Evaluation of local cancellous bone amelioration by poly-L-DL-lactide copolymers to improve primary stability of dental implants: a biomechanical study in sheep.

OBJECTIVES: The aim of this study was to evaluate the clinical performance of local cancellous bone amelioration by a 70:30 poly-(L-lactide-co-D,L-Lacide) copolymer with two different implant designs on primary stability and after 4 and 12 weeks of healing time. MATERIAL AND METHODS: In six sheep, n = 36 implants (TH) with a conditioned, sandblasted, thermal acid-etched micro-rough surface and n = 36 implants (NB) with a highly crystalline and phosphate-enriched anodized titanium oxide surface were placed in the pelvic bone. Using an ultrasound-based process named Constant Amelioration Process (CAP), half of peri-implant trabecular bone structures were locally tested with 70:30 poly-(L-lactide-co-D,L-Lacide) copolymer in both implant groups, TH and NB. The CAP technology employs ultrasonic energy to liquefy 70:30 poly-(L-lactide-co-D,L-Lacide) which enters the inter-trabecular space, leading to local reinforcement of the cancellous bone structure after solidification of the copolymer. The CAP test group was compared with reference implants placed with the conventional site preparation according to the manufacturers' description. Primary stability was assessed by the measurement of torque-in values and implant stability quotient (ISQ; n = 18 per group). Secondary stability was analyzed by biomechanical removal torque testing after 4 and 12 weeks (n = 9 per group). RESULTS: Insertion torque value (23.3 N cm ± 13.6) of reference TH implants demonstrated a statistically significant (P = 0.00) difference in comparison with test TH implants (41.9 N cm ± 19.5). Reference NB implants revealed a statistically significant (P = 0.03) lower insertion torque value (23.7 N cm ± 13.5) than test NB implants (39.7 N cm ± 18.6). ISQ values increased for all implants from initial implant placement until sacrifice at 12 weeks. Reference TH implants tended to result in an increase in torque values from 4 weeks (181.9 N cm ± 22.8) to 12 weeks (225.7 N cm ± 47.4). This trend could be also proven for implants of test sites (4 week: 176.8 N cm ± 24.1; 12 week: 201.5 N cm ± 53.4). For reference, NB implants a non-significant increase in removal torque values from 4 weeks (146. 7 N cm ± 18.0) to 12 weeks (170.2 N cm ± 40.4) was observed. Removal torque values of test NB implants did not increase from 4 weeks (153.3 N cm ± 21.5) to 12 weeks (146.1 N cm ± 37.5). CONCLUSION: Biomechanical data proved significantly enhanced primary stability of dental implants after local amelioration without long-term sequelae and irrespective of implant design. After 4- and 12-week healing time, removal torque of locally test implants was as high as for control implants, and osseointegration was therefore not influenced by the CAP process. No correlation between ISQ values and torque values was found.

Early bone apposition to hydrophilic and hydrophobic titanium implant surfaces: a histologic and histomorphometric study in minipigs.

OBJECTIVE: The first objective of this pilot study was to evaluate the impact of the hydrophilicity on the early phases of osseointegration. The second objective was to compare two hydrophilic implant surfaces with different geometries, surface roughness, and technologies achieving hydrophilicity. MATERIAL AND METHODS: Twelve weeks after extraction, all four quadrants of nine minipigs received three dental implants, alternating between hydrophilic microrough surfaces (INICELL and SLActive) and a conventional hydrophobic microrough surface. After 5, 10, and 15 days of submerged healing, ground sections were prepared and subjected to histologic and histomorphometric analysis. RESULTS: The histologic analysis revealed a similar healing pattern among the hydrophilic and hydrophobic implant surfaces, with extensive bone formation occurring between day 5 and day 10. With BIC values of greater than 50% after 10 days, all examined surfaces indicated favorable osseointegration at this very early point in healing. At day 15, the mean new bone-to-implant contact (newBIC) of one hydrophilic surface (INICELL; 55.8 ± 14.4%) was slightly greater than that of the hydrophobic microrough surface (40.6 ± 20.2%). At day 10 and day 15, an overall of 21% of the implants had to be excluded from analysis due to inflammations primarily caused by surgical complications. CONCLUSION: Substantial bone apposition occurs between day 5 and day 10. The data suggest that the hydrophilic surface can provoke a slight tendency toward increased bone apposition in minipigs after 15 days. A direct comparison of two hydrophilic surfaces with varying geometries is of limited relevance.

