SATB2 Enhances Osseointegration Of Titanium Implant

Titanium dental implants are widely used in dentistry and orthopaedic surgery for replacement of teeth and restoration of the maxillary and mandibular bone defects. Dental implants have become a standard and routine procedure in oral health care.Osseointegration plays a key role in the fixation and anchoring of the implants. It is a phenomenon in which intimate contact between bone and implant at the optical microscopy level and enables dental implant to restore intraoral function. Nevertheless, bone regeneration around implants is relatively slow after placement. The bone support to the implants after initial healing time is limited due to the low density of the newly formed bone tissue around the implants. So, osseointegration at the dental implant surface still remains a challenge. Much effort has been done to reduce the healing time of the dental implants osseointegration and decrease the treatment time by scientists and clinicians. To gain this purpose, implant design parameters are modified including implant material, macrogeometry and surface modification. It is generally accepted that osseointegration of dental implants is achieved by the activity of osteoblasts. Most of these researchs were aimed at host response. The recruitment and differentiation of osteoprogenitor cells are the key in bone formation. It is generally accepted that the bone forming cells at the dental implants surface are from the neighboring host bone. Besides these modifications of the design parameters, application of some bone-related molecules and factors have been adopted to increase bone-to-implant contact (BIC) systemically or locally. It was recently found that special AT-rich sequence-binding protein 2 (SATB2) plays a pivotal role in craniofacial development and osteolbast differentiation. It can regulate skeletal development and osteoblast differentiation by synergizing, amplifying and thus exponentially augmenting the activity of multiple osteogenic factors including Runx2, Osx and Atf4. Satb2 can bind to the promoters of BSP and OC and up-regulate their expression. Satb2 represses Hoxa2 gene expression and regulates multiple genes in osteoblasts. SATB2 gene knockout mouse shows multiple craniofacial defects that include a significant truncation of the mandible, a shortening of the oral maxillofacial bones, malformations of the hyoid bone. The Satb2-/-mice die immediately after birth. The defects in osteoblast differentiation and function were found, and it showed delayed bone formation and mineralization in Satb2-/-mice embryos. The expression level of the collagen in bone matrix decreased dramatically, and the deposition defects of extracellular matrix were also detected in Satb2-/-mice embryos. The expression level of Nanog can be upregulated by Satb2 and play an important role in the regulatory network of embryonic stem cell differentiation. While binding to AT-rich DNA elements, SATB2 modifies chromatin structure and regulates multiple genes. Based on the researches on Satb2, it can tell that Satb2 has a pivotal role in craniofacial development and skeletal patterning, and it is a master gene in osteoblast differentiation. It is suggested that Satb2 can be a robust osteoinductive molecule regulating other transcription factors to form a molecular node for a transcriptional network.Based on researches above, the function of Satb2 on implant osseointegration was studied in this research. The retrovirus RCAS and pBABE-Hygro were used in this study to deliver Satb2 into the cells in the tissue surrounding the implant. And the roles of Satb2 in implant osseointegration were evaluated by the detection of the bone-related genes and bone formation around the implants. The cells of BSP-TVA transgenic mouse infected by RCAS and pBABE-Hygro virus stocks were different from each other. Mammalian cells engineered to express TVA are highly susceptible to be infected by RCAS. While all the dividing mammalian cells can be infected by pBABE-Hygro virus. So the function of the different cells infected on implant osseointegration by these two retrovirus systems was analyzed according to the results. Meanwhile, BMSCs with forced Satb2 expression were used by local administration to detect its function on implant osseointegration.Materials and Methods: Part I:BSP-TVA transgenic mice provided an excellent in vivo model for achieving the purpose of this study. The strategy was based on the receptor for subgroup-A avian aleukosis virus (RCAS), TVA. The retroviral receptor TVA was driven by a 4.9 kb BSP promoter and was selectively expressed in skeleton tissues. The retrovirus construct, RCAS-Satb2, was confirmed by sequencing. The implantation sites were prepared on anterior-distal surface of the femurs.3μl of RCAS or RCAS-Satb2 virus stock of about 108 cfu/ml was applied to the bone defects before the SLA surface implants were press-fitted into slightly undersized holes. The femurs were isolated 1 week and 3 weeks after the surgery. H&E staining was performed to detect the bone formation around the implants. H&E staining, BSP immunohistochemical staining and histomorphometric evaluation were performed to investigate the roles of Satb2 in osseointegration of the implants. Total RNA was extracted from the bone tissues (1 mm mesial and distal to the implantation site) with TRIzol reagent. Quantitative real-time reverse transcription-PCR (qRT-PCR) assay was performed to detect the expression levels of Satb2, Osx, Runx2, BSP, OC and COLI.PartⅡ:To better understand the results from Part I, pBABE-Hygro retrovirus system was used in this part. After produce of the virus stocks of pBABE-Hygro and pBABE-Hygro-Satb2, the viral stocks were used as RCAS and RCAS-Satb2 virus stock in PartⅠ. The same procedure and analysis were performed like what have been done in PartⅠI.PartⅢ:BMSCs labeled by EGFP were infected by pBABE-Hygro-Satb2and pBABE-Hygro viral stocks. The gene expression levels of Satb2, Osx, Runx2, BSP, COLI and OC were detected 2 days,9 days, and 23 days after infection.2 days after