David L. Cochran

Enhanced Bone Apposition to a Chemically Modified SLA Titanium Surface

Increased surface roughness of dental implants has demonstrated greater bone apposition; however, the effect of modifying surface chemistry remains unknown. In the present study, we evaluated bone apposition to a modified sandblasted/acid-etched (modSLA) titanium surface, as compared with a standard SLA surface, during early stages of bone regeneration. Experimental implants were placed in miniature pigs, creating 2 circular bone defects. Test and control implants had the same topography, but differed in surface chemistry. We created the test surface by submerging the implant in an isotonic NaCl solution following acid-etching to avoid contamination with molecules from the atmosphere. Test implants demonstrated a significantly greater mean percentage of bone-implant contact as compared with controls at 2 (49.30 vs. 29.42%; p = 0.017) and 4 wks (81.91 vs. 66.57%; p = 0.011) of healing. At 8 wks, similar results were observed. It is concluded that the modSLA surface promoted enhanced bone apposition during early stages of bone regeneration.

Bone response to unloaded and loaded titanium implants with a sandblasted and acid-etched surface: A histometric study in the canine mandible

Many dental clinical implant studies have focused on the success of endosseous implants with a variety of surface characteristics. Most of the surface alterations have been aimed at achieving greater bone-to-implant contact as determined histometrically at the light microscopic level. A previous investigation in non-oral bone under short-term healing periods (3 and 6 weeks) indicated that a sandblasted and acid-etched titanium (SLA) implant had a greater bone-to-implant contact than did a comparably-shaped implant with a titanium plasma-sprayed (TPS) surface. In this canine mandible study, nonsubmerged implants with a SLA surface were compared to TPS-coated implants under loaded and nonloaded conditions for up to 15 months. Six foxhound dogs had 69 implants placed in an alternating pattern with six implants placed bilaterally in each dog. Gold crowns that mimicked the natural occlusion were fabricated for four dogs. Histometric analysis of bone contact with the implants was made for two dogs after 3 months of healing (unloaded group), 6 months of healing (3 months loaded), and after 15 months of healing (12 months loaded). The SLA implants had a significantly higher (p < 0.001) percentage of bone-to-implant contact than did the TPS implants after 3 months of healing (72.33 +/- 7.16 versus 52.15 +/- 9.19; mean +/- SD). After 3 months of loading (6 months of healing) no significant difference was found between the SLA and TPS surfaced implants (68.21 +/- 10.44 and 78.18 +/- 6.81, respectively). After 12 months of loading (15 months of healing) the SLA implants had a significantly greater percentage (p < 0.001) of bone-to-implant contact than did the TPS implants (71.68 +/- 6.64 and 58.88 +/- 4.62, respectively). No qualitative differences in bone tissue were observed between the two groups of implants nor was there any difference between the implants at the clinical level. These results are consistent with earlier studies on SLA implants and suggest that this surface promotes greater osseous contact at earlier time points compared to TPS-coated implants.

Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells

Titanium (Ti) surface roughness affects proliferation, differentiation, and matrix production of MG-63 osteoblast-like cells. Cytokines and growth factors produced in the milieu surrounding an implant may also be influenced by its surface, thereby modulating the healing process. This study examined the effect of surface roughness on the production of two factors known to have potent effects on bone, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-beta 1). MG-63 cells were cultured on Ti disks of varying roughness. The surfaces were ranked from smoothest to roughest: electropolished (EP), pretreated with hydrofluoric acid-nitric acid (PT), fine sand-blasted, etched with HCl and H2SO4, and washed (EA), coarse sand-blasted, etched with HCl and H2SO4, and washed (CA), and Ti plasma-sprayed (TPS). Cells were cultured in 24-well polystyrene (plastic) dishes as controls and to determine when confluence was achieved. Media were collected and cell number determined 24 h postconfluence. PGE2 and TGF-beta 1 levels in the conditioned media were determined using commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. There was an inverse relationship between cell number and Ti surface roughness. Total PGE2 content in the media of cultures grown on the three roughest surfaces (FA, CA, and TPS) was significantly increased 1.5-4.0 times over that found in media of cultures grown on plastic or smooth surfaces. When PGE2 production was expressed per cell number, CA and TPS cultures exhibited six- to eightfold increases compared to cultures on plastic and smooth surfaces. There was a direct relationship between TGF-beta 1 production and surface roughness, both in terms of total TGF-beta 1 per culture and when normalized for cell number. TGF-beta 1 production on rough surfaces (CA and TPS) was three to five times higher than on plastic. These studies indicate that substrate surface roughness affects cytokine and growth factor production by MG-63 cells, suggesting that surface roughness may modulate the activity of cells interacting with an implant, and thereby affect tissue healing and implant success.

