| BackgroundTotal joint replacement(TJR), one of the greatest advances in orthopaedicsurgery in the twentieth century, has considerably improved the quality of life of patients affected by serious articulate deterioration, ie, osteoarthritis, bone tumor. However, a number of arthoplasties ultimately fail due to the loosening of their components, periprosthetic bone loss standing as the major factor limiting the longevity of prosthetic implants, which is of significant concern to orthopaedic surgeons and an increasing burdon on international health budgets. Most joint prostheses consist of a metal femorl head whose main ingredient is titanium, so titanium wear debris usually derive from the wear of the articulating component. Numerous in vitro and in vivo studies have investigated that phagocytosis of those particulate titanium wear debris is responsible for triggering a serve inflammatory reaction mediated by the release of cytokines and other soluble mediators. These factors favor osteoclasts activity and, consequently, activate focal bone resorption. However, little is kown about the disruptive influence of particulate wear debris on bone formation. Moreover, the decrease in bone mass caused by wear debris-induced bone loss could have been compensated through osteoblasts secreting enough new bone matrix. The normal osteoblastic population depends on the regular differentiation of their progenitor cells, the marrow mesenchymal stem cells (MSC). It is not possible to predict whether wear particles will affect the MSC's adhesion behavior, subsequently their differentiation, and ultimately contribution to the poor periprosthetic bone quality.Attentively, in light of the remodeling concept of adult bone as proposed by Frost et al. and then refined by Rodan and Martin, it is appropriate to consider the effect on periprosthetic osteogenesis in aseptic loosening by wear particles. It is presumed that joint prostheses are held firm by tight apposition of skeletal tissues which initially remodels in a balanced manner. Studies have been recently suggested that it's the net bone loss aroused from the imbalance of the coupling mechanism of bone formation and bone resorption affected by particulate wear debris inducing aseptic loosening around the bone-prosthesis microenviroment. Furthermore, different-sized wear particles relevant to the wear of implant have been documented in the bone-implant interface, but little is known about the extent to which the sizes of the wear particles loading can impact the cells behavior the most. This study has, therefore, concentrated on the potential mechanism for Influence of Different-Sized Titanium Particles Loading (TP) on Predominant Cell's Function of Peri-prosthetic Loosening in vitro, in order to aid the development and application of new bone loss drug therapy and the optimization of design and selection of future orthopaedic biomaterials withimproved long-term compatiblity and stability for arthroplastic patients.MethodsThe present study were divided into pricinpal four parts as the follows:Part I: The net regulation of titanium particles loaded osteoblasts onosteoclasts' function?Rabbit osteoblasts were loaded with different commercially pure titaniumparticles to explored different-sized titanium particles loading on theosteoblastic differentiation by assaying the secretion of alkaline phosphate(ALP),osteocalcin(OCN), N-terminal type I procollagen(PINP), on the osteoblasticmineralization with the use of calcified node number, calcified node area andAlizarin Red S concentration, and on the expression of cytokines (interleukin-6,interleukin-10, Osteoclast differentiation factor); ?Then, the conditioned media(OB-CM) harvested was added to the rabbit osteoclasts culture system for 3 days.The net impacts of conditioned media on osteoclastic function were examinedthrough employing A. tartrate-resistant acid phosphatase (TRAP) positive cellscount with azo-coupling cytochemical test ;B. resorption pit area in bovinecortical bone resorption assay; C.concentration of C-telopeptide of type-Icollagen(CTx) with enzyme-linked immunosorbent assay.Part II: Characterization and function evaluation of rat mesenchymal stemcells(rMSCs)?Rat mesenchymal stem cells (rMSCs) isolated from 3-month-old maleSprague-Dawley rats by combination means of the Percoll intensity gradientcentrifugation method and passaged