An Experimental Study On The Creation Of A Mouse Subcutaneous Ectopic Bone Marrow Model Using A PCL Scaffold

Purpose:To discuss how to use a variety of biological agents to participate in three dimention PCL scaffold induce artificial bone marrow model establishment and evaluation.Material and method:一.To confirm the effect of Novel naphthoquinone analogues that target Wnt/β-Catenin/TCF signalingFirstly,colon cancer carcinoma HCT116 and SW480 cells were cultured and then added final5μM of 41 kinds of naphthoquinone compounds separately to test cell growth inhibition.Then we got preliminary screened compounds with higher cell inhibition.These compounds has been taken cell growth inhibition assay and clone formation experiment from low to high concentration.According to these two kinds of experimental results,we calculated the IC₅₀ of each compound.We transformed and amplied plasmid TOP and plasmid FOP,then extracted and transfected plasmid TOPand plasmid FOP into SW480 and HCT116 cells.After that,added a seriors of concentration of screened compounds to take luciferase reporter gene assay.From results,we got the strongest compounds which inhibit Wnt / beta-Catenin/TCF4 pathway.In Co MFA analysis,the lowest energy compound 23 was used for conformational search,in which was used as a template to establish other analogues and determined the relationship between molecular binding and activity.In above experiments,the most valuable compounds were tested for cytotoxicity assay in normal cells.According to the results,the most promising compounds were applied to the second part of the whole experiment.二.Biomodified PCL scaffold induced ectopic bone marrow formation and its effect（1）firstly,MDA-MB-231/ GFP cells cultured and put on a 96 well plate in which has PCL scaffold.After 48 hours,the scaffold were observed under fluorescence microscope to determine if PCL scaffolds were suitable for cells growth.（2）SR1 and Naphthoquinone compound-N27 respectively,were added in ATDC5 cells containing or not containing10ng/m L of rh BMP-2.ALP activity assay was carried out after 72 hours,and the effect of ALP activity of different biological agents in ATDC5 cells was determined.（3）In mouse abdomen were embeded PCL scaffolds in which added different biological agents subcutaneously.Based on PCL scaffolds of different substances were seperated into seven groups.The first group（blank control group）: only added PBS/0.1%BSA/0.1%DMSO in the PCL scaffolds.The second group: 10μg of rh BMP-2 in the PCL scaffolds,the third group: 10μL of Matrigel+10μg of rh BMP-2 in the PCL scaffolds.The four groups: 2mg of HA+10μL of Matrigel+10μg of rh BMP-2 in the PCL scaffolds.The five groups: 20μg of SR1+2mg of HA+10μL of Matrigel+10μg of rh BMP-2 in the PCL scaffolds.The six groups: 40μg of icariin +2mg of HA+10μL of Matrigel+10 μg of rh BMP-2 in the PCL scaffolds,the seven groups: 40μg of N27+2mg of HA+10μl of Matrigel in the 10μg of rh BMP-2 in the PCL scaffolds.（4）Eight weeks later,mice were anesthetized in vivo,and were took micro CT measurement to check bone formation.（5）Mice were euthanized,removed ossicles.Some of them were fixed,decalcified,sectioned,stained with H&E staining and Masson trichrome staining.Histological stain were determined the composition of each bone tissue structure and cell,and carries on the comparative analysis.6)Some of them were removed from mice,washed repeatedly and then collected cells in bone to take hematopoietic colony formation assay.Using hematopoietic colony formation assay to determine the frequency of neonatal ossicle hematopoietic progenitor cells and precursor cells.（7）Collecting cells within ossicles,using anti Sca-1-FITC antibody in flow cytometry Sca-1 + cells to determine the ossicles of HSCs.Results:一.The effect of new type of compounds on the Wnt/ beta-Catenin/TCF signaling pathway was confirmed.1.To screen compounds for effective inhibition of colon cancer cells When the concentration of 41 compounds was 5μM,the growth inhibition percentages of the two kinds of cells were screened out.With higher cell growth inhibition（inhibition rate >50%）,and tested their IC₅₀ values,in which compounds 23 and 27 with sulfonamide structure has a lower IC₅₀ values（<2μM）).Compounds 23 and 27 had stronger cell inhibitory in HCT116 and SW480 cell lines.The sulfonamide derivatives of compounds 24 and 25 had selective inhibition for SW480 cell line.2.Biological characteristics of 2 representative compounds in the beta-Catenin/TCF signaling pathway The six compounds 4,27,31 and 23-25 were testes in SW480 and HCT116 cells for luciferase activity inhibition.Sulfonamide compounds 27 showed the strongest cell inhibitory.The IC₅₀ values were 2.1μM and 2.5μM in SW480 and HCT116 cells,respectively.3.Experimental results of representative compounds inhibiting colony formation in colon cancer cells with high expression of beta-catenin.Six compounds（4,23,24,25,27,31）significantly inhibited colony formation in a dose-dependent manner,all of the IC₅₀ values of these compounds were less than 2μM（Table 2,figure 2）.The sulfonamide structure of compounds 23 and 27 were the most effective.The results showed IC₅₀ values of compounds 23 and 27 in SW480 cells were346 n M and 343 n M,their IC₅₀ values were 390 n M and 348 n M in HCT116 cells.4.CoMFA analysis The biological activity and Co MFA analysis present the preliminary SARs,as shown in Table1-2 and Figure 3.（1）Steric contours show the main region of favored（green）contribution to the activity that surrounds 2-amines.Di-substituted 2-amines（compounds 4 and 32）were more potent than mono-substituted ones（compound 3）.