Patterned 3D Printed Scaffolds Loaded With Urine Stem Cells For Bone Defect Repair

Background and Objective:Urine-derived stem cells(USCs),isolated from the urine,is a type of adult stem cells with the biological characteristics of mesenchymal stem cells(MSCs).In recent years,USCs have been used in tissue regeneration due to non-invasive isolation process.As a new kind of scaffold manufacturing technology,3D printing technology can be used in the construction of personalized scaffolds for the treatment of bone defects.As one of the key characteristics of scaffolds,the topological structure of the scaffold surface can regulate the biological behaviors of seed cells,including adhesion,proliferation,migration,and differentiation.In this study,we isolated USCs from the urine,detected surface markers of USCs,and evaluated cell proliferation and multidirectional differentiation of USCs.We also used 3D printing technology to fabricate a 3D printed scaffold,and constructed regularly patterned nanosheets on the surface of 3D printed scaffolds by surface epiphytic crystallization.In addition,we observed the biological behaviors of USCs on the surface of patterned 3D printed scaffolds.The bone repair ability of patterned 3D printed scaffolds loaded with USCs in vivo was also discussed in this study.Materials and Methods:1.USCs were isolated from the 200-250 ml urine of healthy adults by double centrifugation.The surface markers of USCs were detected by flow cytometry.The cell proliferation and multidirectional differentiation ability of USCs were also evaluated in this study.2.We used 3D printing technology to fabricate the 3D printed Poly(ε-caprolactone)(PCL)scaffolds.Patterned nanosheets were constructed on the surface of 3D printed PCL scaffolds by surface epiphytic crystallization.We used scanning electron microscope(SEM)and atomic force microscopy(AFM)to observe the topological morphology of patterned 3D printed PCL scaffolds and 3D printed PCL scaffolds.Fourier transform infrared spectrometer(FTIR)and X-ray photoelectron spectroscopy(XPS)were used to detect the elemental composition and functional groups of the patterned nanosheets on the surface of the patterned 3D printed PCL scaffold.The biocompatibility of patterned 3D printed PCL scaffolds and 3D printed PCL scaffolds in vitro were also evaluated in this study.3.USCs were seeded on the surface of patterned 3D printed PCL scaffold and3 D printed PCL scaffold respectively.The cell proliferation and cell morphology of USCs on the surface of two kinds of scaffolds were observed.The expression of osteogenic genes of USCs on the surface of scaffolds was also compared in this study.4.Patterned 3D printed PCL scaffolds loaded with USCs were used in the treatment of rabbit’s bilateral cranial bone defects with a diameter of 8mm.Five groups were conducted in this study,including group of patterned 3D printed PCL scaffolds loaded with USCs,group of 3D printed PCL scaffolds loaded with USCs,group of patterned 3D printed PCL scaffolds,group of 3D printed PCL scaffolds,and blank group.Micro-CT,HE staining,and Masson staining were used to evaluate the bone regeneration of each group in vivo after 6 and 12 weeks.Results:1.The USCs separated by double centrifugation can be observed on the bottom of cell culture dishes as obvious cell colonies on day 5 to day 7.The cell morphology of USCs exhibits a spindle shape.The flow cytometry demonstrated that USCs positively expressed CD29,CD90,and CD44,and negatively expressed CD31,CD45,and HLA-DR.The cell proliferation curve of USCs exhibited typically S-shaped and can be divided into a slow growth period(days 1-3),a logarithmic growth period(days 3-8),and a plateau period(starting on day 8).After osteogenic differentiation of USCs,the formation of calcium nodules can be observed.Additionally,the BMP-2,RUNX-2,COL-1,and ALP m RNA of USCs increased.After adipogenic differentiation of USCs,the formation of lipid droplets can be observed in USCs.After the chondrogenic differentiation of USCs,a large amount of mucopolysaccharides can be observed in USCs.2.In this study,3D printed PCL scaffolds were constructed by using 3D printing technology.Patterned nanosheets were fabricated on the surface of3 D printed PCL scaffolds by surface epiphytic crystallization.SEM found that the surface of 3D printed PCL scaffolds was smooth,while the surface of 3D printed PCL scaffolds was rough.AFM demonstrated that the roughness of patterned 3D printed PCL scaffolds was significantly higher than that of 3D printed PCL scaffolds.In addition,no significant differences were observed between patterned 3D printed PCL scaffolds and 3D printed PCL scaffolds,in terms of porosity,pore size,and mechanical properties.The element composition and functional groups of patterned nanosheets were the same as 3D printed PCL scaffolds.The hemolysis test and animal skin stimulation test of the two groups of scaffolds were negative.In addition,compared with 3D printed PCL scaffolds,patterned 3D printed PCL scaffolds have a higher ability of protein adsorption and mineralization in vitro.3.USCs can survive on the surface of patterned 3D printed PCL scaffolds and3 D printed PCL scaffolds.The number of living cells and cell survival rate of patterned 3D printed PCL scaffolds were higher than that of 3D printed scaffolds.In addition,the length of USCs on the surface of patterned 3D printed PCL scaffolds was longer than that of 3D printed scaffolds.After osteogenic differentiation of USCs on the surface of scaffolds,the RUNX-2and COL-1 m RNA expression of USCs on the surface of the patterned 3D printed PCL scaffolds were significantly higher than that of the 3D printed PCL scaffolds on day 3.The RUNX-2,COL-1,ALP,BMP-2,TAZ m RNA expression of USCs on the surface of patterned 3D printed PCL scaffolds on day 7,were significantly higher than that of 3D printed PCL scaffolds.The RUNX-2,COL-1,ALP,BMP-2,OCN,OPN,and TAZ m RNA expression of USCs on the surface of patterned 3D printed PCL scaffolds on day 14 and day 28,were significantly higher than that of 3D printed PCL scaffolds.4.The results of the general view showed that scaffolds were tightly integrated with the edge of the bone defect.The new bone formation can be observed in the edge of the bone defect.In addition,Micro-CT showed that group of patterned 3D printed scaffolds loaded with USCs has the highest BT/TV of all groups.The blank group is lowest of all groups.The HE and Masson staining results showed that all scaffolds were tightly integrated with the edge of the bone defect.The new bone formation of the group of patterned3 D printed PCL scaffolds loaded with USCs was significantly more than that of the other groups.In addition,a large number of bone lacunas were observed in the group of patterned 3D printed PCL scaffolds loaded with USCs.Conclusion:1.The isolation process of USCs is non-invasive.USCs have a good ability of proliferation and multidirectional differentiation,and can also stably express surface markers of mesenchymal stem cells.USCs can be used as an ideal kind of seed cells in the field of bone regeneration.2.Surface epiphytic crystallization is a safe and effective method for surface patterning of 3D printed PCL scaffolds.Compared with 3D printed PCL scaffolds,patterned 3D printed PCL scaffolds have a higher ability of protein adsorption and mineralization in vitro.3.The patterned 3D printed scaffolds can effectively promote the proliferation and osteogenic differentiation of USCs.4.Patterned 3D printed PCL scaffolds loaded with USCs exhibited a good ability to repair cranial bone defects of rabbits,which provides the theoretical basis for the clinical application of 3D printed bone substitutes in bone regeneration.