Preliminary Study On Bone Mesenchymal Stem Cell Alginate Microspheres Prepared By Microfluidic Technology/Calcium Phosphate Cement

Objective:To study the method of microfluidic technology to prepare bone marrow mesenchymal cell-loaded calcium alginate microspheres;To explore the feasibility of BMSC-loaded calcium alginate microspheres combined with calcium phosphate cement.This provides a new basis for the development strategy of bone substitute.Methods:1.Preparation of fluorescent alginate microspheres and BMSC-loaded calcium alginate microspheres by microfluidic technology,observe their morphology and explore their survival,proliferation and osteogenic differentiation potential of he internal BMSCs.SD rat BMSCs,were isolated and purified by the whole bone marrow,to fabricate fluorescent alginate microspheres and bone mesenchymal cell-loaded alginate microspheres by microfluidic approach.The morphology and dispersion of the fluorescent empty spheres were observed under microscope;The control group(BMSC group),multicellular microgel group(M-BMSC-Gel group)and single-cell microspheres group(S-BMSC-Gel group)were set up.We take advantage of optical microscope to observe the morphology of the microspheres,measure the maximum diameter and area of the microspheres.And we take advantage of laser confocal microscopy and optical biological microscope to observe the growth conditions of cells in microspheres,Using Live/Dead stain to observe the survival of BMSCs;Using CCK-8 to detect the metabolic activity of BMSCs and Using DAPI nuclear staining to observe the proliferation of BMSCs in microspheress.The ALP activity detection,alizarin red staining,scanning electron microscopy and energy spectrum analysis,RT-PCR were used to detect the osteogenic differentiation ability of BMSC after BMSCs were coated with calcium alginate microspheres.2.Cytotoxicity and physical properties of BMSC-loaded calcium microspheres/CPC compundCPC and water were mixed in a mass-volume ratio of 2.5g:1ml as A group(CPC group),CPC and alginate microgels were mixed with 2.5g:1ml,2g:1ml,1.5g:1mlrespectivelyforBgroup:2.5gCPC/1mlGelgroup,C group:2gCPC/1ml Gel group,D group:1.5gCPC/1ml Gel group.Putingthe four groups of paste into a 5ml syringe,3 samples per group,apply the Universal Testing Machine to press the syringe,weigh the paste mass before and after injection,and calculate the extrusion rate.The four group pastes were separately loaded into the mold,6 samples per group,and solidified at 37℃for24 hours,the hardened specimen was demolded.Appling Universal Testing Machine to press CPC specimens,record the maximum pressure,relationship between pressure and displacement,and calculate the compressive modulus and compressive strength.The four group pastes were separately loaded into the mold,3 samples per group,and solidified at 37°C for 24 hours,then dried in a vacuum oven at 60°C for 24h.scanning electron microscopy was used to examine the pore size and morphology in CPC specimens.Results:1.Results of preparation of fluorescent alginate microspheres and BMSC-loaded calcium alginate microspheres by microfluidic technology,the surface morphology of microspheres and survival,proliferation and osteogenic differentiation potential of he internal BMSCs(1)The Results of optical microscopy and microsphere size measurement:The fluorescent alginate microspheres prepared by microfluidic technology showed uniform size,same fluorescence intensity and concentration under the microscope,and were dispersed.BMSC-loaded calcium alginate microspheres prepared by microfluidic technology are uniform in size and controllable in size.The BMSC multi-cell calcium alginate microspheres are ellipsoidal,with the largest diameter(163.54±4.03)μm and the area(17343.33±341.39)μm~2.The BMSC single-cell calcium alginate microspheres are regular in shape and spherical.The diameter is(70.03±0.60)μm,and the area is(3707.85±114.66)μm ~2.(2)The Results of Live/Dead assay:A small number of cell nucleus were stained red in the BMSC group over time,and some apoptosis occurred;In M-BMSC-Gel group and S-BMSC-Gel group,a small number of cells were stained red at 1d.And Almost no cells were stained red at 4d,7d,and 14d,but green fluorescence diminished slightly over time.The results of Live/Dead staining showed that the persent of live cells of BMSC group decreased slowly with time.The persent of live cells of M-BMSC-Gel group and S-BMSC-Gel group were lower than that of BMSC group,but the persent of live cells was still high,reaching more than 89%.There was no significant difference in the persent of live cells e between BMSC-Gel and BMSC group at 4d、7d、14d,P>0.05.