（2）The unfavorable（yellow）region of Co MFA map indicated steric bulks result in decreasing activity（e.g.compounds 11 and12）.（3）The Blue contour（electropositive substituent favored）is distributed surrounding sulfamines.The activity could be enhanced if sulfonamide were substituted by aromatic rings carrying π electrons（e.g.22 vs.23-25）.（4）The 3-chloro group of naphthoquinone core was preferred for the inhibition.3-Hydro substituent led to loss of activity（e.g.23 vs.30,4 vs.29）.5.ATDC5 cytotoxicity test When the doses of Compound 23 were 5μM,2.5μM,1.25μM,0.625μM and 0.3125μM respectively,viability of ATDC5 cells were2.85±1.49%,16.36±10.9%,89.13±3.59%,95.15±1.98% and 95.36±6.14%,respectively.When compound 27 follwed the some doses,the viability of ATDC5 cells were 79.34±2.67%、85.57±2.55%、96.45±4.18%、98.35±1.33%and 99.37±0.53%.So compound 27 has a less cytotoxicity for normal cell line.二.The formation and effect of ectopic bone marrow induced by biological modified PCL scaffold.1.Identification if PCL biological scaffold were suitable for cell growth After 48 hours,MDA-MB-231/GFP cells in the PCL scaffold were observed under fluorescence microscope with green fluorescence.Most of the cells attached to the cell scaffold wall with spindle shaped.2.ALP activity test Compared with the rh BMP-2 group,the ratio of rh BMP-2+ SR1 group increased,P<0.05.The ratio of N27 group and rh BMP-2 +N27 group were lower than that of the rh BMP-2 group,P<0.05.Compared with rh BMP-2 +SR1 group,the value of other groups were lower than rh BMP-2 +SR1 group,P<0.05.Compared with SR1 group,Icariin group and N27 group were no significant difference,P>0.05.Compared with N27+ rh BMP-2 group,SR1+ rh BMP-2group and Icariin+ rh BMP-2,significant difference,P < 0.05（see Table 3 and Figure 6）.3.Micro-CT test Compared with control group（Group1）,the bone mineral density of the other 6 groups were significantly increased,P<0.05.The fifth group was the most obvious visible histogram.While other groups compared with group 5,the difference were significant,P<0.05.However,every group of bone mineral density did not reach the level of mouse spine.When all groups compared mouse spine density,which showed that there were significant differences（P<0.05）（see Table 4 and Figure 8）.4.Histological staining Figure 9 A-G are H&E staining results of group1 to group7 after decalcification.the blank control group（Goup1）didn’t form new bone.Groups formed different new bone,in which the PCL scaffolds added SR1+HA+ Matrigel+ rh BMP-2（group5）formed more new bone and the morphologywas similar to shape of PCL scaffolds,which showed wrapped structure.Scale bars:500μm,and Figure 10 is the Masson Scale staining of group5.Scale bars:500μm.Figure11A-C are the result of H&E staining,in which the figure 11 A is blank control group（group1）,visible red blood cells,proliferation of mesenchymal cells and some blood vessels.Figure 11B-C are group5,in which presents the hematopoietic cells of newly formed bone and hematopoietic cells,including megakaryocytes,mononuclear cells,granulocytes,red blood cells.The scale bars:20μm.5.Hematopoietic colony formation Besides bone marrow formed the highest various types of hematopoietic colony,group5 was the second one.Statistical analysis found that in CFU-GEMM,compared with the group5,the number of CFU-GEMM of group5 formation were significantly higher than other groups,P<0.05.In BFU-E,compared with group5,group2,group3,group4 and group7 were significantly lower than group5,P<0.05.In CFU-GM,compared with group5,group2,group3 and group7 were significantly lower than group5,P<0.05.In CFU-E,compared with group5,the formation of group5 CFU-GEMM was significantly higher than other groups,P<0.05（see Table 5 and figure 12）.Figure 13A-D are colony formation in the process of the formation of different colonies,in which figure 13 A is CFU-GEMM,visible around the center of a cluster of red blood cells around the granulocytes and macrophages,central cluster because of contains more hemoglobin of nucleated red blood cells,regional centers appear red.Figure 13 B are composed of a number of BFU-E,but because the cluster has higher hemoglobin,clusters showed red.Figure 13 C is a cluster of CFU-GM,which is scattered around a number of cells,which contain granulocyte and monocyte/macrophage cells.And compared to the granular cell,the single nucleus / giant cell is more irregular.Figure 13 D is CFU-E（x10）.6.Flow cytometry Ossicles of Group1,group5 and femur of bone marrow were analyzed by flow cytometric.It is found that the group5 vs group1:12.6% vs.0.23%,P < 0.01;group5 vs FBM: 12.6% + 0.94%vs10.2% + 1.21%,P > 0.05（Figure 14）.Conclusion:1.A variety of biomodified PCL scaffold may induce ectopic bone / bone marrow formation.2.Biomodification of PCL scaffolds with matrigel,HA,and SR1 enhances De Novo ectopic bone marrow formation induced by rh BMP-2 with higher hematopoietic function and more bone density.3.In the colon cancer carcinoma,41 kinds of novel synthetic naphthoquinone compounds were screened with low cytotoxicity and inhibitor of Wnt/catenin pathway.Compound N27 in the construction of artificial bone marrow model has obviously inhibition bone/bone marrow formation.4.Biomodification of PCL scaffolds with matrigel,HA,and icariin enhances De Novo ectopic bone marrow formation induced by rh BMP-2 with good hematopoietic function and bone density.5.Through this study to create 3 dimental model of artificial bone marrow has lower technical difficulties,simple operation,high repeatability,easy popularization compared with other research.It offers a new approach for further clinical and basic research.