| BackgroundOsteoporosis(OS)is a kind of systemic bone disease characterized by delayed fracture healing,bone non-union and bone defect due to decreased bone healing and regeneration caused by abnormal bone metabolism.Across the world,the annual medical costs,as well as the social and economic investments associated with osteoporosis,are huge and the input is increasing as the average life expectancy of human beings increases.Osteoporosis with bone defects is one of the difficult problems in the clinical work.Although many types of tissue engineering scaffolds have been developed by experts and scholars at home and abroad,an ideal scaffold has not been found yet.The ideal bone repairing scaffold should have the following characteristics,scaffold surfaces should allow for cell adhesion and growth,scaffold degradation products do not cause inflammation or toxicity,the scaffold should be able to provide suitable mechanical support to avoid destructive compression and stretching of the cells,the scaffold should have the capability to enhance osteoblast activity to promote osteogenesis.At present,the commonly used bone repair materials include Natural organic polymer,such as Collagen,fibrin,chitosan;or Natural inorganic polymer,such as Coral hydroxyapatite,ceramic bone;Synthetic organic polymer: Polylactic acid,polyglycolic acid;Synthetic polymer: Hydroxyapatite,tricalcium phosphate;Metallic material: Titanium,tantalum,etc.Chitosan,made of de-acetyl chitin,is the only kind of alkaline glycosaminoglycan in the polysaccharide.Its structure and certain nature are very similar to glycosaminoglycan as the main component in extracellular matrix.As a scaffold material,the chitosan has a good biocompatibility and controllable degradation,and its degradation products have no toxic effects.But the shortcomings of chitosan are: common steoporotic effect and poor repair effect of osteoporotic bone defect;ordinary the mechanical property and difficult to be applied in the bone defect at the load-bearing site.In order to solve these problems,many scholars have sttempted to prepare composite materials.Bayer et al prepared the polymer composed of calcium phosphate and chitosan,and formed a bone tissue-like structure that can modify some of its properties by adjusting the compositional proportions of the material.However,such scaffold is of low elasticity modulus and insufficient mechanical support.Moreover,the osteogenic capability of such scaffold material has not been further studied.Therefore,the materials that constitute the polymer with chitosan should have a higher elasticity modulus and better osteogenic capability.Tantalum has a good biocompatibility and stronger osteogenic capability.However,the dense tantalum materials cannot be directly implanted into the human body due to large specific gravity and high elasticity modulus.We have modified the chitosan scaffold and prepared the tantalum-containing chitosan composite scaffold.Can the larger elasticity modulus and stronger osteogenic capability of the tantalum material make up for the deficiency of the chitosan material in the mechanical properties and osteogenesis capability? Whether the composite scaffold can provide the mechanical support and osteogenic capability required for the repair of osteoporotic bone defects? In the present study,the mechanical properties and biologic characteristics of the tantalum-containing chitosan composite scaffolds and the repairing capability of osteoporotic bone defect in the animals are analyzed and discussed.Objectives1.Study the mechanical properties of the tantalum-containing chitosan composite scaffolds.2.Test the effects of the tantalum-containing chitosan complex scaffolds on the adhesion,proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells in order to evaluate the biological characteristics of scaffolds.3.Use the osteoporotic rabbit models to detect the capability of tantalum–containing chitosan composite scaffolds to repair osteoporotic bone defects in animals.Methods1.To investigate the mechanical properties of the tantalum-containing chitosan composite scaffolds,the load-displacement curves of the scaffolds were measured by ElectroForce 3100 Test Instrument.At the initial stage of the load-displacement curve,the relatively smooth linear part of the curve is taken as the analysis object to calculate the elasticity modulus(E)of the scaffold.2.To investigate the biological characteristics of the tantalum-containing chitosan scaffolds,the rat BMSCs were cultured in ?-MEM medium containing 10% fetal bovine serum and cultured in an incubator.Fresh medium was exchanged every other day until the confluence was reached,it was digested with 0.25% trypsin and the cell suspension was suspended and prepared in serum-containing medium.3.To investigate the effect of the tantalum-containing chitosan scaffolds on the adhesion of rat BMSCs,the cultured rat BMSCs were inoculated into the chitosan scaffold and tantalum-containing chitosan scaffold respectively.CCK-8 method was used to measure the OD value at a wavelength of 450 nm at 1h,2h,4h and 8h respectively,indirectly reflecting the effect of the scaffold on the cell adhesion.4.To investigate the effect of the tantalum-containing chitosan scaffolds on the proliferation of rat BMSCs,the cultured rat BMSCs were inoculated into the chitosan scaffold and tantalum-containing chitosan scaffold respectively.CCK-8 method was used to measure the OD value at a wavelength of 450 nm at 24 h,48h,72 h,96h and 120 h respectively,indirectly reflecting the effect of the scaffold on the cell adhesion.5.To investigate the effect of tantalum-containing chitosan complex scaffold on the osteogenic differentiation of rat BMSCs,the cultured rat BMSCs were inoculated on the chitosan scaffold and tantalum-containing chitosan composite scaffold respectively and the induction culture was carried out with osteogenic induction medium including ascorbic acid,sodium ?