| BACKGROUNDEpimedium sagittatum is a traditional Chinese medical herb and widely used in the therapies of fractures,bone and joint diseases,impotence and senility in China for hundreds of years.Icariin(C33H40O15,molecular weight:676.67),a typical flavonol glycoside,is considered to be the major pharmacological component of Epimedium sagittatum.Recent evidences have indicated icariin can improve the osteogenesis from mesenchymal stem cells and suppress the activities of osteoclasts in vitro, thereby it exerts its bone-protective functions by increasing bone formation and inhibiting bone resorption.Additional studies have demonstrated that icariin has the ability to enhance the expression of osteogenic-related mRNA level in osteoblasts, and has a direct stimulatory effect on the proliferation and differentiation of pre-osteoblastic MC3T3-El cells in a BMP-and Runx2-dependent manner.Taken together,such results indicate that icariin is a potential osteogenic inductive agent and can be used in bone repair.What is more,icariin is chemically stable,and has high melting point,thus benefiting its extraction from raw herb and combination to form artificial bone material usually used for bone defect repair and/or drug-loading scaffolds. OBJECTIVE1.To investigate effects of icariin on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells(hBMSCs),and study the mechanism of promoting osteogenic differentiation action.2.To construct biomimetic icariin-chitosan/hydroxyapatite(icariin-CS/HA) scaffolds,and study physicochemical properties and biocompatibility.3.To study icariin release behavior from icariin-CS/HA scaffolds in vitro.4.To investigate bone repair capability of icariin-CS/HA scaffolds in vivo.METHODS1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanismIsolation and culture of hBMSCs Cells were obtained from the posterior iliac crests of 3 healthy adult volunteers following informed consent.After being cultured and expanded,the second generation cells were identified by phenotypic analysis and induced by adipogenic,osteogenic and chondrogenic media.hBMSCs in the third generation were used in the investigations.Cytotoxicity test of icariin The hBMSCs were seeded at a density of 5 000 cells/well in a 96-well plate and incubated for 24 h prior to the addition of 150μl icariin(icariin dosages were 10-9,10-8,10-7,10-6,10-5, 10-4and 2×10-4M separately)media and 0.05%(v/v)DMSO media,while control cells were with fresh DMEM.After 48 h,the number of survival cells was detected by MTT method.hBMSCs proliferation test hBMSCs were cultured in 96-well plates at a density of 2 000 per well,and were treated with icariin(10-9M～10-4M) and DMSO media.At 1,3,5,7 and 9d,cell proliferation curves were drawn based on OD values which were measured by MTT method.Osteogenic differentiation test of hBMSCs hBMSCs were plated on 6-well plates,2×107 cells/well,and were treated with icariin(10-9～10-4M),DMSO(negative control)and rhBMP-2(positive control) media separately.At 3,7 and 11d,cells were lysed in 100μl deionized water and homogenized by ultrasound at 4℃.Alkaline phosphatase(ALP)activity and total protein content in cell lysates were measured using an ALP activity kit and a micro-BCA Assay kit separately,and ALP activity was normalized for the corresponding total protein concentration(U/g).At 7,14 and 21d,osteocalcin(OCN) content in cell lysates were measured using an OCN Elisa kit.In addition,ALP staining of hBMSCs cultured for 11d were exerted by NBT/BCIP,and calcified nodules of hBMSCs cultured for 28d were stained with Alizarin red S,then Quantitive analyses of Alizarin red S were exerted.The mechanism of icariin promoting hBMSCs osteogenic differentiation Cells were cultured on 6-well plates at density of 2×l07cells/well,and were treated with DMSO,10-6M icariin,rhBMP-2 and 10-6M icariin+rhBMP-2 media separately.At 1,4 and 7d,RT-PCR was used to examined mRNA expression of osteogenic genes(ALP,OCN,OPN,Col-â… ,Cbfa1, BMP-2,BMP-4 and BMP-7)in hBMSCs.After being seeded onΦ10cm dishes and treated as above for 14d,osteogenic proteins(Cbfa1,OCN and BMPs)in hBMSCs were detected by Westernblot,at the same time,the OCN protein expression of climbing-cells was examined by immunofluorescence detection.2.Construction of biomimetic icariin-CS/HA scaffoldsThe construction,characterization and biocompatibility of biomimetic CS/HA scaffolds CS/HA scaffolds were prepared by in situ hybridization and freeze-dried methods.The micro-structure of scaffolds was examined by scanning electron microscopy(SEM)and HE staining,the density,porosity and pore diameter of CS/HA scaffolds were evaluated with the normal methods,and the physicochemical properties were detected by X-ray diffraction(XRD)examination and Fourier transformed infrared spectroscopy(FTIR).Effects of CS/HA conditional media,prepared with normal method,on proliferation of hBMSCs were tested by MTT method.At 3d and 10d,SEM was used to observe hBMSCs which were seeded on the surface of