The Study On Tissue Engineering Scaffold Constructed By Collagen/True Bone Ceramic Of Different Particle Sizes And Its Physicochemical Properties

Objective:To construct cartilage tissue engineering scaffolds using true bone ceramic particles and collagen of different particle sizes,and explore the effect of the sizes of bone ceramic particles on the physical and chemical properties of the composite scaffolds.Methods:1.Preparation of composite scaffolds: separately mix large bone ceramic(1-2mm in diameter)and small bone ceramic(0.25-1mm in diameter)with 20mg/ml collagen according to a mass ratio of 3:2.Then prepare the large-and small-particle bone ceramic composite collagen scaffolds using freeze-drying method.At last,number the large-particle true bone ceramic composite type I collagen scaffold as group A,and the small-particle true bone ceramic composite type I collagen scaffold as group B.2.Detection of composite scaffold material performance: observe the general shapes of the two sets of scaffolds,and observe the structure of the scaffolds with electron microscope,including pore size,pore connectivity,and bone ceramic particle distribution.Measure the porosity,the water-absorption swelling rate and mechanical properties of the two groups of scaffolds,and analyze and compare the differences in the physical and chemical properties of the two groups of scaffolds.3.Extraction of primary chondrocytes: separate and culture rabbit ear chondrocytes,inoculate the second-generation chondrocytes on two sets of composite scaffolds for culture in vitro,and test the biocompatibility and toxicity of the scaffold materials to make clear the possibility of using composite scaffold materials to construct cartilage scaffolds.Results:1.General observation: the scaffolds in group A are generally white,loose and spongy,with regular forms,regular surface pores,and good elasticity;in group B,the small-particle true bone ceramic composite collagen scaffolds are generally white,loose and spongy,with irregular pores on the surface,and they’re soft and have good elasticity.2.Observation by scanning electron microscopy: the collagen in group A showed a dense and porous structure with uniform pore shape and uniform pore size.The pores are cross linked.The pore sizes range from 180 to 400 μm,with an average of 257 ± 79 μm.The large-particle true bone ceramicis scattered and embedded in the collagen,and the distribution is uneven.There are pores in the middle of the large-particle true bone ceramic,and the pores are connected to the outside world.The particle sizes of large-particle bone ceramic range from 1000 to 2000μm,with an average of 1139±42μm;the pore sizesare 360-622μm,and the average is 474±62μmμm.The scaffolds in group B show a dense and porous structure of collagen,which is not much different from that in group A.The small-particle true bone ceramic is embedded in the collagen and distributed evenly,and there are no pores on the surface of the small-particle bone ceramic.The particle sizes of small-particle bone ceramic are 250-1000μm and the average is 592±68μm.3.The porosity of the scaffolds in group A is(79.91±1.44)%,and the porosity of group B is(67.66±3.68)%,so the difference in porosity between the two groups is significant(P < 0.05);the water-absorbing swelling rate of the composite scaffolds in group A is(244.12±17.03)%,and that of group B is(193.83±5.83)%,so the difference in water-absorbing swelling rate between the two groups is significant(P<0.05).The elastic modulus of the scaffolds in group A is(0.672 ±0.022)MPa,while the scaffolds elastic modulus of group B is(0.502±0.047)MPa,so the difference in elastic modulus between the two groups is significant(P<0.05).4.The results of CCK-8 show that as the number of days of culture increase,the OD value gradually increases.The OD value of the scaffolds in group A is higher than that in group B,and the number of cells on the two types of scaffolds increase with the prolonged culture time.On the 3rd and 7th day,the number of cells on the scaffolds in group A is more than that on the scaffolds in group B,and the difference between the two groups has high statistical significance(p<0.001).The live/dead cell staining assay shows that both the two groups of scaffolds have no cytotoxicity.Conclusions:1.Two types of scaffolds are successfully prepared,namely,large-particle true bone ceramic composite collagen scaffold and small-particle true bone ceramic composite collagen scaffold.2.The cartilage tissue engineering scaffold made from large-particle calcined bone powder and collagen is superior to the small-particle true bone ceramic/collagen composite scaffold in terms of porosity,mechanical properties and biocompatibility,and is more suitable for research as a tissue engineering scaffold.