Experimental Study Of Attapulgite/type â…  Collagen/poly(caprolactone) Composite Scaffold Repaired Bone Defect

Bone defect is a common clinical disease, the traditional treatment methods such as bone graft have flaws about limited source and caused complications, especially for large bone defect. Development of materials science and tissue engineering technology have changed the traditional model of bone defects and improve the shortcomings of traditional therapy, it is expected to become an effective means to solve clinical bone defect repair. Bone defect repair scaffold is one of the hotspots of bone tissue engineering. Good bone substitute materials should have three-dimensional porous structure, mechanical strength, biodegradability,biocompatibility and osteoinductivity etc. A single material is difficult to fully meet the ideal requirements, so composite the materials of different properties to make up the shortcoming of a single material.This study constructed a new type composite scaffold materials of attapulgite/type I collagen /poly(caprolactone)(ATP/Col I/PCL) for bone defect repair. Through a series of experiments in vitro and in vivo to evaluate the characterization, physical and chemical properties, biocompatibility, osteoinductivity and bone defect repair effect of materials.Specific contents are as follows:1、Preparation and characterization of ATP/Col I/PCL composite scaffold materials: The ATP/Col I/PCL composite scaffold materials of different ATP contents(0% wt, 10% wt, 30%wt) were prepared by the solution perfusion-salt-leaching process and freeze drying technique.The characterizations of materials were evaluated by scanning electron microscopy, infrared spectroscopy, water absorption expansion rate,the surface of the water contact angle, porosity,permeability,three-point bending and compression test. The degradation ability of materials was tested by SD rat experiments in vivo. Comprehensive evaluated the physico-chemical properties of the composite materials.2、The biocompatibility and osteoinductivity of ATP/Col I/PCL composite scaffolds in vitro: The ATP/Col I/PCL scaffolds materials of three different content ATP were co-cultured with D1 cells, the biocompatibility was evaluated by scanning electron microscopy, CCK-8,IV phalloidine and H&E staining. The relative expression of bone-related genes including Runx-2,Osterix, ALP, Col I, OPN, and OC were measured by RT-q PCR after D1 cells co-cultured with materials for 7 d, 14 d, and 21 d, and evaluated osteogenic effect comparison of three scaffolds respectively.3、The effect of ATP/Col I/PCL composite scaffold materials repaired bone defect: built animal models of rabbit radius defect, twenty four experimental rabbits were made the bilateral radius bone defects of 15 mm and divided into groups A(no plant), B(Col I/PCL scaffolds)and C(ATP/Col I/PCL scaffolds). The repairing effect was observed by X-ray after 4,8, 12 W. The degradation of scaffold materials and bone repair effect were further evaluated by general observation, histology, immunohistochemical staining of regular decalcified samples. The contents of new bone in defect parts were tested by Micro CT of non-decalcified samples.The results showed that:1、The materials had high plasticity, hardness and three-dimensional porous structure,SEM showed that 0% ATP surface was smooth with C, N, O elements, while composite ATP,the surface was rough with more elements, including Si, Mg, Fe et al. The porosity,water absorption, permeability of 30%ATP were decreased significantly compared with0%,10% ATP, maximal compressed stress and bending stress were improved with the increase of ATP content, significantly difference compared with 0%,10% ATP(P<0.05). Subcutaneous30% ATP materials implanted SD rats after 21 d, materials abounded in the inflammatory cells invasion and pore forming organization, there was tiny part of the degradation, compared with0%, 30%ATP.2 、D1 cell growth well,there were calcium deposits after cultivated of osteogenesis induction medium. After co-culture D1 cell with materials, SEM, phalloidine and H&E staining showed that three scaffolds had suitable cell adhesion and CCK-8 method showed that cell appreciated in materials, especially in 30% wt ATP materials. The results of RT-q PCR showed that Runx-2 and ALP expression of 30%wt ATP was significant increased in earlier stage, but decreased in later stage; while Osterix, Col Ⅰ, OPN and OC expression was significant increased with the increase of the culture time and the ATP content(P<0.01).3、Animal model experimental results showed that the scaffold in the organisms with good biocompatibility, did not cause inflammation. X-ray and Micro CT indicated that ATP/Col I/PCL scaffold of new bone volumes was significantly higher than Col I/PCL scaffold. Histology and immunohistochemical staining results showed that ATP/Col I/PCL material of bone repair effect was superior to the Col I/PCL material, the degradation rate was lower.In conclusion, ATP/Col I/PCL composite scaffolds prepared by the solution perfusion-salt-leaching process and freeze drying technique in experiment, with three-dimensional porous structure, good physical and chemical properties, strong mechanical properties, good biocompatibility, appropriate degradation rate, osteoinductivity and the high potentiality of bone repir, it may be used as an ideal scaffold for bone substitute material,provide new scaffolds and ideas for the future development of bone tissue engineering and bone defect repair in clinical.