CS/HA.After being implanted in dorsal muscle pockets of New Zealand rabbits,CS/HA scaffolds were obtained at 1,4,8 and 12w,and histocompatibility and degradation of scaffolds were observed by histomorphology. The construction,characterization and biocompatibility of biomimetic icariin-CS/HA scaffolds We prepared 10-7,10-6,10-5mol icariin-CS/HA scaffolds with the same procedures as the preparation of CS/HA scaffolds but without touching icariin.Physicochemical properties of icariin-CS/HA were analyzed as above.And the mechanical properties of scaffolds in wet state were detected with universal testing machine.Influences of icariin-CS/HA on proliferation and osteodifferentiation (ALP activity)of hBMSCs were tested by MTT and ALP kit.And cells were observed using SEM after seeding for 10d.Biocompatibility of icariin-CS/HA in vitro and in vivo was evaluated by hemolysis test and pyrogen test separately.3.Icariin release behavior of icariin-CS/HA scaffolds in vitroIcariin-CS/HA scaffolds were soaked in 5 ml phosphate-buffered solution(PBS) and maintained at 37℃and kept shaking gentally at 10 rpm.The samples of 1,2,3, 5,10,15,20,30,60 and 90 d were analyzed by ultra performance liquid chromatography(UPLC).The precision and sensitivity of UPLC were evaluated by precision test,repeatability test,icariin recovery test and stability test.Icariin released from scaffolds was calculated according to standard curve and the percentage of icariin released was accumulated.4.Bone repair capability of icariin-CS/HA scaffolds in vivo60 Male White New Zealand rabbits were allocated into groups of icariin-CS/HA,CS/HA and control(no treatment)randomly(n=12).After anesthetized by pentobarbitol sodium,a 1.5 cm segment defect was made in the right radius of the animals.The bone defect areas were filled with icariin-CS/HA with different icariin dosages(the icariin-CS/HA groups),CS/HA scaffolds only(the CS/HA group)or no scaffolds(the control group).Emission computed tomography (ECT)Four weeks after surgery,four rabbits in every group were selected randomly for ECT examination.3 h after administration of mTc-MDP,the right forelimb was scanned with a single photon emission computed tomography.Thereafter,region of interesting(ROI)of the same size was chosen and quantitative counting was performed,the mean of ROI=value/areaselected.Gross specimen observation and X-ray examination Gross specimen observation and X-ray images of right forelimb were taken 4,8 and 12 weeks after implantation.Histological observation All of radius specimens were fixed in buffered formalin,and decalcified in 10%(v/v)nitric acid solution.Following routine histological processing 5-um-thick tissue slices were obtained and stained with haematoxylin and eosin(H&E)and observed under a light microscope.RESULTS1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanismPhenotypic analysis of hBMSCs in the second generation showed that CD29 (93.98±6.32)%,CD44(85.98±3.87)%,CD71(72.19±4.66)%,CD105(79.28±7.37)%, CD166(97.42±7.43)%,CD14(0.95±0.06)%,CD34(1.45±0.38)%and CD45 (0.73±0.11)%;Cells could be inducted into osteoblasts,chondrocytes and adipocytes by induction of osteo-,chondro-and adipogenesis.The data suggested that with no higher than 10-4M concentration,cytotoxicity of icariin was low.0.05%(v/v)DMSO was safe for cell and could be used as a cosolvent for icariin.10-8M icariin could promote proliferation of hBMSCs,and osteo-induction(ALP activities)related with dose:low dose(＜10-8M)icariin could not increase hBMSCs osteogenic differentiation,high dose(＞10-5M),however,inhibited osteigenic differentiation. The concentration between 10-8M and 10-5M could promote OCN expression;ALP staining and Alizarin red S staining also indicated 10-8～10-5M icariin could accelerate osteodifferentiation of hBMSCs,and 10-6M was the best concentration.On the whole,osteo-induction of icariin was not better than rhBMP-2.10-6M icariin could increase osteogenic genes mRNA(ALP,OCN,OPN,Col-â… ,Cbfa1,BMP-2, BMP-4 and BMP-7)and proteins(Cbfa1,OCN and BMPs)expression,and synergistic effects were observed when combined rhBMP-2 with icariin.2.Construction of biomimetic icariin-CS/HA scaffoldsBiomimetic CS/HA scaffolds could be constructed by in situ hybridization and freeze-dried method.CS/HA composite had abundant homogeneous pores with the diameter(112.63±20.47)μm and porosity(88.65±2.34)%.HA parcels were distributed on the pore walls homogeneously with nanoscale(200～700nm).The XRD and FTIR results showed that the HA crystals were carbonate-substituded and not well-crystallized.The cytocompatibility test showed that the seeded hBMSCs could adhere the scaffolds,and the proliferation ability was not effected by CS/HA composite and its leaching liquor.In addition,histocompability test found that tissue inflammatory reactions of CS/HA composite implanted were decreased significantly at 4 w,the composite was degraded mostly and was substituted by new tissue at 12 w.As we expected,icariin-CS/HA