(3)The Results of CCK-8 cell metabolic activity detection:the alginate microspheres-encapsulated BMSCs had lower metabolic activity at 3d,then there was an upward trend,but the metabolic activity was significantly lower than that of the unencapsulated cells;the M-BMSC-Gel group had a slightly higher than the S-BMSC-Gel group.(4)The Results of DAPI staining:at 6d and 8d,the cell mass became larger,showing a multinuclear state.(5)The results of osteogenic differentiation potential of BMSCs encapsulated by microspheres:(1)Observing the BMSC-loaded alginate microgel by microscope,the formation of calcium nodules was observed in the BMSC group at 14d and 21d,and the alizarin red staining was stained by red.We could observe thatcolor deepens around the cells at 7d in the M-BMSC-Gel group and the S-BMSC-Gel group.And at 14d,there had been formed opaque calcifications in the whole microgel.the deposition of calcium salt increased at 21d,the empty gel also mineralized.The microgel were stained red by alizarin red,and the M-BMSC-Gel group was darker than the S-BMSC-Gel group.(2)The results of ALP activity:the BMSC group increased at 1d to 7d,reached the peak at 7d,and decreased at 14d and 21d.The M-BMSC-Gel group and the S-BMSC-Gel group increased at 1d to 4d,reached the peak at 4d,and decreased at 7d to 21d.At 4d,M-BMSC-Gel group and the S-BMSC-Gel group were significantly higher than the BMSC group,P<0.05,M-BMSC-Gel group was higher than S-BMSC-Gel group,P<0.05;At 7d,BMSC group was higher than that in the M-BMSC-Gel group and the S-BMSC-Gel group,P<0.05.(3)The results of Scanning electron microscopy:the alginate microspheres were smooth before osteogenic induction.And the surface of BMSC alginate microgel was deposited Calcium phosphate crystals after 21d and 28d.The results of energy spectrum analysis showed that the content of calcium and phosphorus increased gradually before osteogenic induction to after osteogenic induction 28d.At the same time point,the M-BMSC-Gel group had more calcium and phosphorus than the S-BMSC-Gel group.(4)The results of Real-time PCR:The expression of ALP mRNA in BMSC group was the highest at 7d,and decreased at 14d and 21d.The expression level of ALP mRNA in BMSC microsphere group decreased first and then increased at 7d,14d and 21d,and the trend is consistent with the ALP activity test results;The relative expression of OCN mRNA in the BMSC-Gel group was significantly higher than that in the BMSC group at three time point,P<0.05.At7d,the up-regulated expression of theOCN gene in the BMSC-Gel group was136 fold.At 14d、21d,BMSC-Gel group up-regulated the expression of OCN gene were less than that at the 7d,but it was still over 49 fold.2.The results ofthe study on BMSC-loaded calcium microspheres/CPC composite(1)Live/Dead staining results:at 1d,the persent of live cells of CPC+BMSC-Gel group was slightly lower than that of BMSC-Gel group(P<0.05),but there was no significant difference between the two groups at4d,7d and 14d(P>0.05).The persent of live cells were slowly reduced with time,but the persent of live cells were both above 92%.(2)Injectability of CPC/BMSC alginate microgel compound results:The extrusion rate of the CPC group was(32.19±6.53)%,the extrusion rate of the 2.5gCPC/1mlGel group was(7.12±0.653)%,the 2.0gCPC/1mlGel group was(20.51±0.855)%,The 1.5gCPC/1mlGel group was(43.45±0.890)%.Pairwise comparison between the four groups showed significant statistical difference,P<0.05.(3)The mechanical test results show:(1)compression modulus results:There was no significant difference between 2.5gCPC/1mlGel group(181.50±48.87)MPa and CPC group(188.23±48.06)MPa,P>0.05.The2.0gCPC/1mlGel Group(86.09±12.45)MPa and 1.5gCPC/1mlGel group(65.40±11.20)MPa was significantly lower than CPC group,P<0.05.(2)Compressive strength results:the compressive strength of 2.5gCPC/1mlGel group(3.10±0.37)MPa and CPC group(3.14±0.33)MPa,there was no significant difference,P>0.05;2.0gCPC/1mlGel group(2.04±0.39)MPa and 1.5gCPC/1mlGel group(1.30±0.29)MPa was lower than the CPC group,P<0.05.(4)Scanning electron microscopy results:the surface of the CPC group was rough,dense and without obvious pores.The 2.5gCPC/1mlGel group had a small amount of scattered small pores.The 2.0gCPC/1mlGel group was looserthan the 2.5gCPC/1mlGel group,with more pores,and1.5gCPC/1mlGel group showed obvious large pores,and the pores communicated with each other.Conclusion:(1)The alginate microspheres prepared by microfluidic technology have regular morphology and the size is controllable;(2)Alginate microspheres do not affect the survival and osteogenic differentiation potential of the encapsulated BMSC;(3)During the curing of CPC,the alginate microspheres have a certain protective effect on the encapsulated BMSC;(4)The BMSC alginate microsphere/CPC composite increases the injectability,and the alginate microspheres have a certain pore-forming effect on CPC.