adjustable shape.In this experiment,we chose to construct micro-scaffolds.The diameter of micro-scaffolds is small,generally around a few millimeters.Not only can the shape be adjusted by arranging multiple micro-scaffolds,but also the number of seed cells is much lower than that of general scaffolds,which are suitable for the construction of craniofacial cartilage scaffolds.Type I collagen accounts for 80%-90% of all collagen and is expressed in almost all connective tissues.The wide sources of type I collagen,simple extraction and good biocompatibility make it one of the most promising biomaterials in cartilage tissue engineering.However,the poor mechanical properties of type I collagen limit its use alone.Therefore,it is necessary to add other biological materials to increase the mechanical strength of the scaffold.True-bone-ceramic（TBC）is a calcium phosphate-based biomaterial obtained by twice calcining fresh bovine cancellous bone at high temperature.Large particle TBC has a small diameter of about 1-2 mm,has a porous structure similar to human bone tissue,has good mechanical strength and no cytotoxicity,and can be used as the skeleton structure of the scaffold.Using large particle TBC as the main body of the scaffold has the following advantages:（1）the need for seed cells is less,（2）the shape can be flexibly adjusted,and（3）the connected porous structure is conducive to cell growth.However,due to its lack of cell adhesion,it is rarely used alone,and other materials are often added to compensate for this weakness.Therefore,in this study,we constructed a type I collagen-coated largeparticle TBC（COLI/TBC）micro-scaffold.The large-particle bone TBC was used as the main body of the scaffold to ensure the mechanical strength,and the shape of the scaffold could be flexibly adjusted.In the meanwhile,the biocompatibility of micro-scaffolds could be increased by type I collagen coating.In order to explore the appropriate concentration of type I collagen,we set up three concentration of type I collagen（1mg/ml,5mg/ml,12mg/ml）and evaluated the physicochemical properties of the micro-scaffolds in detail in vitro and vivo.And the possibility of constructing tissue engineered cartilage with this micro-scaffold was evaluated by in vivo implantation experiments.Objective1.To evaluate the physical and chemical properties of large-particle TBC micro-scaffolds coated with different type I collagen concentrations.2.To evaluate the cartilage formation ability of the large-particle TBC micro-scaffolds coated with different type I collagen concentrations in vitro.3.To evaluate the cartilage ability of the large-particle TBC microscaffolds coated with different type I collagen concentrations in nude mice.MethodsPart 1 Characterization of COLI/TBC micro-scaffolds1.Use light and electron microscopes to observe the structure of the micro-scaffolds and the coating of type I collagen.2.Detect the swelling performance,degradation performance and chemical structure of the micro-scaffolds,and analyze the differences in physical and chemical properties between different micro-scaffolds.3.Isolate and culture chondrocytes,and evaluate the possibility of each group of micro-scaffolds being applied to chondrocyte experiments via CCK8 assay.Part 2 Cartilage formation of large-particle TBC micro-scaffolds coated with type Ⅰ collagen in vitro.1.Use the live/dead cell staining kit to detect the growth and distribution of cells on the micro-scaffolds at different culture time.2.Electron microscope was used to observe the growth of cells cultured on the scaffold at different culture time.3.HE staining,Safranin O staining were used to detect the growth and cartilage ability of cultured cells in vitro.4.Histochemical staining was used to detect the expression of cartilage-related proteins in vitro.Part 3 Study on cartilage formation of large-particle TBC microscaffolds coated with type Ⅰ collagen in vivo1.The scaffolds-cell complexes cultured were implanted in nude mice in vitro2.Observe the general changes in implants at specific time.3.HE staining,Safranin O staining and immunohistochemistry were used to observe cartilage formation at specific time.ResultsPart 1 Characterization of large-particle TBC Micro-scaffolds coated with type Ⅰ collagen1.Both the light microscope and the electron microscope showed the uniform attachment of type I collagen on the large particle bone powder.With the increase of collagen concentration,the collagen fibers on the surface of the large particle bone powder were arranged more closely.2.With the increase of collagen concentration,the swelling rate of the COL1/TBC scaffolds increased,but there was no significant difference in the degradation rate.FTIR analysis showed obvious characteristic functional groups of type I collagen and characteristic functional groups of TBC.3.The chondrocyte viability cultured by the experimental groups has no significant difference with the control group.It can be seen that the micro-scaffolds have no cytotoxicity and can be used for cell experiments.Part 2 Cartilage formation of the large-particle TBC micro-scaffolds coated with type Ⅰ collagen in vitro1.With the increase of in vitro culture time,the number of cells in each group increased to varying degrees.And with the increase of collagen concentration,the number of cells increased significantly.2.Chondrocytes cultured for 4 weeks secreted a large amount of extracellular matrix,and the increase in collagen concentration significantly promoted the secretion of extracellular matrix and the expression of chondrogenesis-related proteins.Part 3 Study on cartilage formation of large-particle TBC microscaffolds coated with type Ⅰ collagen in vivoThe three groups of micro-scaffolds-cell complexes were intact when implanted in nude mice for 4 weeks,and the results of immunohistochemistry showed strong positive expression of COLII.The increase in collagen concentration significantly improves the level of cell proliferation,cartilage formation and protein expression.Conclusions1.Type I collagen-coated large-particle TBC micro-scaffolds have good physicochemical properties and biocompatibility,and can be used as scaffolds for tissue-engineered cartilage.2.The type I collagen coating increases the cell adhesion of the microscaffolds.3.The higher the collagen concentration,the better the cartilage formation performance of the composite scaffold in vivo and in vitro,and the collagen concentration of 12mg/ml is more suitable as a tissue engineering cartilage scaffold. |
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