| Joint is vital for the movement function of the bone animals such as human,which plays an important role in activities,the normal bone & articular cartilage structure presents obvious gradient progression and complex physiological characteristics.Articular cartilage is a termin al differentiation multiphase connective tissue contained a small number of cells and had no blood vessels or nerves coated on the joint surface.Among articular cartilage,the cell and collagen fibers also have complex physiological gradient progression.Articular cartilage is frequently used in various activities,which can easily lead to lesions and injuries.At present,the effect of several repair methods is very limited;therefore,looking for innovative repair method is the effort direction of scientific research and medical workers.In recent years,the rapid development of tissue engineering technology has provided new ideas for cartilage repair.Most of the existing tissue engineering studies focused on the problem of biocompatibility of scaffold materials.It is very rare for studies that combine establishing the physiological stratified structure and preparing scaffold materials with good biocompatibility.Natural materials with complementary properties of Chitosan(Cs),?-Sodium Glycerophosphate(GP),Gelatin(Gel)as raw material are used in this paper.We bases on the multi-coupling biomimetic concept,gives full play to the functional advantage of each raw materials,uses the permeator-type reaction device to form the gradient pore structure,adjusts the ratio of raw materials to adjust the aperture of scaffolds,uses freeze-drying technology to generate pores.Firstly,Cs/GP/Gel biomimetic cartilage gradient scaffold materials which are similar to the natural structure of cartilage and have good biocompatibility and biodegradability are prepared.The results show that the gradient scaffolds have many commonalities: the three-dimensional porous structure is excellent,the aperture is between 20 ~ 180 ?m,the pore channel is interconnected,the pore wall have many fold,and the scaffolds present obvious physiological stratified structure and gradient progression of aperture in the height direction.With the content increase of Cs,the aperture of gradient scaffolds gradually increases,until the large aperture is visible,and the gradient phenomenon becomes unobvious.The aperture of control group scaffolds is uniform,and has no gradient above,which proves the gradient scaffolds prepared to be a good simulation of the physiological stratified structure of real cartilage from the side.To prepare scaffolds which have more similar structure and performance to natural cartilage and have good biocompatibility and physicochemical properties,nano-Hydroxyapatite(nHAp)is composited in Cs/GP/Gel hydrogel to build gradient scaffold materials(served as cartilage calcified layer).The biomimetic cartilage layer in the upper of the whole gradient scaffold commendably maintains the good pore characteristics of Cs/GP/Gel gradient scaffold materials,indicating that the gradient phenomenon has a good repeatability.The biomimetic cartilage calcified layer in the middle of the whole gradient scaffold is well connected with the biomimetic cartilage layer in the upper of the whole gradient scaffold;The aperture of biomimetic cartilage calcified layer at the bottom of the whole gradient scaffold is very smal,which can effectively obstruct chondrocytes and osteoblasts on both sides.To explore the effect of adding in graphene(Gr)on the biocompatibility of scaffolds and the effect of electromagnetic stimulation on BMSCs in scaffolds,Gr is doped into Cs/GP/Gel hydrogel to give scaffold a certain electromagnetic conduction function to prepare Cs/GP/Gel/Gr electromagnetic scaffold materials.The results indicate that the scaffolds present excellent three-dimensional porous structure microscopically and the pore wall is gray,pleated and the Gr lamellas can't be observed in the pore wall,which is the same as the control group.Water absorption rate and porosity are between 250.28±13.16 ~ 424.27±19.32% and 74.81±1.21 ~ 85.42±2.59% respectively,which is slightly lower than the control group under the same condition,but the elastic modulus was generally higher than the control group with the same ratio.The Gr lamellas are continuously and uniformly inlaid on the wall of scaffolds,which will not change the pore distribution of scaffolds and will provide greater support for pore wall to reinforce it.To test the biocompatibility of scaffolds above and to test the effect of electromagnetic stimulation on cells in scaffolds,bone marrow mesenchymal stem cells(BMSCs)extracted from SD rat are inoculated into each piece of scaffolds in the same cell density.(1)Cs/GP/Gel biomimetic cartilage gradient scaffold materials: The number of cells also presents gradient arrangement on the gradient direction of aperture,the regions which have smaller aperture and the more coherent pore channel survives more cells.A large number of cells have adhered to the scaffold walls in 24 h,and the cell synapses have a tendency to begin to extend.In 72 h,BMSCs have most of spread out on the scaffold surface.Scaffolds in the control group with the same proportion doesn't appear gradient phenomenon above,and the number of cells trapped in 24 h and the spread status of cells in 72 h are also significantly inferior to which of gradient scaffold.Therefore,gradient scaffolds with bionic physiological stratified structure have good biocompatibility,which are closer to the real structure of natural cartilage,and are more suitable to be used as biomimetic scaffolds for cartilage repair.(2)Biomimetic cartilage gradient scaffold materials(including calcified layer): The cells fully spread and have been extended into the scaffold in the biomimetic cartilage calcified layer;The biocompatibility of the biomimetic cartilage layer has good repeatability,calcified cartilage layer which simulated from Cs/GP/Gel/nHAp scaffold can effectively connect biomimetic cartilage layer and biomimetic subchondral bone layer,which can play a good function of transition,communication and cohesion.(3)Cs/GP/Gel/Gr electromagnetic scaffold materials: Doping Gr will increase the cell adhesion rate of scaffolds.A large number of cells adhered on the pore wall of scaffolds and in the pore channel,and the cells have a tendency to start spreading in 72 h.BMSCs have most of spread out on the scaffold surface and different cells have synaptic connection in 48 h.The number of cells in the control group scaffolds with the same proportion is significantly smaller and the growth process was obviously slower than that of gradient scaffolds.The results above suggest that uniformly inlaying a moderate amount of Gr into the pore walls of scaffold in nanoscale can regulate the hydrophobic property and hydrophilic performance,at the same time,increase the surface roughness to provide cells with larger area to spread and more locus to adhere so that accelerate the growth behavior of cells on it,and satisfy the experimental requirement of magnetic properties.(4)Intermittent electromagnetic stimulation of BMSCs-Cs/GP/Gel/Gr electromagnetic scaffolds: Electromagnetic stimulation can increase the adhesion rate of BMSCs on scaffold,and the cell adhesion rate on the electromagnetic scaffold which is exerted electromagnetic stimulation increases to 111.66% to that of normal incubator.In the electromagnetic stimulation environment,there can be found many cells had spread out on Cs/GP/Gel/Gr scaffolds surface in 24 h,and the integrate process is more quickly than that of the construct system in normal incubator,BMSCs fully spread o ut on scaffold pore walls in 72 h,and there is rarely found ball cells which didn't spread out similar to cells in a normal incubator.Therefore,electroma gnetic stimulation with appropriate intensity is beneficial to the adhesion and growth of BMSCs on the scaffolds. |
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