Study Of Effect And Mechanism Of Cathepsin K On Jaw Bone Marrow Mesenchymal Stem Cells And Alveolar Bone Regeneration

No matter a small alveolar bone defect resulted from periodontitis or periapical periodontitis,or a large jaw defect caused by jaw cyst,tumor,or trauma,it will seriously impair the patients’ oral function.Conventional surgical treatment is always complicated,and also difficult to obtain ideal repair effect.Promoting endogenous jaw regeneration is a potential strategy for the treatment of jaw defects.Bone marrow mesenchymal stem cells(BMMSC)play an important role in the regulation of bone regeneration through self-renewal and multi-directional differentiation.Comparing with long bone derived BMMSC,jaw bone marrow mesenchymal stem cells(JBMMSC)have higher osteogenic ability in situ,and are the most potential seed cells in the field of jaw regeneration.Therefore,improving the proliferation and osteogenic differentiation of JBMMSC is the key to promote endogenous jaw regeneration.Cathepsin K(CTSK),a key regulator of bone metabolism,was initially considered to be specifically expressed in osteoclasts and played an important role in regulating bone resorption.Studies had shown that CTSK was also expressed in bone formation related cells,such as fibroblasts,osteoblasts,BMMSC and periodontal ligament stem cells(PDLSC).It was reported that CTSK positive periosteal stem cell could participate in fracture healing process.But whether endogenous CTSK could regulate proliferation and osteogenic differentiation of JBMMSC has not been reported,and the mechanism of endogenous CTSK regulating the regeneration of MSC remains unclear.In this study,we used tooth extraction models of Ctsk knockout mice(Ctsk-/-)and their wildtype(WT)littermates to compare their alveolar bone regeneration.We further investigated the effects of Ctsk deficiency or inhibition on the proliferation and osteogenic differentiation of JBMMSC in vitro.At last,we explored mechanism of endogenous CTSK regulating the regeneration of JBMMSC.This study will be helpful to find out the new mechanism about how to regulate the JBMMSC regeneration through CTSK and will provide a new strategy for the treatment of jaw bone defects.Part 1 Effect of Ctsk knockout on alveolar bone regenerationPurpose:To study the effect of CTSK on alveolar bone regeneration.Methods: Ctsk-knockout(Ctsk-/-)mice in a C57BL/6J background were generated and genotypes were determined by agarose electrophoresis.Then general observation,Micro-CT scanning and immunohistochemistry were performed to identify whether Ctsk was successfully knocked out.Eight-week-old Ctsk+/+ and Ctsk-/-were selected to extract the bilateral maxillary first molars and 3 mice were sacrificed on the 3,7,10,14,21 and 28 days after tooth extraction.Then samples were subjected to stereo microscope,Micro-CT,HE and Masson staining to observe the effect of Ctsk knockout on the tooth extraction healing process.TRAP staining was performed to investigate the effect of Ctsk knockout on osteoclasts.At last,Osterix immunohistochemistry was used to study the effect of Ctsk deficiency on bone formation ability.Results: Micro-CT and immunohistochemistry showed that Ctsk was successfully knocked out in C57BL/6 mice.Stereo microscope results showed that: The healing process of tooth extraction sockets in mice could be divided into two stages: bone filling stage and bone remodeling stage.Ctsk deficiency did not affect the mucosa healing.Micro-CT,HE and Masson staining showed that it needed 10 days for new bone to fill the socket in Ctsk-/-mice and 14 days in Ctsk+/+ mice.TRAP staining showed that the number of osteoclasts was not affected by Ctsk deficiency during the bone filling stage,and was increased in bone remodeling stage.Number of Osterix-positive cells in Ctsk-/-mice was higher than that of the Ctsk+/+ mice on 7 days post-extraction which was the active phase of bone formation,and then was lower than that of the Ctsk+/+ mice which was the period of bone remodeling(P<0.05).Conclusion: Our datas indicated that Ctsk knockout could significantly promote alveolar bone regeneration,and this promotion may be related to the enhancement of osteogenic ability.Part 2 Expression pattern of CTSK in the alveolar bone regeneration processPurpose:To observe the expression and distribution of CTSK in the alveolar bone regeneration process.Methods: Eight-week-old Ctsk+/+ mice were used to extract the bilateral maxillary first