The Effect Of HAPLN1 On Fracture Healing In Children

Fracture is a common orthopedic disease in which the continuity and integrity of bone structure are interrupted.The latest research shows that nearly 10%of fracture patients have poor fracture healing due to various reasons,which seriously affects the living quality of patients.In the process of fracture healing,the capillaries at the damaged site grow faster,transporting nutrients and bringing many undifferentiated mesenchymal cells at the same time.These cells can differentiate into osteoblasts or chondrocytes,which can promote the formation of callus and accelerate fracture healing.The healing process of fracture in children is basically similar to adult fracture healing.Studies have shown that the fracture rate of children and adolescents is higher than that of adults,and it has a greater impact on their health and physical development.Therefore,it is of great significance to explore the potential targets for the treatment of fracture patients,especially children.Osteoblasts play a key role in the process of fracture healing and are essential for the bone formation of intramembranous and endochondral during fracture healing.After fracture,preosteoblasts recruit and migrate to sites of bone formation can accelerate bone formation and repair.Mouse osteoblastic MC3T3-E1 is an important cell model for the study of bone formation which derived from mesenchymal stem cells of the multipotent bone marrow matrix,and is responsible for the synthesis,secretion and mineralization of bone matrix.Hyaluronan and proteoglycan link protein 1(HAPLN1)is an essential component of the extracellular matrix required for normal cartilage development.HAPLN1 was originally found to maintain the aggregation and binding activity of hyaluronic acid(HA)to proteoglycans.Studies have shown that down-regulated expression of HAPLN1 is closely associated with osteoarthritis(OA)and Kashin-Beck disease(KBD).In contrast,the expression of HAPLN1 was significantly increased in induced pluripotent stem cells(ipscs)which promoted osteogenic differentiation.However,the osteogenic properties of HAPLN1 have rarely been investigated.Currently,comprehensive gene expression databases show that HAPLN1 is highly expressed in osteogenic differentiation of MC3T3-E1 cells induced by bone morphogenetic protein(BMP)9 and ascorbic acid.According to databases,BMP4/Smad 1/5/8 pathway plays an important role in preventing bone loss and promoting osteogenic differentiation.Based on these findings and current knowledge,we attempted to experimentally investigate the expression and the possible cellular signalling mechanisms of HaplN1 in fracture healing.In the present study,the relationship between HAPLN1 expression and fracture healing process was first investigated by examining the expression of HAPLN1 in blood samples from children with fracture and in the chondrocytes of callus in a rat model of right femoral fracture.The expression of HAPLN1 during the osteogenic differentiation of MC3T3-E1 cells and the effect of knocked-down HAPLN1 on bone formation were studied.Finally,we investigated whether this process was related to the activation of bone morphogenetic protein 4(BMP4)/Smad 1/5/8 signaling pathway.The following figure is a graphic summary of the experimental concept.Objective1.Investigate the expression change of HAPLN1 during fracture healing process.2.To investigate the endogenous expression changes of HAPLN1 in osteogenic differentiation of MC3T3-E1 cells.3.To investigate whether HAPLN1 affects osteogenic differentiation through the BMP4/SMAD1/5/8 signaling pathway.Methods1.Human Fracture Specimen Collection and ELISA AnalysisWe collected 20 blood samples from patients with femoral fractures(bone healed at 2 weeks)and normal people separately.Blood samples were drawn by a specialized orthopedic nurse and all participants provided written informed consent.The level of HAPLN1 in serum was detected by Human HAPLN1 ELISA Kit(MyBioSource,China)according to the manufacturer’s instructions and then the standard curve method is used for the quantitative analysis.All the samples were collected from The First Afliated Hospital of Anhui Medical University.2.Rat Model of Femoral FractureMale Sprague-Dawley(SD)rats[6 weeks old]were randomly divided into two groups:the sham group and the fracture group.Rats in the sham group were only exposed to the right femur,and rats in the fracture group experienced middle femoral shaft fracture surgeries.Briefy,the right femur of rats was subjected to blunt dissection.A transverse osteotomy was then performed using an osteotome and hammer in the mid-shaft of the femur.Then,Kirschner wires were inserted in the femur medullary cavity to fx fracture bone sites.We observed the bone formation of fractured rats at 1,2,4,and 6 weeks after model establishment,and we collected bone tissues at the site of a fracture(with bone callus)after euthanasia.In animal experiments,we set up 6 biological replicates and 3 technical replicates for each test.All animal protocols were approved by the Ethics Committee of Anhui Medical University(LLSC20221090).3.To investigate the endogenous expression changes of HAPLN1 in osteogenic differentiation of MC3T3-E1 cellsThe murine pre-osteoblast cell line MC3T3-E1 was purchased from iCell Bioscience(China).MC3T3-E1 cells were cultured in alpha-modifed eagle’s medium(MEM-α medium;iCell