Inflammation and Bone Loss in Periodontal Disease

Inflammation and bone loss are hallmarks of periodontal disease (PD). The question is how the former leads to the latter. Accumulated evidence demonstrates that PD involves bacterially derived factors and antigens that stimulate a local inflammatory reaction and activation of the innate immune system. Proinflammatory molecules and cytokine networks play essential roles in this process. Interleukin-1 and tumor necrosis factor-alpha seem to be primary molecules that, in turn, influence cells in the lesion. Antigen-stimulated lymphocytes (B and T cells) also seem to be important. Eventually, a cascade of events leads to osteoclastogenesis and subsequent bone loss via the receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) axis. This axis and its regulation are not unique to PD but rather are critical for pathologic lesions involving chronic inflammation. Multiple lines of evidence in models of PD clearly indicate that increases in RANKL mRNA expression and protein production increase the RANKL/OPG ratio and stimulate the differentiation of macrophage precursor cells into osteoclasts. They also stimulate the maturation and survival of the osteoclast, leading to bone loss. OPG mRNA expression and protein production do not generally seem to be increased in the periodontitis lesion. Studies of RANKL and OPG transgenic and knockout animals provide further support for the involvement of these molecules in the tissue loss observed in PD. Ironically, periodontal practitioners have focused on the bacterial etiology of PD and believed that plaque removal was aimed at eliminating specific bacteria or bacterial complexes. However, it seems that the reduction of inflammation and attenuation of the hosts immune reaction to the microbial plaque, eventually leading to a decrease in the ratio of RANKL/OPG and a decrease in associated bone loss, are the actual and desired outcomes of periodontal therapy. Future therapeutic options are likely to have regulation of the RANK-RANKL-OPG axis as their goal.

Interface shear strength of titanium implants with a sandblasted and acid-etched surface: A biomechanical study in the maxilla of miniature pigs

The purpose of the present study was to evaluate the interface shear strength of unloaded titanium implants with a sandblasted and acid-etched (SLA) surface in the maxilla of miniature pigs. The two best documented surfaces in implant dentistry, the machined and the titanium plasma-sprayed (TPS) surfaces served as controls. After 4, 8, and 12 weeks of healing, removal torque testing was performed to evaluate the interface shear strength of each implant type. The results revealed statistically significant differences between the machined and the two rough titanium surfaces (p <.00001). The machined surface demonstrated mean removal torque values (RTV) between 0.13 and 0.26 Nm, whereas the RTV of the two rough surfaces ranged between 1.14 and 1.56 Nm. At 4 weeks of healing, the SLA implants yielded a higher mean RTV than the TPS implants (1.39 vs. 1. 14 Nm) without reaching statistical significance. At 8 and 12 weeks of healing, the two rough surfaces showed similar mean RTVs. The implant position also had a significant influence on removal torques for each implant type primarily owing to differences in density in the periimplant bone structure. It can be concluded that the interface shear strength of titanium implants is significantly influenced by their surface characteristics, since the machined titanium surface demonstrated significantly lower RTV in the maxilla of miniature pigs compared with the TPS and SLA surfaces.