screening were cultured in DMEM medium(low glucose ) supplemented with 10% fetal bovine serum ,100 U/mlpenicillin ,and 100 ug/ml streptomycin in a humidified incubator with 5% CO2 at37°C .?Identified rMSCs through morphologic (TEM, SEM) and phenotypemolecule (Stro-1 antigen, CD44/CD55, CD29/CD14) assessments ?evaluatedrMSCs' proliferation ability through DNA- content- FCM test and differentiationfunction by osteogenesic, adipocytic, chondrocytic and neron-like- cells induction.Part III Alteration of adhesion and cellular viability of rMSCs loaded bytitanium particles?rMSCs were seeded in the 6 well culture plates and exposed to three differentcircle diameters. Unexposed rMSCs were used as control. In the given periods ofTi loading, fluid shear stress(FSS) was applied to each group cells . By means ofconfocal laser scanning microscopy and flow cytometry, The expression ofF-actin , DNA , HCAM, integrin pi of the rMSCs at the indicated time weredetermined to determine rMSCs' adhesion ability. ?Using the SEM, FCM, immunofluorescence technique and quantitative RT-PCR, analyzed change of ultrastructure , cell cycle distribution / proliferation index/ apoptosis percentage , terminal dexoynucleotidyl transferase-catalyzed dUTP-fluorecein nick-end labeling (TUNEL), iNOS and Caspase-3 mRNA expressions to determine impact of titanium particles loading on cellular viability of rMSCs. Part IV Influence of different sized titanium particles on osteogenic and adipocytic differentiation of rMSCs?Upon in vitro culture in the presence of osteogenic induction, osteogenic induced rMSCs(OSI-rMSCs) were loaded with titanium particles at varied concentrations, respectively in order to explored different-sized titanium particles loading on the osteoblastic differentiation by assaying the secretion of alkaline phosphate(ALP), osteocalcin(OCN), N-terminal type I procollagen(PINP), and on the osteoblastic mineralization with the use of calcified node number, calcified node area and Alizarin Red S concentration; ?And in the presence of adipocytic supplement, rMSCs(ADI-rMSCs) were challenged with titanium particles in order to explored different-sized titanium particles loading on the adipocytic differentiation by explore items tests, i.e., lipid droplets formation, lipoprotein lipase(LPL) mRNA and Triglycerides(TG) secretion in rMSCs. Results1. Titanium particles loading suppressed differentiation and mineralization ability of OBs Upon in vitro culture in the absence of titanium particles, we observed that cultures of osteoblasts isolated from newborn Japanese rabbits' cranium were excellently capable of differentiation and mineralization. 6.9 urn titanium particles did not obviously alter osteoblastic differentiation and mineralization. In comparison, 2.7 um and 0.9 um titanium particles, especially 0.9 um (submicron), suppressed ALP expression ,reduced PINP production , decreased OCN secretion and inhibited matrix mineralization. Transmission electron microscopy (TEM) of titanium particles-loaded osteoblastic cultures revealed that osteoblasts phagocytosed titanium particles and exhibited ultrastructional changes consistent with cellular dysfuction.2. Titanium particles loading stimulated OBs express both bone resorption factors and anti-bone resorption factors The present study showed that titanium particles loading activated osteoblasts to produce not only IL-6, ODFbut also IL-10 in a time- and particles dose-dependent manner. Time course study demonstrated that the submicron titanium particle (0.9 um) loading had earlier and sharper up-regulation of IL-6 production and ODF expression, and persistently stimulated OBs release IL-10. The concentration scale study demonstrated that 2.7 um particle loading intensively occurred IL-6 production of OBs concentrated at 0.05%,while 6.9 um stimulated OBs release IL-6 at higher concentration(0.25%) and powerfully upregulate ODF mRNA , but these elaborations relatively late for 24 hours. In contrast, no significant changes as those were seen in the control.3. Different net regulations of OCs function by OBs loaded various sized titanium particles The conditioned media harvested from osteoblasts culture loaded with titanium particles(OB-CM) increased the number of TRAP-positive cells(recruitment and differentiation of precursors into mature multinucleated OCs), resorption pit area(activation of mature OCs) as