| Background:The surface morphology of the implant can regulate cellular protein adsorption,change intracellular signaling,and affect osteoblast differentiation and final osteogenesis.The micro-nano composite morphology can better simulate the natural bone structure.Compared with the simple micro-or nano-surface morphology,it has a better effect on promoting osteoblast maturation and mesenchymal stem cell differentiation.At present,many scholars' research on material modification focuses only on its promotion of osteoblast proliferation and differentiation,but ignores the impact of early immune response of implant implantation.After the implant is implanted,tissue damage results in extravasation of blood,and molecules such as proteins-in the blood will be released and attached to the surface of the implant in a short time,causing blood-material interactions.The expression and release of damage-related molecules can be activated The host immune response causes infiltration and activation of inflammatory cells.Macrophages have a high degree of plasticity and dynamics.They can transform each other under the influence of signals in the tissue microenvironment.They can be transformed into pro-inflammatory M1 or anti-inflammatory M2,secreting IFN-?,TNF-?,and IL-1? Inflammatory factors or anti-inflammatory factors such as VEGF,TGP-? and IL-10,which have a beneficial or harmful effect on bone healing and regeneration,are the central regulators of the entire bone regeneration microenvironment.Macrophage polarization is affected by a variety of factors.Among them,the surface morphology of the implant plays a leading role in regulating the immune cell response.Currently,many studies have shown that the immunomodulation effect of nanotopography is better than microtopography.There are obvious differences in the immunoregulatory ability of nanotopography;however,there are few studies on the immunomodulatory ability and immune-promoting bone formation ability of microtopography on micro-nano composite surface.Purposes:This experiment intends to treat smooth titanium wafers by sandblasted,large-grit,acid-etched(SLA),and alkali thermal reaction,and finally obtain smooth,micron,nano and micro-nano titanium wafers.Observe the expression of four M1 and M2 type marker factors,inflammatory factors,and morphological changes of macrophages to investigate the immunoregulatory ability of micro-nano titanium tablets;observe cell migration experiments and co-culture to observe the immune environment of different titanium tablets Effects on osteoblast migration,extracellular matrix mineralization and osteogenic gene expression,so as to explore the mechanism of micro-nano composite morphology of bone immunity and osteogenesis,which is a micro-nano surface structure implant to promote osteointegration The molecular biological mechanism provides a theoretical basis and a meaningful theoretical basis for the clinical treatment of micro-nano implants.Methods:1.Preparation of smooth,micron,nano and micro-nano titanium sheets,the surface topographies of different specimens were characterized by a cold-field scanning electron microscope,the roughness average(Ra)were characterized by 3D laser scanning microscope.Contact angle measurements of different specimens were carried out using sessile drop method with a fixed volume of 2 uL every time using a contact angle goniometer.2.Using cold-field scanning electron microscope to observe the individual morphological changes of RAW264.7 cells on the four titanium films at 5 and 7 days of culturing.3.CCK-8(Cell Counting Kit-8)technology was used to detect the proliferation activity of MC3T3-E1 cells and RAW264.7 cells on the surface of four titanium disks.4.qRT-PCR was used to detect the expression of CD206,CD86,IL-1?,IL-10,TNF-?,and VEGF mRNA levels in RAW264.7 cells on the surface of the four titanium plates;different RAW264.7 conditioned media were used to stimulate Corresponding to MC3T3-E1 cultured on titanium plate,the expression level of BMP2,VEGF in MC3T3-E1 cells on the surface of four titanium discs was detected by qRT-PCR after 7-d stimulation.5.ELISA(Enzyme Linked Immunosorbent Assay)technology was used to detect the expression levels of IL-1? and IL-10 in the supernatants of RAW264.7 cultured on four kinds of titanium tablets for 1d,3d,and 7d.6.Transwell chamber system was used for MC3T3-E1 migration chemotaxis experiments;the number of cells migrated from.the upper chamber to the lower chamber was detected by CCK8 after 24h stimulation with different titanium conditioned media.7.Different RAW264.7 conditioned media were used to stimulate MC3T3-E1 of the corresponding titanium plate culture.Alizarin red staining was performed at 7Dand semi-quantitative analysis was performed by elution method;8.Cell mineralization was assessed semi-quantitatively by alizarin red staining.The RAW264.7 culture media on the four groups of specimens were used to stimulate MC3T3-E1 cells cultured on the corresponding specimens.The culture was stopped after 7d stimulation.Meanwhile,a-MEM culture media containing only 10%FBS were used to culture MC3T3-E1 cells on the four groups of specimens for 14 d.After termination of the culture,the cells were fixed with 4%paraformaldehyde for 30 min and then washed with PBS.After PBS washing,staining was performed with 0.1%alizarin red in Tris-HCL buffer,followed by PBS washing for removal of floccule.After that,photos were taken and 1 ml of eluent(hexadecyl pyridine chloride-sodium phosphate aqueous solution)added to each well.The solution was shaken on a shaker at 120 rpm until the dye on the titanium sheets was fully dissolved.The absorbance of the cells was measured at 620 nm.Results:1.The scanning electron microscopy(SEM)images at different magnifications and the superficial 3D microstructure and roughness are shown in Fig.1.The polished titanium sheet surface was flat and had no obvious scratches;after the sandblasted,large-grit and acid-etched(SLA)treatment,a large number of pits that overlapped and connected with each other were visible on the surface of titanium sheets at low magnification(10000X).Further observation at high magnification with 3D microstructures showed that on the primary depressions with a diameter of 30-50 um were several secondarily depressions with a diameter of 3-8 um.On the alkali-treated titanium sheets,an interconnected network structure of nanopores was visible,and the nanopores had a diameter of about 50-200 nm.Micro-nano titanium sheets were produced by