Response of human bone marrow stromal cells, MG-63, and SaOS-2 to titanium-based dental implant surfaces with different topography and surface energy.

OBJECTIVES: Osseointegration is dependent on different parameters of the implant surface like surface roughness and physicochemical properties. In vitro studies using a wide variety of surface parameters and cell lines make it difficult to address the influence of a single parameter. With this study the influence of surface topography and energy on different osteoblast derived cell lines, namely MG-63 and SaOS-2 and of human mesenchymal stromal cells (hMSC) were investigated. MATERIAL AND METHODS: Cells were cultured on polished (POL) and sandblasted/hot acid etched (SBA) titanium surfaces which were partly alkaline treated (SBA NaOH). Cell morphology, metabolic activity, tissue non-specific alkaline phosphatase (TNAP) activity and prostaglandin E(2) (PGE(2) ) formation were determined. RESULTS: Impaired spreading was found on both SBA surfaces. Proliferation after 4 and 7 days increased on POL compared to both SBA surfaces. TNAP activity of hMSC and MG-63 was increased on POL compared to both SBA surfaces whereas SaOS-2 did not discriminate between the three surfaces. PGE(2) formation of hMSC and MG-63 was on both SBA surfaces after 2 days significantly higher than on POL. CONCLUSIONS: The results of this study show that surface roughness has a distinct influence on proliferation and differentiation of osteoblasts. However, variations in physicochemical properties seem to have little influence under the used experimental conditions. It is suggested that more sever and long-lasting modifications of surface chemistry would have an influence on osteoblastic cells.

Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation.

Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA) titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm), patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.

Mechanical anchorage and peri-implant bone formation of surface-modified zirconia in minipigs.

AIM: To test the hypothesis that peri-implant bone formation and mechanical stability of surface-modified zirconia and titanium implants are equivalent. MATERIALS AND METHODS: Twelve minipigs received three types of implants on either side of the mandible 8 weeks after removal of all pre-molar teeth: (i) a zirconia implant with a sandblasted surface; (ii) a zirconia implants with a sandblasted and etched surface; and (iii) a titanium implant with a sandblasted and acid-etched surface that served as a control. Removal torque and peri-implant bone regeneration were evaluated in six animals each after 4 and 13 weeks. RESULTS: The titanium surface was significantly rougher than both tested zirconia surfaces. Mean bone to implant contact (BIC) did not differ significantly between the three implant types after 4 weeks but was significantly higher for titanium compared with both zirconia implants after 13 weeks (p<0.05). Bone volume density (BVD) did not differ significantly at any interval. Removal torque was significantly higher for titanium compared with both zirconia surfaces after 4 and 13 weeks (p<0.001). The sandblasted and etched zirconia surface showed a significantly higher removal torque after 4 weeks compared with sandblasted zirconia (p<0.05); this difference levelled out after 13 weeks. CONCLUSIONS: It is concluded that all implants achieved osseointegration with similar degrees of BIC and BVD; however, titanium implants showed a higher resistance to removal torque, probably due to higher surface roughness.

Response of osteoblast-like SAOS-2 cells to zirconia ceramics with different surface topographies.