well as the concentration of CTx (survival of OCs). While 2.7 um and 6.9 um especially 6.9 um OB-CM , significantly augmented OCs' pseudopodium formation (maturation and survival of OCs)and cytoskeletion assembly(activation of mature OCs)function , whose impacts than that of 0.9 um OB-CM, in spite of the time late to 24h. Attentively, we observed the effect of OB-CM on [Ca2+]i of OCs and the result showed that three OB-CM stimulated OCs appear upregulated peak: 0.9 um OB-CM had earliest up-regulation peak value , the highest level was at 55-75 seconds, and that of 2.7 um OB-CM obviously emerged at 115-125 seconds. While 6.9 um OB-CM had two peak value of [Ca2+]i, at 120-135 seconds and 195-215 seconds ,which lower than that of 0.9 um.4. Achievement of target stem cells for titanium particles loading The morphologic feature showed the rMSCs had the general appearance of stem cells , i.e., larger nuclei and minor plasm. By means of FCM and IHC, rMSCs had the phenotype of positive-CD44/CD29/Stro-l and negative-CD45/CD14, and passaged rMSCs showed active proliferation ability, which identify that our isolated cells are bone marrow mesenchymal stem cells distinct form the hemopoietic stem cells. Moreover, upon in the presence of induction media, rMSCs differentiated into osteoblasts, adipocytes, chondrocytic and neron-like- cells, respectively. Combination analysis of passaged rMSCs , we concluded that P4 rMSCs was available to the target stem cells for the following TP experiment.5. Titanium particles loading reduce the adhesive ability The results showed that there were up-regulation expression of F-actin , higher adhesive percentage (about 90%) and adhesion molecules scale-up to approximate two folds in the rMSCs without Ti particles loading but in the presence of FSS ,which suggested rMSCs have favorable mechanics responsibility .Flow cytometry analysis revealed that Ti particle loading leaded to a decrease in the adhesion of the cells, suppresses the expression of the MSC adhesion molecules, integrin Pi. The down-regulation response varied with the size of the Ti particles, their concentration and the duration of loading. The smallest Ti particles (0.9 um) exhibited the earliest and largest suppression of MSC adhesion molecules, with the lowest expression occurring at 0.1 wt% after 24 h. In the case of the 6.9 urn particles, significant inhibition of adhesion did not occur until 24 h, and even then was lower than that of the 0.9 um particles. Confocal imaging demonstrated attenuation of adhesion behavior of MSC was also related with an abnormality of cytoskeleton.6. Titanium particles loading inhibit rMSCs' proliferation and induce rMSCs' apoptosis The results showed that unloaded Ti particles, rMSCs had excellent cellular viability, i.e., higher percentage of S phase, proliferation index reached to 87.4%, no TUNEL-positive cells and basal level of expression of iNOS and Caspase-3 mRNA. Three TP inhibited rMSCs' proliferation and induce rMSCs' apoptosis, but different sizes appeared various effects, depended in a manner of concentration and duration : with the higher at concentration and longer at duration , the more suppressive in proliferation and stronger induction of apoptosis. Among the three particles, 0.9 um TP was the most restraining effect on the proliferation and the most powerful induction on the apoptosis. Those apoptosis effect was association with the abnormal accentuation of iNOS activity.7. Titanium particles loading decrease the osteogenic differentiation of rMSCs Our results showed that the cultured rMSCs could be induced to osteoblasts, which was testified by, under osteogenic induction condition, the increases of ALP activity and OCN and COL-I proteins production, as well as the formation of calcified bone nodules. Titanium particles loading could influence ALP activity and OCN and COL-I proteins production, but this regulation was various in different circle diameter, varied concentration and various loading durations of Ti particles , depended in a manner of concentration and duration. With the augmenting of concentration andduration (except for 6.9 um), 0.9 urn and 2.7 um TP decreased ALP activity of rMSCs, but the lowest value higher than that of the control. TP obviously suppressed the production of OCN and COL-I, but the down-regulation was related with size, concentration and duration: 0.9 um TP