| Objective:To prepare biomimetic porous CMC/ALG/HA（CAHA）composite hydrogel scaffold with different parameters by freezing casting method,and evaluate the effects of HA addition amount,freezing temperature and polymer ratio on the structural characteristics and physical properties of the scaffold,and optimize the preparation parameters.The effects of HA addition on the surface morphology,degradation rate and swelling behavior of CAHA scaffolded were evaluated.The cytotoxicity,biocompatibility and biofunction of the scaffolds were evaluated by in vitro cell assay and in vivo implantation.Methods:（1）The central cold source mold was self-made,and the bionic porous CAHA scaffold with different parameters was prepared by freezing casting method.（2）Process parameter optimization:the microstructure of the scaffold was observedby SCANNING electron microscopy（SEM）,porosity was measured by Archimedes drainage method,compressive strength was tested by universal mechanical testing machine,and process parameters were optimized according to the pore structure and mechanical properties of the scaffold.（3）The effect of HA supplementation on the physical properties of CAHA scaffold:the surface structure of the scaffold lamella was observed by scanning electron microscopy and contour scanner;The swelling rate of the scaffold in simulated body fluid（SBF）was measured by weighing method,and the size change rate of the scaffold was measured when it was completely swollen.The weight loss rate of the scaffold in simulated body fluid（SBF）was measured by weighing method,and the release concentration of Ca2+in the immersion solution was measured by inductively coupled plasma spectrometer（ICP）.（5）In vitro evaluation of biological effects of HA supplementation on CAHA scaffolds:scaffold extracts were co-cultured with L929,cytotoxicity of scaffolds was detected by CCK-8 method,extracts were co-cultured with MC3T3-E1 cells,and the effects of materials with different HA contents on cell activity were evaluated by live and death staining method.The effects of scaffolds on the proliferation and adhesion morphology of MG63 cells were evaluated by co-culture of scaffolds and cells.The m RNA expressions of ALP,OC and OPN in MC3T3-E1 cells were detected by PCR,and the effect of different HA contents on osteogenic differentiation of MC3T3-E1 cells was evaluated.（6）In vivo implantation evaluation of the effect of HA supplementation on bone induction of CAHA scaffold:Different HA contents of CAHA scaffold were implanted into bone marrow cavity defect near the epiphysis of the proximal tibia of New Zealand rabbits,and the control group was a blank control.The experimental animals were sacrificed at 4 weeks and 8 weeks respectively,tibial specimens were taken out,and the possible degradation of the scaffold in vivo and the repair of bone defects were evaluated by micro-CT.Results:（1）Preparation parameters affect the pore structure of scaffolds:SEM and porosity tests of CAHA scaffolds show that scaffolds have central macropores and open pore structure like surrounding radioactive lamellar.Preparation parameters affect the lamellar distance and thickness of scaffolds:When the freezing temperature and polymer ratio remained unchanged,the addition of HA affected the pore structure,porosity and lamellar thickness of the scaffold.When the addition of HA increased from0 to 60wt%,the pore size decreased from 116.63±41.18μm to 56.38±11.16μm.The lamellar thickness increased from 2.00±0.44μm to 63.11±7.06μm,and the difference was statistically significant（p<0.05）;When the HA content remained unchanged,the decrease of freezing temperature reduced the pore size and lamellar thickness.When the temperature decreased from-15℃to-35℃,the pore size decreased from 74.89±11.08μm to 33.44±6.32μm,and the lamellar thickness decreased from 46.68±6.58μm to 19.01±4.79μm.The difference was statistically significant（p<0.05）,but has no significant effect on porosity;However,when HA content and freezing temperature were controlled and only the polymer Belgium was changed,it was found that the thickness of scaffolds with high ALG content decreased,and there was no significant difference in total porosity and pore size.（2）Preparation parameters affect the mechanical properties of scaffolds:When thefreezing temperature and CMC/ALG ratio were the same,the solid content affected the compressive strength of the stent.When the solid content increased from 15 wt%to 60wt%,the compressive strength increased from 1.50±0.16k Pa to 21.19±1.42 k Pa,and the difference was statistically significant（p<0.05）;When the solid content was 45wt%and CMC/ALG ratio was the same,the freezing temperature affected the compressive strength of the stent.When the freezing temperature decreased from-15℃to-35℃,the compressive strength of the stent increased from 11.82±0.87 k Pa to 14.75±1.19k Pa,and the difference was statistically significant（p<0.05）.CMC/ALG ratio affects the pore structure and mechanical strength of the scaffold.When the concentration of ALG is too high,the homogeneity of the overall structure of the scaffold may deteriorate,and the compressive strength of the scaffold may be reduced.