OBJECTIVES: Zirconia is a suitable biomaterial for use in medicine (stomatology, orthopaedics) due to its good biocompatibility and outstanding mechanical properties. This study compares the effect of (i) zirconia to the widely used titanium and (ii) zirconia with two different surface topographies (sandblasted and sandblasted/etched) on the adhesion, proliferation and differentiation of SAOS-2 osteoblasts. METHODS: SAOS-2 cells were cultured on either sandblasted or sandblasted/etched zirconia and compared with sandblasted/etched titanium. 2 and 24 h after plating, cell morphology was investigated by scanning electron microscope (SEM) and fluorescence imaging. At 24 and 48 h, cell number-relevant parameters were determined. Alkaline phosphatase (ALP) activity and mineral accumulation were measured at days 8, 11, 15 and day 22 of culture, respectively. RESULTS: SEM and fluorescence images revealed a faster spreading as well as higher number of adherent cells after 24 h incubation on zirconia compared with titanium. Also, the cellular metabolic activity after 24 h and the proliferation rate after 48 h is higher with zirconia compared with titanium. Zirconia had a more pronounced effect compared with titanium on the differentiation of SAOS-2 cells: ALP activity, an early differentiation marker increased earlier and mineralization, a late differentiation marker was increased. Only minor differences were found between zirconia with two different surface topographies; etched zirconia promoted slightly greater the differentiation of SAOS-2 cells. CONCLUSIONS: These data indicate that zirconia mediates a pronounced stronger effect on the adhesion, proliferation and differentiation compared with titanium; and that topographical differences of zirconia have minor effects on osteoblast biology.

Preparation of superhydrophilic microrough titanium implant surfaces by alkali treatment.

A new strategy to render intrinsically hydrophobic microrough titanium implant surfaces superhydrophilic is reported, which is based on a rapid treatment with diluted aqueous sodium hydroxide solutions. The physicochemical characterization and protein interaction of the resulting superhydrophilic implant surfaces are presented. The superhydrophilicity of alkali treated microrough titanium substrates was mainly attributed to deprotonation and ion exchange processes in combination with a strong enhancement of wettability due to the roughness of the used substrates. Albeit these minor and mostly reversible chemical changes qualitative and quantitative differences between the protein adsorption on untreated and alkali treated microrough titanium substrates were detected. These differences in protein adsorption might account for the enhanced osseointegrative potential of superhydrophilic alkali treated microrough implant surfaces. The presented alkali treatment protocol represents a new clinically applicable route to superhydrophilic microrough titanium substrates by rendering the implant surface superhydrophilic "in situ of implantation".

Surface-conditioned dental implants: an animal study on bone formation.

AIM: The aim of this study was to determine whether bone formation around surface-conditioned implants is enhanced compared with non-surface-conditioned sandblasted acid-etched titanium implants. MATERIALS AND METHODS: One hundred and forty-four implants were placed in the mandible of 18 minipigs. Before placement, implants were either surface conditioned in a solution containing hydroxide ions (conSF) or assigned to controls. Animals were euthanized after 2, 4 and 8 weeks of submerged healing, the 8-week group receiving polyfluorochrome labelling at week 2, 4, 6 and 8. One jaw quadrant per animal was selected for histological and histomorphometrical evaluation of mineralized bone-implant contact (mBIC), osteoid-implant contact (OIC) and bone volume (BV) analysis. RESULTS: Polyfluorochrome labelling showed no general differences in bone dynamics. mBIC showed the most pronounced differences after 2 weeks, reaching 65.5% for conSF compared with 48.1% for controls, p=0.270. Differences levelled out after 4 weeks (67.4% control, 65.7% conSF) and 8 weeks (64.0% control, 70.2% conSF). OIC levels were initially comparable, showing a slower decline for conSF after 4 weeks. BV was higher for conSF at all times. No significant differences could be found. CONCLUSION: A tendency towards increased mBIC was shown for surface-conditioned implants after short-term healing.

Biomechanical comparison of different surface modifications for dental implants.