Bioscience,China)supplemented with 10%fetal bovine serum(FBS;Tianhang Biotechnology,China),7-mM β-glycerol phosphate(Macklin,China),and 50-mg/mL ascorbic acid(Aladdin,China)at 37℃ in an incubator with 5%CO2.MC3T3-E1 cells were preferred to differentiate into osteocytes on days 0,3,7 and 14 using this medium,and the expression of HAPLN1 in cells was detected by Real-time PCR,Western blot,and immunofluorescence.Comparative analysis of HAPLN1 expression changes in cells before and after 3,7,and 14 days in differentiation.Subsequently,we committed HAPLN1 silencing by HAPLN1 shRNA lentivirus infection for 48 hours,and MC3T3-E1 cells were culturedin osteogenic medium for 7 days,14 days,and 21 days to detect several biochemical indexes such as Col1A1,OCN,and ALP.The experiments were divided into infected and uninfected groups for comparative control analysis.4.HAPLN1 affects osteogenic differentiation through the BMP4/SMAD1/5/8 signaling pathwayIn order to silence the expression of HAPLN1,it is preferred to infect MC3T3-E1 cells with lentivirus carrying HAPLN1 shRNA for 48 hours,and then the cells was incubated with osteogenic medium for 7 days,and Western blot detects the expression of BMP4,p-SMAD1S463+S465,p-SMAD5S463+S465,SMAD1+SMAD5 in cells.The biochemical indexes of BMP/SMAD pathway were different between the infected and uninfected groups.Then,After 48 h post infection,we induced osteogenic differentiation of cells,and added or without the BMP/SMAD pathway inhibitor 0.2μM LDN193189(Far Field Biotechnology,China)to detect the expression of Col1A1,OCN and ALP after 7,14 and 21 days and cell mineralization.In this step,we set up four groups,namely the blank control group(Control group),the inhibitor group(LDN193189 group),the inhibitor+independent sequence group(LDN193189+NC group)and the synergy group(LDN193189+HAPLN1-shRNA group).Results1.The Fluctuant Expression of HAPLN1 in the Progress of Fracture HealingThe expression of HAPLN1 in blood samples collected from fracture patients is higher than that of normal individuals.In addition,we used SD rats to establish a right femur fracture model for in vivo measurement.Accordingly,the expression of HAPLN1 increased firstly and then decreased.It was observed that the expression of HAPLN1 was consistent with the progress of bone formation,indicating that HAPLN1 was related to enhancing the porosity of bone tissue at the fracture site.In general,we found that there was a large amount of HAPLN1 expression in the process of fracture healing in humans and rats.2.Increased Endogenous Expression of HAPLN1 in the Osteogenic Diferentiation of MC3T3-E1 CellsMC3T3-E1 cells were selected as in vitro model to evaluate their osteoblastic function and several osteoblastic cytokines were detected.The results showed that the increased expression of HAPLN1 was consistent with Runx2 and OCN,and its endogenous expression level was increasedin osteogenic differentiation of MC3T3-E1 cells.These results indicate that Hapln is upregulated in the early osteogenic process of MC3T3-E1 cells.Meanwhile,the effect of HAPLN1 on osteogenic differentiation was detected by establishing HAPLN1 silenced MC3T3-E1 cells.After shRNA2 silenced HAPLN1,the protein expression of Runx2,Col1A1,OCN,and OPN was significantly downregulated(Figure 3c).In addition,compared with normal MC3T3-E1 cells,the osteogenic markers of ALP activity were reduced by approximately 50%(Figure 3b).Similarly,the mineralization of osteoblasts in cells was significantly reduced(Figure 3d).These results indicate that HAPLN1 can promote osteogenic differentiation and matrix mineralization.3.HAPLN1 activates BMP4/SMAD1/5/8 signaling pathwayThe BMP4/SMAD1/5/8 pathway plays an important role in the induction of bone formation.WB and IF staining assays were used to evaluate the sensitivity of BMP4/SMAD 1/5/8 signaling cascade after inhibiting HAPLN1 expression.After HAPLN 1 silencing,the protein expression of BMP4 was significantly inhibited.The function of BMP4 is achieved through intracellular signaling of Smads proteins.The expression of p-Smad 1(Ser463/465)/Smad5(Ser463-465)/Smad9(8)(Ser465/467)protein was significantly downregulated,while there was no significant change in Smad 1/5/9 protein(Figure 4a).These results indicate that HAPLN1 promotes BMP4 mediated phosphorylation of Smad 1/5/8 in osteoblasts.the inhibitor LDN193189 was used to specifically block the BMP4/Smd 1/5/8 signaling pathway.When cells were induced to differentiate in the presence of LDN193189 for 7 days,the protein expression of osteogenic markers Col1A1 and OCN were significantly reduced compared to the carrier group(Figure 5a).During the 14 day induction period,the osteogenic markers ALP was inhibited(p<0.005)(Figure 5b).Compared with treated cells,stronger alizarin red staining was observed in cells without LDN193189,indicating that the silencing effect of LDN193189+on HAPLN1 hindered the mineralization process(Figure 5c).The results indicate that HAPLN1 activates osteogenic differentiation through the BMP-4/Smad 1/5/8 signaling pathwayConclusions(1)There is a significant expression of HAPLN1 during the fracture healing process in children and rats.(2)The endogenous expression of HAPLN1 in MC3T3-E1 cells during osteogenic differentiation is increased,can promote osteogenic differentiation and matrix mineralization.(3)HAPLN1 activates osteogenic differentiation through the BMP-4/Smad 1/5/8 signaling pathway.