-glycerophosphate and fetal bovine serum.The degree of mineralization of mesenchymal stem cells was evaluated by alizarin red staining on 7th day and 14 th day and by ALP staining on 21 st day and 28 th day.On the 7th,14 th,21st and 28 th day,the concentrations of calcium ion absorbed in the mineralization of the samples were quantitatively analyzed,indirectly reflecting the effect of scaffold on osteogenic differentiation.Osteogenesis-associated gene expression was determined by Real-time PCR method on the 28 th day after osteogenesis induction,and the expression of osteoblast-associated target protein was determined by Western blot method.6.To investigate the capability of tantalum–containing chitosan composite scaffold to repair osteoporotic bone defects,the female New Zealand white rabbits whose osteoporosis models had been successfully established were randomly divided into two groups,the bone defects were made in lumbar vertebrae and then the chitosan scaffold and tantalum–containing chitosan composite scaffolds were implanted in the bone defect sites.After 12 weeks,the vertebral bodies were removed,the specimens were scanned by Micro-CT and the bone volume fraction,the number of bone trabecula,the thickness of bone trabecula and the bone density around the scaffold were detected and statistically analyzed.The hard tissue sections observed under microscope afetr stained with 1% toluidine blue and the area ratio of bones around the scaffold was detected and statistically analyzed.Results1.Mechanical properties of the composite scaffoldsThe tantalum-containing chitosan composite scaffolds are of good mechanical properties.The elasticity modulus of the composite scaffold was 3.07 ± 0.12 GPa and the elasticity modulus of the chitosan scaffold was 2.38 ± 0.69 GPa.The elasticity modulus of the composite scaffold was higher than that of the chitosan scaffold,and the difference was statistically significant(P <0.05).2.Effect of the complex scaffold on adhesion of rat BMSCsTantalum-containing chitosan complex scaffold had a better promoting effect on adhesion of BMSCs.At 1h,2h,4h and 8h,the adhesive capabilities of the cells on the composite scaffold surface were higher than that on chitosan scaffold,and the difference gradually increased.Effect of the complex scaffold on proliferation of rat BMSCsTantalum-containing chitosan complex scaffold had a better promoting effect on proliferation of rat BMSCs.At the 24 h,48h,72 h,96h and 120 h,the proliferation capability of the cells on the surface of the composite scaffold was higher than that of the chitosan scaffold,and the difference was gradually increased.3.Effect of the complex scaffold on differentiation of rat BMSCsTantalum-containing chitosan complex scaffold had a better promoting effect on osteogenesis differentiation of BMSCs.The positive level of alkaline phosphatase in cells on the composite scaffold was significantly higher than that on the chitosan scaffold.The number of calcium nodules on the composite scaffold was significantly higher than that on the chitosan scaffold,from the 7th day after the osteoinductive differentiation medium was changed,the calcium ion absorption degree of the cells on the composite scaffold surface was greater than that on the chitosan scaffold,and the difference was gradually increased.After the osteogenic induction for 28 days,the expression level of osteoblast-associated gene of the cells on the surface of the composite scaffold determined by Real-time PCR method was higher that of the chitosan scaffold,the expression level of osteoblast-associated target protein of the cells on the surface of the composite scaffold determined by Western blot method was 3-5 times higher than that of the chitosan scaffold.4.The capability of the composite scaffold repairing osteoporotic bone defect.Tantalum-containing chitosan composite scaffolds have a better repair effect on osteoporotic bone defects.Micro-CT showed that the volume fraction of bones around the composite scaffold,the number of bone trabecula,the thickness of bone trabecula and the bone density were higher than those of the chitosan scaffold and the hard tissue section indicated that the area ratio of the composite scaffold was higher than that of the chitosan scaffold.ConclusionOur study results show that tantalum-containing chitosan composite scaffold not only has good mechanical properties,but can also facilitate the adhesion,proliferation and osteoblastic differentiation of cells on the scaffold surface and obviously promote the repair of the osteoporotic bone defects.The tantalum-containing chitosan composite scaffold is expected to be a kind of bone repairing material with a good application prospect. |
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