sandblasting and acid-etching polished titanium sheets followed by alkali treatment.With the help of an electron microscopy and 3D structure diagrams,a network of nanopore structures was superimposed on the entire micropit surface.Fig.2A presents the roughness average(Ra)of the four types of specimens.Compared with P specimens,S and SN specimens had an increased Ra from 0.054 to 1.701 and 1.641,respectively.Although N specimens had a lower Ra than SN specimens,they were still significantly higher in Ra than P specimens(P<0.05).Fig.2 BC presents the hydrophobic test images of the four groups of specimens.2 ul of artificial saliva was used as the wetting agent.Compared with the polished surface,SN specimens had a significantly reduced contact angle(P<0.05).2.The cell viability grown on different samples was measured using CCK8 assay after 1,3,5,and 7 days of culturing.The results are shown in Fig.3A.The RAW264.7 cells cultured on the four groups of specimens demonstrated a similar proliferation curve trend;MC3TE-E1 on the SN and S specimens performed less well in proliferation than those on the P and N specimens on day 3,but the SN specimens had had a much higher count than the other three groups of specimens on day 7.3.Fig.3B presents the morphological changes of RAW264.7 cells cultured on the four groups of specimens cultivated after 5 and 7 days.The RAW264.7 cells on P specimens were disc-shaped as a whole,with little difference in morphology on day 5 and 7,and their pseudopods were thin and short.The RAW264.7 cells on S specimens were similar to those on P specimens in morphology,characterized by a disc shape and no obvious pseudopods The RAW264.7 cells on N specimens were mainly in a star shape with multiple bulges and long pseudopods that extended toward the surrounding and connected to the adjacent cells.The cells on SN specimens were mostly disc-shaped and have unapparent short pseudopods on day 5,but they changed to be similar to those on N specimens,manifest as polygons and long pseudopods that extended toward the surrounding and connected to the adjacent cells.4.The expression of M2-related genes is shown in the left of Fig.4.It can be seen that the expression of IL-10 on SN specimens after 1,3 and 7days of culturing.were better than that on the other three groups.The expression of CD206 and VEGF on SN specimens after 1 and 3 days of culturing was higher than that on P,S,and N specimens(P<0.05),but its expression on SN specimens after culturing for7 days was not statistically different with that on N specimens.The M1 gene expression is shown in the right of Fig.6.indicating that CD86 genes on S specimens had a significantly higher expression than those on the other groups of specimens after culturing for 1 day(P<0.05),followed by the CD86 genes on SN specimens;after culturing for 3 days,CD86 genes on S and SN specimens had no significant difference in expression,both slightly higher than that on P and N specimens(P<0.05);after culturing for 7 days,the CD86 genes on the four groups of specimens were not significantly different in expression,but the CD86 genes on N specimens had the lowest expression.N specimens demonstrated the highest expression of inflammatory factor TNF-? after 1,3 and 7days of culturing.followed by SN specimens,and the expression of TNF-? on these two groups of specimens were significantly higher than that on P and S specimens(P<0.05);the expression of IL-1? on S and N specimens was significantly higher than on P and SN specimens after 1 and 3 days of culturing(P<0.05);and the expression was highest on S specimens after culturing for 7 days(P<0.05).5.The contents of IL-10 and TNF-? in cell supernatants are shown in Fig.5.It can be seen that SN specimens had a higher secretion volume of IL-10 at day 1,3,and 7 than the other three groups of specimens(P<0.05);N specimens had the highest secretion volume of TNF-? at day 1,3,and 7(P<0.05),with no significant difference between SN and P specimens and S specimens having the lowest volume.6.The migration of MC3T3-E1 induced by conditioned medium was detected using CCK8 assay,as shown in figure 6.Transwell 24 h results showed that SN and N specimens outperformed P and S specimens in promoting cell migration,and SN specimens demonstrated the best performance in promoting migration of MC3T3-E1(P<0.05).7.The qRT-PCR technique was adopted to detect the effects of different surface topographies and conditioned media on the expression of MC3T3-E1 BMP2 and VEGF,as shown in figure 7.Under the combined effect of surface microtopography and conditioned medium,BMP2 and VEGF gene expression on SN specimens was higher than that on the other three groups of specimens(P<0.05).8.The effects of surface topography and conditioned medium on the mineralization of extracellular matrix of MC3T3-E1 were analyzed semi-quantitatively by alizarin red staining.The results are shown in the figure 8.7 days after the stimulation of conditioned medium,the MC3T3-E1 cells on all four groups of specimens showed significantly increased mineralization of extracellular matrix,and the cells on SN specimens showed significantly higher mineralization of extracellular matrix than those on the other three groups of specimens(P<0.05).After 14 days of purely culturing,the extracellular matrix mineralization of the MC3T3-E1 cells cultured on N and SN specimens was not significantly different,but significantly higher than that on P and S specimens(P<0.05).Comparative results between the MC3T3-E1 cells co-cultured for 7 days and those singly cultured for 14 days showed that there was no significant change in mineralization in the cells cultured on P and S specimens,but the cells cultured on N and SN specimens experienced significantly increased mineralization,and it was especially true for the cells cultured on SN specimens(P<0.05).Conclusion:The three-dimensional nano-network structure obtained by SLA and alkaline thermal treatment can promote macrophages to polarize toward the M2 phenotype,thereby realizing the anti-inflammatory effect of local microenvironments.Titanium sheets in a micro/nano-textured topography can promote the migration,proliferation,and extracellular matrix mineralization of osteoblasts.The micro/nano-textured topography can induce bone regeneration and vascular regeneration by promoting the secretion of BMP2 and VEGF in osteoblasts.However,the signal pathways and specific mechanisms involved therein need to be further studied. |
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