PURPOSE: A satisfactory clinical outcome in dental implant treatment relies on primary stability for immediate load bearing. While the geometric design of an implant contributes to mechanical stability, the nature of the implant surface itself is also critically important. Biomechanical and microcomputerized tomographic evaluation of implant osseointegration was performed to compare alternative structural, chemical and biochemical, and/or pharmaceutical surface treatments applied to an identical established implant design. MATERIALS AND METHODS: Dental implants with the same geometry but with 6 different surface treatments were tested in vivo in a sheep model (pelvis). Peri-implant bone density and removal torque were compared at 2, 4, and 8 weeks after implantation. Implant surfaces tested were: sandblasted and acid-etched titanium (Ti), sandblasted and etched zirconia, Ti coated with calcium phosphate (CaP), Ti modified via anodic plasma-chemical treatment (APC), bisphosphonate-coated Ti (Ti + Bisphos), and Ti coated with collagen containing chondroitin sulfate (CS). RESULTS: All dental implants were well integrated at the time of sacrifice. There were no significant differences observed in peri-implant bone density between implant groups. After 8 weeks of healing, removal torque values for Ti, Ti + CaP, Ti + Bisphos, and Ti + collagen + CS were significantly higher than those for zirconia and Ti + APC. CONCLUSIONS: Whereas the sandblasted/acid-etched Ti implant can still be considered the reference standard surface for dental implants, functional surface modifications such as bisphosphonate or collagen coating seem to enhance early peri-implant bone formation and should be studied further.

Clot-entrapped blood cells in synergy with human mesenchymal stem cells create a pro-angiogenic healing response.

Blood clots stop bleeding and provide cell-instructive microenvironments. Still, in vitro models used to study implant performance typically neglect any possible interactions of recruited cells with surface-adhering blood clots. Here we study the interaction and synergies of bone marrow derived human mesenchymal stem cells (hMSCs) with surface-induced blood clots in an in vitro model by fluorescence microscopy, scanning and correlative light and electron microscopy, ELISA assays and zymography. The clinically used alkali-treated rough titanium (Ti) surfaces investigated here are known to enhance blood clotting compared to native Ti and to improve the healing response, but the underlying mechanisms remain elusive. Here we show that the presence of blood clots synergistically increased hMSC proliferation, extracellular matrix (ECM) remodelling and the release of matrix fragments and angiogenic VEGF, but did not increase the osteogenic differentiation of hMSCs. While many biomaterials are nowadays engineered to release pro-angiogenic factors, we show here that clot-entrapped blood cells on conventional materials in synergy with hMSCs are potent producers of pro-angiogenic factors. Our data might thus not only explain why alkali-treatment is beneficial for Ti implant integration, but they suggest that the physiological importance of blood clots to create pro-angiogenic environments on implants has been greatly underestimated. The importance of blood clots might have been missed because the pro-angiogenic functions get activated only upon stimulation by synergistic interactions with the invading cells.

Primary stability and osseointegration of dental implants in polylactide modified bone - A pilot study in Goettingen minipigs.

The present study aimed to evaluate primary stability (PS) and osseointegration of dental implants in polylactide [70/30 poly(l-lactide-co-d, l-lactide); (PLDLA)] modified bone in 30 Goettingen minipigs. Each animal received three implants per jaw quadrant. In a split-mouth design, one side of the maxilla and mandible was randomly allocated to the experimental treatment (PLDLA applied into the drill hole before implantation), while the contralateral sides served as intraindividual controls (no PLDLA applied). The required insertion torque and the implant stability quotient (ISQ) were measured during implantation. ISQ, volume density (VD) of new bone formation (NBF), and the bone-implant contact (BIC) were evaluated at the end of the observation period (1, 3, 6, 12, and 24 months, respectively) in six animals each. Across all study groups, the PLDLA treatment resulted in a) a comparable insertion torque, b) an equivalent ISQ, c) a reduced BIC, and d) a reduced VD of NBF, as opposed to the untreated controls. In conclusion, the PLDLA treatment did not affect the PS, but rather led to an impaired osseointegration, which was particularly strong in the compact mandibular bone, and decreased in the spongious maxillary bone. PLDLA induced anchoring in spongious bone should be evaluated in further investigations.

Synergistic interactions of blood-borne immune cells, fibroblasts and extracellular matrix drive repair in an in vitro peri-implant wound healing model.