infection, BMSCs were locally administrated in implant sites of B6D2F1 mice. The same procedure and analysis were performed like that in PartⅠ. Immunohistochemical staining of GFP was performed to trace labeled BMSCs.Results:PartⅠ:1 week after the surgery, the formation of woven bone could be detected. It was found that the cells in contact with the implants were well organized. The cells lined in several layers surrounding the implants. The inflammatory cells could be detected in the tissue around the implants. The bone area/total implant area was higher in the test group than that in the control group, but there was no significant difference. About 30%of the tissue in contact with the implants was bone relative to 70%soft tissue in test group. And the%BIC in Satb2 group was significantly higher than that in control group. The real-time RT-PCR results showed that the expression level of SATB2 was up-regulated about 3 times in RCAS-Satb2 group compared with control group. The expression levels of OSX, Runx2, BSP, COLI and OC were significantly up-regulated in RCAS-Satb2 group than that in control group. The results of BSP immunohistochemical staining were in coincidence with the results of H&E staining. There were more BSP positive cells in test group than that in control group.3 weeks after implantation, the implants were surrounded by woven bone and lamella bone. There were no detectable inflammatory cells in the tissue surrounding the implants. The bone area/total implant area was higher in the test group than that in the control group 3 weeks after implantation. About 80%of the tissue in contact with the implants was bone. And the%BIC in Satb2 group was significantly higher in the test group than that in control group. It showed in real-time RT-PCR results that 3 weeks after the operation, there were no significant difference in the expression levels of Satb2, Osx, Runx2, BSP, COLI and OC in RCAS-Satb2 group and control group. Immunohistochemical staining of BSP showed that there was no difference between control group and test group.PartⅡ:H&E staining, immunohistochemical staining and histomorphometric analysis of the pBABE-Hygro and pBABE-Hygro-Satb2 group 1 week after the implantation were almost the same with that in RCAS and RCAS-Satb2 group 1 week after the operation. Quantitative real-time RT-PCR analysis results implied that the expression levels of Satb2, Osx, Runx2, BSP and COLI were significantly higher in pBABE-Hygro-SATB2 group than that in control group 1 week after implantation. The expression level of OC was upregulated, but there was no significant difference between test group and control group. 3 weeks after implantation, organized bone around the implants could be detected in pBABE-Hygro group and PBABE-Hygro-Satb2 group. In pBABE-Hygro-Satb2 group, the mature and organized lamellar bone in contact with the implant could be seen. While in pBABE-Hygro group, there was immature bone in contact with the implant site. Histomorphometric analysis showed that the percentage of the bone at implantation site was significantly higher in pBABE-Hygro-Satb2 group than that in the control group 3 weeks after implantation. And the percentage of bone in contact with the implant was significantly higher in pBABE-Hygro-Satb2 group than that in the control group. The percentage of bone in direct contact with implant was 1.6 times in Satb2 group than that in control group.21 days after surgery, the expression levels of Satb2, Osx, Runx2, BSP, COLI and OC were significantly higher in pBABE-SATB2 group than that in control group. Cell counting results showed that there were more BSP positive cells in pBABE-Hygro-Satb2 group than that in pBABE-Hygro group at 3 weeks after surgery.PartⅢ:EGFP labeled BMSCs scattered in the tissues surrounding the implants 1 week after implantation. The GFP positive BMSCs were still detectable 3 weeks after the surgery. Local administration of BMSCs with forced expression of Satb2 enhanced osseointegration of the implants 3 weeks after the implantation and promoted bone formation. The real-time RT-PCR results from in vivo smples coincided with that of in vitro results. The expression levels of Satb2, Osx, Runx2, BSP, COLI, and OC were significantly higher in the Satb2 group than that of control group, which was the same pattern with the 3-week results of PartⅡ.Conclusions:1. In this presented study, it can be found that Satb2 can be delivered into the cells of the tissue surrounding the implant successfully by both pBABE-Hygro and RCAS retrovirus system when virus stocks are used locally. And the expression level of Satb2 in the tissue surrounding the implant can be up-regulated.2. Overexpression of Satb2 upregulates the expression levels of transcriptional factors Osx and Runx2 in implantation site. Satb2 overexpression enhances the expression levels of bone matrix proteins and osteogenic factors, such as BSP, COLI, and OC. While RCAS-Satb2 viral stocks are locally used, implant osseointegration can be enhanced 1 week after implantation.3 weeks after operation, Satb2 overexpression enhances osseointegration of the implants and promotes bone formation around the implants.3. The mechanism on bone formation between pBABE-Hygro-Satb2 and RCAS-Satb2 is different from each other after local administration. Local use of RCAS-Satb2 viral stocks can improve bone-to-implant contact significantly 1 week after implantation. The different effects on bone formation between RCAS-Satb2 and pBABE-Hygro-Satb2 may lie in the different cells infected when the viral stocks were locally used. Only BSP positive cells can be infected by RCAS retrovirus. While all mammalian dividing cells can be infected by pBABE-Hygro retrovirus, and the cells infected by pBABE-Hygro-Satb2 virus get involved in bone formation and remodeling of the tissue surrounding the implants.4. Overexpression of Satb2 can be detected in BMSCs infected with pBABE-Hygro-Satb2 viral stocks at 2,9 and 23 days after infection. Satb2 overexpressed BMSCs enhance osseointegration of implants and promote bone formation around the implants.