Titanium surface roughness alters responsiveness of MG63 osteoblast‐like cells to 1α,25‐(OH)2D3

Surface roughness has been shown to affect dif- ferentiation and local factor production of MG63 osteoblast- like cells. This study examined whether surface roughness alters cellular response to circulating hormones such as 1a,25-(OH)2D3. Unalloyed titanium (Ti) disks were pre- treated with HF/HNO3 (PT) and then were machined and acid-etched (MA). Ti disks also were sandblasted (SB), sand- blasted and acid etched (CA), or plasma sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were: PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on standard tissue culture polystyrene (plastic) or the Ti surfaces and then treated for 24 h with either 10 ˛8 M or 10 ˛7 M 1a,25-(OH)2D3 or vehicle (control). Cellular re- sponse was measured by assaying cell number, cell layer alkaline phosphatase specific-activity, and the production of osteocalcin, latent (L) TGFb, and PGE2. Alkaline phospha- tase activity was affected by surface roughness; as the sur- face became rougher, the cells showed a significant increase in alkaline phosphatase activity. Addition of 1a,25-(OH)2D3 to the cultures caused a dose-dependent stimulation of al- kaline phosphatase activity that was synergistic with the

Persistent Acute Inflammation at the Implant-Abutment Interface:

The inflammatory response adjacent to implants has not been well-investigated and may influence peri-implant tissue levels. The purpose of this study was to assess, histomorphometrically, (1) the timing of abutment connection and (2) the influence of a microgap. Three implant designs were placed in the mandibles of dogs. Two-piece implants were placed at the alveolar crest and abutments connected either at initial surgery (non-submerged) or three months later (submerged). The third implant was one-piece. Adjacent interstitial tissues were analyzed. Both two-piece implants resulted in a peak of inflammatory cells approximately 0.50 mm coronal to the microgap and consisted primarily of neutrophilic polymorphonuclear leukocytes. For one-piece implants, no such peak was observed. Also, significantly greater bone loss was observed for both two-piece implants compared with one-piece implants. In summary, the absence of an implant-abutment interface (microgap) at the bone crest was associated with reduced peri-implant inflammatory cell accumulation and minimal bone loss.

Peri-implant Inflammation Defined by the Implant-Abutment Interface

An implant-abutment interface at the alveolar bone crest is associated with sustained peri-implant inflammation; however, whether magnitude of inflammation is proportionally dependent upon interface position remains unknown. This study compared the distribution and density of inflammatory cells surrounding implants with a supracrestal, crestal, or subcrestal implant-abutment interface. All implants developed a similar pattern of peri-implant inflammation: neutrophilic polymorphonuclear leukocytes (neutrophils) maximally accumulated at or immediately coronal to the interface. However, peri-implant neutrophil accrual increased progressively as the implant-abutment interface depth increased, i.e., subcrestal interfaces promoted a significantly greater maximum density of neutrophils than did supracrestal interfaces (10,512 ± 691 vs. 2398 ± 1077 neutrophils/mm2). Moreover, inflammatory cell accumulation below the original bone crest was significantly correlated with bone loss. Thus, the implant-abutment interface dictates the intensity and location of peri-implant inflammatory cell accumulation, a potential contributing component in the extent of implant-associated alveolar bone loss.

Osteoblast-mediated mineral deposition in culture is dependent on surface microtopography

