Covalently-Linked Hyaluronan versus Acid Etched Titanium Dental Implants: A Crossover RCT in Humans.

Biochemical modification of titanium surfaces (BMTiS) entails immobilization of biomolecules to implant surfaces in order to induce specific host responses. This crossover randomized clinical trial assesses clinical success and marginal bone resorption of dental implants bearing a surface molecular layer of covalently-linked hyaluronan in comparison with control implants up to 36 months after loading. Patients requiring bilateral implant rehabilitation received hyaluronan covered implants in one side of the mouth and traditional implants in the other side. Two months after the first surgery, a second surgery was undergone to uncover the screw and to place a healing abutment. After two weeks, the operator proceeded with prosthetic procedures. Implants were evaluated by periapical radiographs and the crestal bone level was recorded at mesial and distal sites-at baseline and up to 36 months. One hundred and six implants were positioned, 52 HY-coated, and 48 controls were followed up. No differences were observed in terms of insertion and stability, wound healing, implant success, and crestal bone resorption at any time considered. All interventions had an optimal healing, and no adverse events were recorded. This trial shows, for the first time, a successful use in humans of biochemical-modified implants in routine clinical practice and in healthy patients and tissues with satisfactory outcomes.

Surface chemistry and effects on bone regeneration of a novel biomimetic synthetic bone filler.

The paper presents results of physico-chemical and biological investigations of a surface-engineered synthetic bone filler. Surface analysis confirms that the ceramic phosphate granules present a collagen nanolayer to the surrounding environment. Cell cultures tests show that, in agreement with literature reports, surface-immobilized collagen molecular cues can stimulate progression along the osteogenic pathway of undifferentiated human mesenchymal cells. Finally, in vivo test in a rabbit model of critical bone defects shows statistically significant increase of bone volume and mineral apposition rate between the biomimetic bone filler and collagen-free control. All together, obtained data confirm that biomolecular surface engineering can upgrade the properties of implant device, by promoting more specific and targeted implant-host cells interactions.

Collagen type I coating stimulates bone regeneration and osteointegration of titanium implants in the osteopenic rat.

PURPOSE: To investigate the effects of titanium implants functionalised with collagen type I (TiColl) on bone regeneration and osteointegration in a healthy and osteopenic rat animal model. METHOD: TiColl screws were implanted into the femoral condyles of healthy and osteopenic rats and compared with acid-etched titanium (Ti) screws. The osteointegration process was evaluated by a complementary approach combining microtomographic, histological, histomorphometric and biomechanical investigations at four and 12 weeks. RESULTS: The TiColl screw also ensured a greater mechanical stability; the push-out values for TiColl screws increased from four to 12 weeks (+28 %). The energy necessary to detach the bone from the screw was significantly higher for TiColl-functionalised screws in comparison to Ti screws (+23 %) at 12 weeks. Histomorphometric investigation revealed that total bone-to-implant contact was higher in TiColl screws in comparison to Ti screws (P < 0.05) and at epiphyseal level, increased bone-to-implant contact was found with TiColl screws in comparison to Ti screws (P < 0.05) in an ovariectomy (OVX) condition. A significant increase in the measured total bone ingrowth from four to 12 weeks was detected for both materials, but more significant for the TiColl material (P < 0.0005). Finally, bone ingrowth in the TiColl group was significantly higher (P < 0.005) in comparison to that of Ti screws in the SHAM condition at metaphyseal level at 12 weeks. CONCLUSION: The present results showed that TiColl is effective in promoting implant osteointegration even in compromised bone.

Adherent endotoxin on dental implant surfaces: a reappraisal.

Osteoimmunology is the crosstalk between cells from the immune and skeletal systems, suggesting a role of pro-inflammatory cytokines in the stimulation of osteoclast activity. Endotoxin or bacterial challenges to inflammatory cells are directly relevant to dental implant pathologies involving bone resorption, such as osseointegration failure and peri-implantitis. While the endotoxin amount on implant devices is regulated by standards, it is unknown whether commercially available dental implants elicit different levels of adherent-endotoxin stimulated cytokines. The objective of this work is to develop a model system and evaluate endotoxin-induced expression of pro-inflammatory cytokine genes relevant to osteoclast activation on commercially available dental implants. Murine J774-A1 macrophages were cultured on Ti disks with different level of lipopolysaccharide (LPS) contamination to define the time-course of the inflammatory response to endotoxin, as evaluated by reverse transcription polymerase chain reaction analysis. The developed protocol was then used to measure adherent endotoxin on commercially available packaged and sterile dental implants in the "as-implanted" condition. Results show that tested dental implants induce variable expression of endotoxin-stimulated genes, sometimes above the level expected to promote bone resorption in vivo. Results are unaffected by the specific surface treatment; rather, they likely reflect care in cleaning and packaging protocols. In conclusion, expression of genes that enhance osteoclast activity through endotoxin stimulation of inflammatory cells is widely different on commercially available dental implants. A reappraisal of the clinical impact of adherent endotoxins on dental (and bone) implant devices is required in light of increasing knowledge on crosstalk between cells from the immune and skeletal systems.

