Study On The Differentiation Of Rabbit BMSCs Into Osteoblasts Induced By Light-cured GelMA Hydrogel Combined With BMP-2 In Vitro And In Vivo

Background:As the global population aging intensifies,the rapid growth in the number of patients with intervertebral disc degeneration(IVDD)diseases may seriously impact the current social medical resources and socio-economic.Spinal fusion is one of the important methods for the treatment of this disease,but serious complications such as postoperative bone non-fusion have not been effectively resolved.Previous studies have shown that bone marrow mesenchymal stem cells(BMSCs)transplantation and bone morphogenetic protein 2(BMP-2)transplantation can effectively reduce the risk of postoperative bone non fusion,but their viability and utilization rate after transplantation are low,and suitable biological carriers are expected to solve this problem.Light curable methacryloyl gelatin(Gel MA)is a safe and effective biological carrier that can provide a good transplantation environment for cells and biological factors,and is considered to be the most promising biomaterial for clinical application in the field of biomedicine.The purpose is to preliminarily explore the characterization and biocompatibility characteristics of different concentrations of light cured Gel MA hydrogel,and observe the ability of Gel MA hydrogel combined with BMP-2 to induce osteoblast differentiation of BMSCs,and further explore the effect of Gel MA hydrogel combined with BMP-2 and BMSCs transplantation on spinal fusion in rabbits through in vivo experiments,in order to lay a theoretical foundation for the faster application of tissue engineering in the treatment of bone non-fusion after spinal fusion.Objective:1.Discuss the influence of concentration on the characterization and biocompatibility of UV curable Gel MA hydrogel.2.Explore the ability of light curable Gel MA hydrogel combined with BMP-2 to induce BMSCs into osteoblasts in vitro.3.To investigate the effect of Gel MA hydrogel combined with BMP-2 and BMSCs transplantation on interbody bone fusion at spinal fusion level in rabbits.Methods:Part I: prepare different concentrations of photocurable Gel MA hydrogels,and observe the microstructure and pore size,swelling performance,degradation performance,release performance and cell compatibility of the hydrogels.Part II: We detect the activity and expression levels of osteogenic associated protein alkaline phosphatase(ALP)and run related transcription factor 2(Runx2)in BMP-2 induced BMSC cells by immunofluorescence staining.The proliferative activity of the induced cells was detected using a Cell Counting Kit 8(CCK8).Quantitative real-time PCR(q PCR)and Western Blot were used to detect the gene and protein expression levels of ALP,Runx2,Osteocalcin(OCN),and Collagen-1(COL-I)in the cells after induction.The results of the experimental data were statistically analyzed using Origin software.Single factor analysis of variance was used for pairwise comparisons between groups.The LSD method or Games-Howell method of repeated measurement data was used for analysis of different groups.The difference was statistically significant with a P<0.05.Use Graphpad Pism 9.5 software for drawing.Part III: Select 12 rabbits,including 6 New Zealand white rabbits in the BMP-2-BMSCs group and 6 New Zealand white rabbits in the Gel MA-BMP-2-BMSCs group.Preparing BMP-2-BMSCs bone particles and Gel MA-BMP-2-BMSCs bone particle grafts.Strictly standardize the operation and transplantation of spinal fusion in rabbits.Manual palpation,intervertebral height measurement,and Micro-CT radiology were used to evaluate the stability of the intervertebral fusion segment and the status of intervertebral fusion in the two groups after surgery.Results:Part I: 1.The microscopic structure of 5%,7.5% and 10% light cured Gel MA hydrogels is "honeycomb",and the pore size is 329.60 ± 15.10 μm 、270.8 ± 2.44 μm and 247.2 ± 5.94 μm.There is a significant difference between the two pairs(P<0.001).There was a significant difference between the two groups(P<0.001).2.The 5%,7.5% and 10% light cured Gel MA hydrogels showed a swelling trend from fast to slow to equilibrium,and the time to reach the swelling equilibrium was 24 h,30h and 30 h,respectively.The maximum swelling rates were 1430.56 ± 34.49%,1239.26 ± 41.92% and 1097.72 ± 53.27%,respectively.3.The degradation of UV curable Gel MA hydrogel in PBS solution is generally slow.On the 30 th day,the cumulative degradation rates of 5%,7.5% and 10% hydrogels are 14.42 ± 0.70%,11.43 ± 0.74% and 9.02 ± 0.69% respectively.The degradation rate of 5%,7.5% and10% hydrogels in collagenase II solution was 86.96 ± 3.17%,74.24 ± 4.59% and62.30 ± 4.47% respectively at the 10 th hour.4.With the extension of time,BMP-2 in5%,7.5% and 10% hydrogels generally showed a trend of fast release first and then slow release,in which the release of BMP-2 was relatively fast in the first five days,and relatively slow after that.On the 30 th day,the cumulative release rates of BMP-2in 5%,7.5% and 10% hydrogels were 24.08 ± 0.49%,19.27 ± 1.76% and 14.08 ±0.94%,respectively.5.On the first day,the OD values of BMSCs after proliferation of 0%,5%,7.5% and 10% light cured Gel MA hydrogels measured by CCK8 were0.62 ± 0.03,0.61 ± 0.02,0.55 ± 0.04 and 0.57 ± 0.02 respectively;On the second day,the OD values measured by CCK8 were 1.38 ± 0.05,1.16 ± 0.04,1.16 ± 0.04,1.06 ±0.09,respectively.BMSCs could grow well in the hydrogels of the above concentrations.On the third day,the measured OD values of CCK8 were 2.18 ± 0.04,2.09 ± 0.06,2.03 ± 0.07,and 1.75 ± 0.04,respectively.Part II: The immunofluorescence results showed that the expression levels of osteoblast related proteins ALP and Runx2 in cells varied with the concentration of BMP-2 and the intervention time.At the same intervention time,100 ng/ml of BMP-2induced the highest expression of ALP and Runx2 proteins in BMSCs.At the same concentration,the highest expression levels of ALP and Runx2 proteins were observed on the 7th day of BMP-2 intervention.The results of CCK8 showed that the proliferative activity of BMSCs in BMP-2 group was significantly increased compared to Control group(P<0.001).Compared with BMP-2 group,the proliferation ability of BMSCs in BMP-2+Gel MA group was significantly enhanced(P<0.001).The immunofluorescence results showed that the intracellular ALP and Runx2 activities in BMP-2 group were significantly increased compared to those in Control group.Compared with BMP-2 group,the intracellular ALP and Runx2 activities in BMP-2+Gel MA group were increased;After adding BMP-2 to induce BMSCs,the expression of osteogenic related proteins ALP and Runx2 in the cells significantly increased compared to the control group(P<0.001).When the induction process was placed on 5% Gel MA hydrogel,the expression of ALP and Runx2 protein in cells increased compared with BMP-2 group(P<0.01).Western blot results showed that the expression level of ALP protein in the experimental group was significantly higher than that in the control group(P<0.01).The expression level of Runx2 protein in the experimental group was significantly higher than that in the control group(P<0.01);The expression level of OCN protein in the experimental group was significantly higher than that in the control group(P<0.01).The expression level of COL-I in the experimental group was significantly higher than that in the control group(P<0.01).The results of q PCR showed that the m RNA level of intracellular ALP in BMP-2+Gel MA group was significantly higher than that in BMP-2 group(P<0.001),while the m RNA level of intracellular ALP in BMP-2 group was higher than that in Control group(P<0.01).The m RNA level of Runx2 in the BMP-2+Gel MA group was significantly higher than that in the BMP-2 group(P<0.01),while the m RNA level of Runx2 in the BMP-2 group was higher than that in the Control group(P<0.05).The m RNA level of OCN in BMP-2+Gel MA group was higher than that in BMP-2 group(P<0.05),while the m RNA level of OCN in BMP-2 group was significantly higher than that in Control group(P<0.01).The m RNA level of intracellular COL-I in BMP-2+Gel MA group was significantly higher than that in BMP-2 group(P<0.01),while the m RNA level of intracellular COL-I in BMP-2 group was higher than that in Control group(P<0.01).Part III: The results of manual palpation showed that no fusion was touched in both groups of experimental animals 3 weeks after surgery.At 6 weeks after surgery,the intervertebral fusion of experimental animals in the Gel MA-BMP-2-BMSCs group was better than that in the BMP-2-BMSCs group.The measurement results of intervertebral height showed that at 3 and 6 weeks after surgery,the intervertebral height of the rabbit spinal fusion segment in the BMP-2-BMSCs group and the Gel MA-BMP-2-BMSCs group was lower than the preoperative intervertebral height.In the BMP-2-BMSCs group,the intervertebral height(6.55 ± 0.71mm)at 3 weeks postoperatively was significantly higher than that at 6 weeks postoperatively(2.51 ±0.74mm),P<0.001;There was no significant difference in intervertebral height changes between the Gel MA-BMP-2-BMSCs group at 3 weeks(7.43 ± 0.55mm)and6 weeks(7.27 ± 0.64mm)after surgery.Micro-CT imaging results showed that at 3weeks after surgery,there was no intervertebral fusion in both the BMP-2-BMSCs group and the Gel MA-BMP-2-BMSCs group,but the intervertebral height in the BMP-2-BMSCs group was significantly lower than that in the Gel MA-BMP-2-BMSCs group.At 6 weeks after surgery,there was no significant spinal fusion in the BMP-2-BMSCs group rabbits,while spinal fusion was visible in the Gel MA-BMP-2-BMSCs group.Conclusion:1.The biological characterization of light cured Gel MA hydrogel has good controllability,and its pore size,swelling rate,degradation rate,biofactor release and cell proliferation can be controlled by the concentration of hydrogel.2.Gel MA hydrogel combined with BMP-2 has a better effect on inducing BMSCs to differentiate into osteoblasts.3.Gel MA hydrogel combined with BMP-2 and BMSCs in vivo transplantation can significantly promote the segmental bone fusion of spinal fusion in rabbits.