Low correlations of cell culture data with clinical outcomes pose major medical challenges with costly consequences. While the majority of biomaterials are tested using in vitro cell monocultures, the importance of synergistic interactions between different cell types on paracrine signalling has recently been highlighted. In this proof-of-concept study, we asked whether the first contact of surfaces with whole human blood could steer the tissue healing response. This hypothesis was tested using alkali-treatment of rough titanium (Ti) surfaces since they have clinically been shown to improve early implant integration and stability, yet blood-free in vitro cell cultures poorly correlated with in vivo tissue healing. We show that alkali-treatment, compared to native Ti surfaces, increased blood clot thickness, including platelet adhesion. Strikingly, blood clots with entrapped blood cells in synergistic interactions with fibroblasts, but not fibroblasts alone, upregulated the secretion of major factors associated with fast healing. This includes matrix metalloproteinases (MMPs) to break down extracellular matrix and the growth factor VEGF, known for its angiogenic potential. Consequently, in vitro test platforms, which consider whole blood-implant interactions, might be superior in predicting wound healing in response to biomaterial properties.

Response of osteoclasts to titanium surfaces with increasing surface roughness: an in vitro study.

Osteoclasts are responsible for bone resorption and implant surface roughness promotes osseointegration. However, little is known about the effect of roughness on osteoclast activity. This study aims at the characterization of osteoclastic response to surface roughness. The number of osteoclasts, the tartrate-resistant acid phosphatase and matrix metalloproteinase (MMP) activities, the cell morphology and the actin-ring formation were examined on smooth (TS), acid-etched (TA) and sandblasted acid-etched (TLA) titanium and on native bone. Cell morphology was comparable on TA, TLA and bone, actin rings being similar in size on TLA and bone, but smaller on TA and virtually absent on TS. Gelatin zymography revealed increased proMMP-9 expression on TA, TLA, and bone compared to TS. In general, osteoclasts show similar characteristics on rough titanium surfaces and on bone, but reduced activity on smooth titanium surfaces. These results offer some insight into the involvement of osteoclasts in remodeling processes around implant surfaces.

Biomechanical evaluation of a titanium implant surface conditioned by a hydroxide ion solution.

Two groups of titanium dental implants, identical in geometry but different in the treatment of their surfaces, were tested in an in vivo minipig model of the mandible. The surfaces that were tested were, first, sandblasted and acid-etched; and secondly, sandblasted, acid-etched, and conditioned. The removal torque was assessed at 2, 4, and 8 weeks after implantation (n=6 animals in each healing period). The interfacial stiffness was also evaluated. All dental implants were well-integrated at the time of death. Removal torque values increased significantly over the course of 8 weeks. Removal torque and interfacial stiffness were increased for conditioned surfaces after 2 weeks, but there were no significant differences between the two surfaces. The sandblasted and acid-etched implants are the standard, and conditioning of the surface showed a tendency to increase early peri-implant formation of bone.

A comparison of osteoclast resorption pits on bone with titanium and zirconia surfaces.

Osteoclasts resorb bone at surfaces, leaving behind pits and trails where both mineral and organic phases of bone have been dissolved. Rough surface structures are deliberately imparted to synthetic implants, in order to improve osseointegration. The aim of this study is to characterize osteoclastic resorption pits on native bone surfaces and to compare these with state-of-the-art titanium and zirconia implant surfaces. The size (i.e. length, width and depth) of resorption pits was compared to the size of surface features of sandblasted and etched titanium and zirconia surfaces. It was found that resorption pits from native bone and surface features of the sandblasted and etched titanium and zirconia surfaces were quite similar in their dimensions. Most structures showed a length between 5 and 40 mum, a width between 2 and 20 mum and a depth between 1 and 8 mum. Additionally, the wavelength-dependent surface roughness was measured, revealing an S(a) value of 60 nm in the resorption pits, 86 nm on zirconia and between 127 and 140 nm on titanium surfaces. The results of this study may provide some insight into structural requirements for the bone-remodeling cycle and help to improve the design of new implant surfaces for osseointegration applications.