Osteoblast phenotypic expression in monolayer culture depends on surface microtopography. Here we tested the hypothesis that mineralized bone nodule formation in response to osteotropic agents such as bone morphogenetic protein-2 (BMP-2) and dexamethasone is also influenced by surface microtopography. Fetal rat calvarial (FRC) cells were cultured on Ti implant materials (PT [pretreated], Ra = 0.6 mm; SLA [course grit blasted and acid etched], Ra = 4.0 mm; TPS [Ti plasma sprayed], Ra = 5.2 mm) in the presence of either BMP-2 (20 ng/ml) or 10?8 M dexamethasone (Dex). At 14 days post-confluence, a homogenous layer of cells covered the surfaces, and stacks of cells that appeared to be nodules emerging from the culture surface were present in some areas on all three Ti surfaces. Cell proliferation decreased while alkaline phosphatase specific activity (ALPase) and nodule number generally increased with increasing surface roughness in both control and treated cultures. There was no difference in cell number between the control and Dex-treated cultures for a particular surface, but BMP-2 significantly reduced cell number compared with control or Dex-treated cultures. Treatment with Dex or BMP-2 further increased ALPase on all surfaces except for PT cultures with Dex. Dex had no effect on nodule area in cultures grown on PT or SLA disks, yet increased nodule number by more than 100% in cultures on PT disks. Though the effect of BMP-2 on nodule number was the same as Dex, BMP-2 increased nodule area on all surfaces except TPS, where area was decreased. Ca and P content of the cell layers in control cultures did not vary with surface roughness. However, cultures treated with Dex had increased Ca content on all surfaces, but the greatest increase was seen on SLA and TPS. BMP-2 increased Ca content in cultures on all surfaces, with the greatest increase on the PT surface. BMP-2 treatment increased P content on all surfaces, whereas Dex only increased P on rough surfaces. Of all cultures examined, the Ca/P weight ratio was 2:1 only on rough surfaces with BMP-2, indicating the presence of bone-like apatite. This was further validated by Fourier transform infrared (FTIR) imaging showing a close association between mineral and matrix on TPS and SLA surfaces with BMP-2-treated cells, and individual spectra indicated the presence of an apatitic mineral phase comparable to bone. In contrast, mineral on the smooth surface of BMP-2-treated cultures and on all surfaces where cultures were treated with Dex was not associated with the matrix and the spectra, not typical of bone apatite, implying dystrophic mineralization. This demonstrates that interactions between growth factor or hormone and surface microtopography can modulate bone cell differentiation and mineralization.

Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation

Although it is well accepted that implant success is dependent on various surface properties, little is known about the effect of surface roughness on cell metabolism or differentiation, or whether the effects vary with the maturational state of the cells interacting with the implant. In the current study, we examined the effect of titanium (Ti) surface roughness on chondrocyte proliferation, differentiation, and matrix synthesis using cells derived from known stages of endochondral development. Chondrocytes derived from the resting zone (RCs) and growth zone (GCs) of rat costochondral cartilage were cultured on Ti disks that were prepared as follows: HF-HNO3-treated and washed (PT); PT-treated and electropolished (EP); fine sand-blasted, HCl-H2SO4-etched, and washed (FA); coarse sand-blasted, HCl-H2SO4-etched, and washed (CA); or Ti plasma-sprayed (TPS). Based on surface analysis, the Ti surfaces were ranked from smoothest to roughest: EP, PT, FA, CA, and TPS. Cell proliferation was assessed by cell number and [3H]-thymidine incorporation, and RNA synthesis was assessed by [3H]-uridine incorporation. Differentiation was determined by alkaline phosphatase specific activity (AL-Pase). Matrix production was measured by [3H]-proline incorporation into collagenase-digestible (CDP) and noncollagenase-digestible (NCP) protein and by [35S]-sulfate incorporation into proteoglycan. GCs required two trypsinizations for complete removal from the culture disks; the number of cells released by the first trypsinization was generally decreased with increasing surface roughness while that released by the second trypsinization was increased. In RC cultures, cell number was similarly decreased on the rougher surfaces; only minimal numbers of RCs were released by a second trypsinization. [3H]-thymidine incorporation by RCs decreased with increasing surface roughness while that by GCs was increased. [3H]-Uridine incorporation by both GCs and RCs was greater on rough surfaces. Conversely, ALPase in the cell layer and isolated cells of both cell types was significantly decreased. GC CDP and NCP production was significantly decreased on rough surfaces while CDP production by RC cells was significantly decreased on smooth surfaces. [35S]-sulfate incorporation by RCs and GCs was decreased on all surfaces compared to tissue culture plastic. The results of this study indicate that surface roughness affects chondrocyte proliferation, differentiation, and matrix synthesis, and that this regulation is cell maturation dependent.