Introduction: A critical stage during osseointegration of a titanium (Ti) implant is the bone remodeling phase, which involves cross talk among osteoclasts (OCs), mesenchymal stem cells (MSCs), and osteoblasts (OBs). During this phase, OCs remove primary bone, enabling formation of mature bone. MSCs and OBs on microstructured Ti surfaces produce osteogenic factors which may serve to regulate osteoclastic resorption. This investigated surface dependent mechanisms that control osteoclastic differentiation and activity. Materials and Methods: 15mm grade 2 smooth (PT), rough (SLA), and hydrophilic-rough Ti (modSLA) disks were provided by Institut Straumann AG (Basel, Switzerland), and were characterized by SEM, CLSM, XPS, and CAM. MSCs and OBs were cultured separately on surfaces and grown to confluence on tissue culture polystyrene (TCPS). At confluence, media were collected and either 1) supplemented with 66ng/mi RANKL and 33ng/ml M-CSF, 2) unmodified, or 3) analyzed for osteoblastic markers. OC precursors (OCPs) were cultured for 7D in OC growth medium (OCGM) with RANKL and M-CSF on an Osteolyse Kit. Differentiated OCs were treated with conditioned media. OCs with M-CSF and RANKL and OCPs with M-CSF were positive and negative controls, respectively. OC activity was measured 2D and 7D after treatment by fluorescence of released collagen. Afterwards, osteoclast mRNA levels were quantified. (n=6 cultures/variable; ANOVA-Tukey p＜0.05). Results and Discussion: Differences in surface microroughness, contact angles, and carbon contamination (PT[SA=1.10Mm;6CA=93°;%C=31], SLA[SA=3.38μm;8CA=126°;%C=35], modSLA[SA=3.52Mm;6CA=0°;%C=17]) were observed. Compared to TCPS, MSCs and OBs produced increased osteoprotegerin, TGFβ1, and osteocalcin, in a surface dependent manner (modSLA＞SLA＞PT＞TCPS). At 2D, OC activity was highest in MSC/OB TCPS media and lowest in MSC/OB modSLA media. At 7D, inhibition was mitigated but recovery was slower in MSC/OB modSLA media. These effects were evident in RANKL conditioned media, although its absence led to reduced OC activity in all cultures with minimal recovery. Additionally, the effects of OB vs MSC conditioned media were more robust. At 2D, MSC/OB media reduced osteoclast activity (CTSK, ATP6V0D2, MMP9, OSCAR), integrins (ITGB3, ITAV), and fusion (OCSTAMP) genes to similar levels. At 7D, all but OCSTAMP mRNA levels recovered past the positive control (modSLA＞SLA＞PT＞TCPS), suggesting the inhibitory effects dissipate allowing OC functional recovery but no new OC fusion. Furthermore, during primary bone formation on Ti substrates, MSCs and OBs regulate at least two remodeling aspects: reduced fusion of new OCs and reduced activity of existing OCs. Conclusion: Ti surfaces regulate MSC and OB production of osteogenic factors. These secreted factors are capable of suppressing OC activity long enough to induce primary bone formation. This inhibition slowly subsides allowing OCs to initiate remodeling.
展开▼
