| BackgroundOsteonecrosis of femoral head(ONFH)is a common orthopedic disorder with high rate of incidence and disability.Although the detailed pathogenesis of ONFH remains unclear,consensus has been reached that disruption of blood supply of the femoral head plays a key role in the onset and deterioration of ONFH.In the management of ONFH,the crucial part is early diagnosis and treatment.The principle of early treatment is to provide mechanical support and promote regeneration of bone tissues.Therefore,core decompression with biomaterials implantation serves as an optimal choice for patients with early-stage ONFH,which debrides the necrotic tissues and slows or reverses the course of disease.Titanium alloy has been one of the most popular biomaterials in clinical application due to its excellent biomechanics properties and biocompatibility.Porous titanium scaffolds are equipped with ideal elastic modulus and provides excellent microenvironment for cell adhesion and growth.VEGF,the most powerful angiogenic factor,has been proved to induce osteogenic differentiation of BMSC in vitro.Chitosan microsphere is an optimal drug carrier with no toxicity,excellent biocompatibility,and spontaneous degradation in vivo.Therefore,we intend to manufacture porous titanium scaffold with chitosan microspheres containing VEGF and verify its effect in the treatment of osteonecrosis of femoral head.MethodsPorous titanium scaffolds were manufactured with 3D printing and combined with chitosan microspheres containing VEGF by freeze-drying.The rate of release of VEGF by the scaffolds was measured.BMSCs were co-cultured with the scaffolds.After 24 hours,48 hours and 72 hours,CCK-8 test was performed to decide the scaffold’s influence on the proliferation of BMSCs.After 3 days,6 days and 12 days,the expression of genes ALP,BSP,OCN and Collagen I were measured with real-time PCR.ALP staining and alizarin red staining were performed at day 6 and day 12 to detect the activity of ALP and calcium deposit.The scaffolds were implanted in New Zealand rabbit ONFH models.The rabbits were evaluated by microCT and sections of the femoral heads.Core decompression with autologous bone graft served as the positive control group,and models without treatment served as the negative control group.ResultsWe successfully designed and manufactured the chitosan microspheres encapsulating VEGF with porous titanium scaffolds.Scaffolds for cell culture were cylinders with a diameter of 0.9cm and a height of 0.5cm.Scaffolds for implantation in vivo were cylinders with a diameter of 0.3cm and a length of 2.5cn.SEM observation of the surface of the scaffolds revealed chitosan microspheres with diameters around 30μm.The scaffold released VEGF continuously for 21 days,and the total amount of VEGF released was 0.42μg,accounting for 84%of the total carrying VEGF,and significantly higher than that of the control group(p<0.05).In the aspect of in vitro experiment,BMSC co-cultured with the scaffolds were considered the experimental group,while BMSC cultured without scaffolds were considered the control group.CCK-8 indicated that after cultured for 24 hours and 48 hours,there was no significant difference in absorbance between the two groups(0.40±0.05 vs 0.38±0.04,0.68±0.03 vs 0.60±0.03,p>0.05).After cultured for 72 hours,the absorbance of the experimental group was significantly higher than that in the control group(1.11±0.08 vs 0.0±0.01,p<0.05).The expression of osteogenesis related genes,ALP,BSP,Collagen I,and OCN,were quantified by real-time PCR,which showed that the expression of the four genes was significantly higher in the experimental group after cultured for 3 days,6 days,and 12 days(p<0.05).ALP staining and alizarin red staining revealed higher ALP activity and more calcium deposition in the experimental group than that in the control group after cultured for 6 days and 12 days.After implantation of the scaffolds,rabbits were not negatively influenced regarding feeding and sleeping.The gait of rabbits in the experimental group improved comparing to the model group.MicroCT revealed regeneration of bone trabecula around and inside the scaffolds,which was similar as the positive control group,and much better than the model group.Quantitative analysis showed higher percentage of trabecula in the experimental group than that of the model group two and three months after implantation(0.56±0.06 vs 0.41 ±0.003,0.59±0.005,p<0.05),and was similar to that in the positive control group(0.56±0.06 vs 0.57±0.05,0.59±0.005 vs 0.59±0.03,p>0.05).Pathological findings included bone regeneration around the scaffolds,and maturation of collagen.In the model group,little bone regeneration was observed.ConclusionPorous titanium scaffolds with chitosan microspheres containing VEGF releases VEGF steadily,and promotes the proliferation and osteogenic differentiation of BMSCs.The treatment of ONFH in rabbit models with the scaffolds was also successful.No toxicity of the scaffolds was detected.Conclusively,the scaffolds provide a promising option of the treatment of early-stage ONFH. |