composite had abundant homogeneous pores with the diameter arround 110μm,which provided appropriate 3-demensional micro-structure for cells.Icariin loading did not change physical structure of CS/HA composite significantly,but decreased mechanical properties of CS/HA composite with higher dosage,10-5and 10-6mol icariin-CS/HA had lower fracture strength and elastic modulus;10-5mol icariin-CA/HA has decreased elastic modulus(P＜0.05 vs blank CS/HA scaffolds).icariin-CS/HA had favorable cell compatibility and promoted osteogenic differentiation of hBMSCs;hemolysis test and pyrogen test separately showed that icariin-CS/HA had food biocompatibility.3.Icariin release behavior of icariin-CS/HA scaffolds in vitroRSD of precision test and repeatability test was 0.636%and 3.245% respectively;average recovery rate of recovery test was 96.667%,RSD was 2.139%; the RSD of stability test was 1.286%.There was a good linear relationship between icariin dose(l～2000ng)and chromatographic peak area,and the regression equation was:Y=7877.3X+202422,R2=0.9976.Icariin releasing from scaffolds was calculated based on standard curve and demonstrated as the accumulated percentage of icariin.At day 1 to day 3,approximately 25%icariin was released;then the speed decreased and about 40%～60%icariin was released by 20 d.90 d later,there was still a certain amount of icariin remained in CS/HA scaffolds.Fitting equations of icariin release from CS/HA scaffolds were as follows:10-7mol icariin-CS/HA scaffold:Y=6.267+13.468 ln(X)R2=0.901;10-6mol icariin-CS/HA scaffold: Y=5.668+16.846 ln(X)R2=0.916;10-5mol icariin-CS/HA scaffold:Y=6.322+18.466 ln(X)R2=0.923.And the icariin release behavior could be fitted with the first-order equation.4.Bone repair capability of icariin-CS/HA scaffolds in vivoThe self-repair ability of bone defect control was low,therefore medullary cavity at both ends of defect site was closed during 4～8 w,and the defect was obviously visible 12 weeks postoperatively.Investigated by ECT,a sensitive index of bone formation at early stage,osteogenesis in situ could be monitored by 99mTc-MDP density.At 4 w,ROI values of three icariin-CS/HA groups(icariin dosages were 10-7, 10-6and 10-5mol each)and CS/HA group were higher than the bone defect control group(P＜0.001).Furthermore,ROIs of the 10-6and 10-5mol icariin-CS/HA groups were higher than that of the CS/HA group(P＜0.01).At the same time,obvious bony callus at defect sites with icariin-CS/HA scaffolds could be seen by gross specimens observation and X-ray.Considering both ECT and X-ray results,icariin-CS/HA composites had osteoinduction functions at early stage.The X-ray examination thereafter showed a large amount of bony callus formed and the healing of defect at 8 w and bone marrow cavity reappeared at 12 w,in icariin-CS/HA groups.The bone mass density(BMD)values of three icariin-CS/HA groups and the CS/HA group were higher than the bone defect control group(P＜0.01),and the 10-6and 10-5mol icariin-CS/HA groups were higher than CS/HA group(P＜0.01).Histological observations at different intervals showed that after being implanted in defect site for 4 w,CS/HA composite were degraded partially with microporous structure lost and inflammatory cell infiltration.Scaffolds kept on degrading at 8 w,with newborn fibrous and cartilage tissues creeping along scaffolds.Residual CS/HA scaffolds were segmented and encapsulated by newborn tissues and bone defect site was substituted by cartilage and bone tissues at 12 w.As icariin dosage increased,the degradation speed of icariin-CS/HA composite was increased and the disintegration and fragmentation could be seen earlier at 4 weeks,and there were large amount of newborn cartilage around the scaffolds.At 8 w,scaffolds degraded mostly and some of the cartilage tissues transformed into bone tissues.And at 12 w,icariin-CS/HA scaffolds were degraded completely and cartilage tissues were substituted by bone tissues which arranged in disorder and small medullary cavities reformed in the center. CONCLUSION1.Icariin could promote proliferation and osteodifferentiation of hBMSCs, osteoinduction function of icariin,however,was inferior to that of rhBMP-2.2.Icariin could induce osteogenic genes and proteins expression,and had synergisitic effect with rhBMP-2.3.CS/HA scaffolds prepared by in situ hybridization and freeze-dried method had satisfactory porosity and biocompatibility.4.Icariin loading did not change physical structure of CS/HA composite significantly,but decreased mechanical properties of CS/HA composite with higher dosage.5.The controlled release of icariin from CS/HA scaffolds were satisfactory and the release retained after 90 d in vitro.And the icariin release behavior could be fitted with the first-order equation.6.Icariin-CS/HA scaffolds had favorable osteoconduction and osteoinduction in vivo,and could fill bone defect sites and stimulate newborn bone tissues formation at early stage. |
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