（3）Surface morphology of CAHA scaffold:The addition amount of HA can affect the surface morphology of the scaffold laminate,and disorderly protrusions can be seen on the surface of the scaffold without HA;The higher HA content（30wt%and 45wt%）could induce the better growth of ice crystals,and the long and parallel protrusions were sculpted on the surface of the scaffold,and parallel grooves were formed between the protrusions.The distance between the two groups of grooves was 27.69±6.55μm and24.48±8.51μm,respectively（p<0.05）;The scaffold with 60wt%HA content was too high,which was not conducive to the formation of ice crystals,and the surface structure was unclear and disorderly.When the HA content increased from 0 to 60wt%,the roughness increased from 2.38±0.213μm to 21.38±4.95μm,and the difference was statistically significant（p<0.05）.（4）Swelling performance of CAHA support:CAHA support soaked in SBF can completely swell within 30min.With the increase of HA addition,the swelling rate of the scaffold decreased significantly and the dimensional stability increased.The swelling rate of the scaffold without HA was 350.59±7.31%,and the size change rate was 23.73±3.41%.When the HA content increased to 60wt%,the swelling rate decreased to 34.84±7%,and the size change rate decreased to 6.51±2.81%,with statistically significant differences（p<0.05）.Under the condition of full swelling,scaffoldings with high HA content（45wt%and 60wt%）remained stable,and no significant changes were observed in the pores and overall structure.（5）Degradation rate of CAHA scaffold:CAHA scaffold can be degraded in SBF.With the increase of HA,the weight loss rate of the scaffold slowed down and its stability increased.When HA content increased from 0 to 60wt%,weight loss rate at 28days decreased from 65.15±7.23%to 15.95±3.62%,the difference was statistically significant（p<0.05）.After soaking for 7 days,the ion concentration of each group increased rapidly,and the release rate slowed down after 7 days.The higher the HA content,the lower the Ca2+release concentration in the solution.The cumulative Ca2+release concentration of the sample without HA after soaking for 28 days was 489.2±5.985mg/L,and when the HA content increased to 60wt%,the cumulative Ca2+release concentration for 28 days decreased to 91.1±3.26mg/L.The difference was statistically significant（p<0.05）.（6）In vitro cell test:CCK-8 method showed that CAHA scaffold had good biocompatibility and no cytotoxicity;Cell adhesion experiments demonstrated that the open pores of the scaffold layer were plate-like for cell migration into the scaffold,and the surface grooves of small size（20-30μm）could influence cell adhesion morphology and promote cell migration through"contact guidance"effect.Cell viability/death staining showed that scaffold with different HA contents did not affect MC3T3-E1 cell viability.PCR showed that 30wt%and 45wt%HA scaffold materials could promote the expression of osteogenic genes ALP,OC and OPN in MC3T3-E1 cells.（7）In vivo implantation experiment:the composite scaffold could degrade in vivo,and all experimental groups could induce new bone formation in rabbit tibial bone marrow cavity,which was better than the blank control.Scaffolds without HA and15wt%HA degraded too quickly in vivo,unable to maintain the pore structure after implantation,and only induced a small amount of new bone around the scaffold.After implantation of 30wt%and 45wt%HA scaffolds,their pore structures could maintain good stability and support the growth of new bone tissues into the scaffolds.Among them,the 30wt%HA scaffold could maintain the scaffold structure well within 4 weeks of implantation and induce new bone formation,while the new bone mass did not increase significantly after 8 weeks of implantation,possibly because the scaffold had been degraded and could not provide support anymore.The scaffold of 45wt%HA maintained good bone regeneration ability after 4 and 8 weeks of implantation,inducing new bone to grow into the scaffold,indicating that it maintained good stability and adaptation to degradation during implantation.The porosity of 60wt%HA scaffold is small,which is not conducive to the proliferation and differentiation of cells inside the scaffold,and it is difficult to complete the replacement of scaffold materials by bone tissue.HA aggregation is prone to occur after implantation.The 45wt%HA scaffold was superior to other groups in pore structure stability,degradation suitability and bone regeneration.Conclusion:（1）The CAHA composite hydrogel scaffold with high porosity prepared by freezing casting method has a large central pore and a radially arranged lamellar open pore around it.（2）The addition amount of HA was the main factor affecting the structure and physical properties of CAHA scaffold.The appropriate technological parameters for the preparation of composite scaffold were 45wt%HA,-15℃freezing,and CMC/ALG ratio of 2:1.（3）The bionic porous CAHA composite hydrogel scaffold has no cytotoxicity,and the structure of the scaffold is conducive to cell migration,proliferation,adhesion and differentiation.（4）Composite scaffolds can degrade in vivo,and HA content affects the osteoinduction ability of scaffolds.Scaffolds with 45wt%HA are superior to other groups in pore structure stability,degradation suitability and bone regeneration.Figure 19,table 11,reference 54. |
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