molars,and 3 mice were sacrificed 3,7,10,14,21 and 28 days after tooth extraction.The expression of CTSK during tooth extraction wound healing process was observed by immunohistochemistry.TRAP staining was performed to observe the distribution of osteoclasts.Then CTSK/CD44,CTSK/CD90 and CTSK/OSX immunofluorescence were performed to observe whether MSC and osteoblasts expressed CTSK during the tooth extraction healing process.Results: CTSK-positive staining was observed on D3,when neither osteoclasts nor newly formed bone was observed.The number of CTSK-positive cells peaked on D10 and then decreased with time.TRAP staining showed that osteoclasts appeared with the beginning of new bone formation on D7 and peaked on D21.Then the osteoclasts decreased with time.And besides osteoclasts,CTSK also expressed in CD44 and CD90-positive cells and osteoblasts during the extraction socket healing process.Conclusion: CTSK expressed in a variety of cells during the extraction socket healing process,which indicated that CTSK not only played a role through bone resorption mediated by osteoclasts,but may also act through bone formation mediated by CD44+/CD90+ mesenchymal stem cells and osteoblasts during alveolar bone regeneration process.Part 3 Study of the effect and mechanism of Ctsk deficiency or inhibition on the proliferation and osteogenic differentiation of JBMMSCPurpose : To explore the potential mechanism of Ctsk deficiency or inhibition on JBMMSC proliferation and osteogenic differentiation.Methods: Six-week-old Ctsk+/+ and Ctsk-/-mice were used to separate and culture JBMMSC,and Western blot and immunocytochemistry were performed to observe the expression and distribution of CTSK in JBMMSC.The effect of Ctsk deficiency or inhibition on proliferation and osteogenic differentiation of JBMMSC was evaluated by CCK-8,Western blot and Alizarin Red staining.Then differentially expressed genes between Ctsk+/+ and Ctsk-/-JBMMSC were checked by RNA-seq and bioinformatics analysis was performed to select the potential mechanism.ECAR,glucose consumption and lactate production were detected to study the effect of Ctsk deficiency or inhibition on glycolysis of JBMMSC.At last,CCK-8,Western blot and Alizarin Red staining were performed to check whether 3PO(inhibitor of glycolytic)could block the effect of Ctsk deficiency or inhibition on proliferation and osteogenic differentiation of JBMMSC.Results: Western blot and immunocytochemistry results showed that CTSK was expressed in JBMMSC.CTSK was not only located in the lysosomal,but also in the cytoplasm.CCK-8,Western blot and Alizarin Red staining results showed that,the OD value of Ctsk knockout or ODN treatment group was significantly higher than that of the Ctsk+/+ JBMMSC(P<0.05).The expression levels of osteogenic related proteins Runx2 and ALP were significantly increased,and the number and area of calcium nodules were also significantly increased(P<0.05).RNA-seq results showed that after Ctsk knockout,the glycolysis-related genes(such as Pfkfb3,HK1)were significantly increased.ECAR results revealed that the glycolysis and glycolysis capacity of JBMMSC in the Ctsk-/-and ODN treatment group were significantly higher than that of control group(P<0.05),while the glucose consumption and lactate production were significantly higher than those of control group(P<0.05).After 3PO treatment to block the glycolysis pathway,OD value of Ctsk-/-+3PO and 3PO+ODN treatment group was significantly lower than that of the control group;the expression levels of osteogenic related proteins and the number and area of calcium nodules were also significant lower than control group(P<0.05).Conclusion: Our data confirmed that endogenous Ctsk deficiency or inhibition could promote JBMMSC proliferation and osteogenic differentiation by glycolysis.In summary,the present study confirmed Ctsk knockout could promote alveolar bone regeneration by tooth extraction model,and showed that CTSK may not only depend on osteoclasts during this process,but may also act through bone formation mediated by MSC and osteoblasts.In vitro studies showed knockout or inhibition of CTSK could enhance the proliferation and osteogenic differentiation of JBMMSC through glycolysis.However,the present study still need further experiments to improve it,such as constructing BMMSC-specific Ctsk knockout mice to exclude the influence of osteoclasts;verifying whether 3PO can block the effect of Ctsk knockout on bone filling in vivo.