was the most extent of inhibition among three sizes , the lowest values sharply lower than that of the control at 36 h. while the reductive impact of 6.9 um TP was weaker than that of 2.7 um and 0.9 um, the lowest value of OCN and COL-I no more than that of the control. Those effects suggested that 0.9 um TP decreased the osteogenic differentiation of rMSCs the most extent. Further investigation of mineralization of rMSCs confirmed this inference.8. Titanium particles loading disturb the adipogenic differentiation of rMSCs The results demonstrated that the cultured rMSCs could be differentiated to adipocytes, which was identified by, upon adipogenic induction media, the increases of lipid droplets formation, expression of lipoprotein lipase (LPL)mRNA and Triglycerides secretion. Titanium particles loading could influence adipogenesis of rMSCs, but this regulation was various in different circle diameter, varied concentration and various loading durations of TP. 2.7 um and 0.9 um TP augmented lipid droplets formation, lipoprotein lipase(LPL) mRNA expression and Triglycerides secretion. Especially, after 0.9 um TP, rMSCs changed to the morphology-like fat cells such as round cell body and marginal nuclei, in addition, values of LPLmRNA and TG higher than that of the corresponding ADI-control. While with the increase of TP concentration and duration, 6.9 um groups appeared gradually down-regulation trend of adipogenesis: values of LPLmRNA and TG lower than that of the corresponding ADI-control. Those difference of the three particles suggested the promotion of 0.9 um to adipogenetic ability of rMSCs was combination with additive effect the induction media, while 6.9 um TP may reduced, even inhibited the osteogenic induction, which inferred that titanium particles cause metabolic disturbance in the bone marrow.Conclusions1. Three size titanium particles loading obviously affected differentiation and mineralization ability of OBs: 6.9 um titanium particles did not obviously alter osteoblastic differentiation and mineralization. In comparison, 2.7 um and 0.9 um titanium particles, especially 0.9 um (submicron ), suppressed differentiation and mineralization ability of OBs.2. Titanium particles loading stimulated OBs express intracellular mediators, but different sizes had various effects: 0.9 urn particles could promote OBs express both of bone resorption factors and anti-bone resorption factors, whose reaction was sharp and temporal; while 2.7 um and 6.9 urn particles, especially 6.9 urn ,mainly enhanced OBs product bone resorption factors, whose response was slow and intensive.3. Different net regulations of OCs function by OBs loaded various sized titanium particles: three of OB-CM enhanced OCs' differentiation , maturation, While 2.7 um and 6.9 um, especially 6.9 um OB-CM , significantly augmented OCs' maturation and survival of OCs , the extent more than that the 0.9 um OB-CM. These responds were related to the change of [Ca2+]j of OCs: Ca2+ overloading cripple the augmented effect of 0.9 urn OB-CM on OCs' function, however appropriate up-regulation of [Ca2+]j especially enhance the bone resorption efficacy of OCs from 6.9 um OB-CM.4. Three size particles loading suppress the adhesion ability of rMSCs , the effect was size-dependent, being greatest for the smallest particles. Because of decrease of adhesive molecules and abnormal expression of cytoskeleton, wear particles jeopardize the mechanic responsibility of rMSCs, inhibit the extra-to-intra signaling transduction, subsequently suppress the cellular viability.5. Titanium particles loading inhibited rMSCs' proliferation and induce rMSCs' apoptosis, the apoptotic impact is dependent in a manner of size, being most promotion for the smallest particles, and was association with the cause that the smallest was prone to be phagocytosized and activate iNOS, ultimately result the local accumulation of NO.6. Three size titanium particles loading obviously influence the differentiation capability of rMSCs: all particles loading can inhibit the osteogenic ability of rMSCs, 0.9 um decreased the osteogenic differentiation of rMSCs the most extent; different sized titanium particles loading could product diverse influence of adipogenesis of rMSCs: the smaller promote while the larger inhibit the adipogenic differentiation of rMSCs.
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