Functionalization with a Polyphenol-Rich Pomace Extract Empowers a Ceramic Bone Filler with In Vitro Antioxidant, Anti-Inflammatory, and Pro-Osteogenic Properties.

Oral diseases and periodontitis in particular are a major health burden worldwide, because of their association with various systemic diseases and with conditions such as peri-implantitis. Attempts have been made over the years to reverse bone loss due to the host disproportionate inflammatory response and to prevent failure of dental implants. To this end, the use of biomaterials functionalized with molecules characterized by anti-inflammatory and antioxidant properties could represent a new frontier for regenerating functional periodontal tissues. In this study, a new ceramic granulated biomaterial, named Synergoss Red (SR), functionalized with a polyphenolic mixture extracted from pomace of the Croatina grape variety, is introduced. Following a preliminary in-depth characterization of the extract by HPLC analysis and of the biomaterial surface and composition, we performed evaluations of cytocompatibility and a biological response through in vitro assays. The anti-inflammatory and antioxidant properties of the identified phenolic molecules contained in SR were shown to downregulate inflammation in macrophages, to stimulate in osteoblast-like cells the expression of genes involved in deposition of the early bone matrix, and to mitigate bone remodeling by decreasing the RANKL/OPG ratio. Thanks to its cytocompatibility and assorted beneficial effects on bone regeneration, SR could be considered an innovative regenerative approach in periodontal therapy.

Polyphenols from grape pomace induce osteogenic differentiation in mesenchymal stem cells.

Polyphenols are increasingly investigated for the treatment of periodontitis and research on their use in dental biomaterials is currently being conducted. Grape pomace extracts are a rich source of polyphenols. In the present study, the polyphenols of two different types of grape pomace were characterized and identified by high­performance liquid chromatography­diode array detector, and the effect of polyphenol­rich grape pomace extracts on mesenchymal stem cell (MSC) osteogenic differentiation was investigated. Solid­liquid extraction was used to recover polyphenols from red and white grape pomace. The two extracts have been characterized through the phenolic content and antioxidant power. Human MSCs (hMSCs) from the bone marrow were cultured both with and without given amounts (10 or 20 µg/ml) of the obtained pomace extracts. Their effects on cell differentiation were evaluated by reverse transcription­quantitative polymerase chain reaction, compared with relevant controls. Results showed that both pomace extracts, albeit different in phenolic composition and concentration, induced multiple effects on hMSC gene expression, such as a decreased receptor activator of nuclear factor κ­Β ligand/osteoprotegerin ratio and an enhanced expression of genes involved in osteoblast differentiation, thus suggesting a shift of hMSCs towards osteoblast differentiation. The obtained results provided data in favor of the exploitation of polyphenol properties from grape pomace extracts as complementary active molecules for dental materials and devices for bone regeneration in periodontal defects.

Evaluation of Cytotoxicity and Antibacterial Activity of a New Class of Silver Citrate-Based Compounds as Endodontic Irrigants.

In the present study, the cytotoxicity and the antimicrobial activity of two silver citrate-based irrigant solutions were investigated. Cytotoxicity of various concentrations (0.25%, 0.5%, 1%, 2.5%, 5%) of both solutions (BioAKT and BioAKT Endo) was assessed on L-929 mouse fibroblasts using the MTT assay. For the quantitative analysis of components, an infrared (I.R.) spectroscopy was performed. The minimum inhibitory and minimal bactericidal concentrations (M.I.C. and M.B.C., respectively) were ascertained on Enterococcus faecalis strain ATCC 4083. For biofilm susceptibility after treatment with the irrigating agent, a minimum biofilm eradication concentration (M.B.E.C.) and confocal laser scanning microscope (C.L.S.M.) assays were performed. Quantification of E. faecalis cell biomass and percentage of live and dead cells in the biomass was appraised. Normality of data was analyzed using the D'Agostino & Pearson's test and the Shapiro-Wilk test. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's test. Both silver citrate solutions showed mouse fibroblasts viability >70% when diluted to 0.25% and 0.5%. Conversely, at higher concentrations, they were extremely cytotoxic. F.T.-IR spectroscopy measurements of both liquids showed the same spectra, indicating similar chemical characteristics. No substantial contrast in antimicrobial activity was observed among the two silver citrate solutions by using broth microdilution methods, biofilm susceptibility (MBEC-HTP device), and biomass screening using confocal laser scanning microscopy (C.L.S.M.) technique. Both solutions, used as root canal irrigants, exhibited significant antimicrobial activity and low cytocompatibility at dilutions greater than 0.5%.

Permanent wettability of a novel, nanoengineered, clinically available, hyaluronan-coated dental implant.

The objectives of this study are to evaluate long-term wettability of novel surface-engineered, clinically available dental implants, featuring a surface nanolayer of covalently linked hyaluronan, and to confirm the relationships between wetting properties and surface nanostructure and microstructure. Wettability measurements were performed on clinically available hyaluronan-coated Grade 4 titanium implants, packaged and sterile, that is, in the "on the shelf" condition, after 1 year from production. Wetting properties were measured by the Wilhelmy plate method. Analysis of the surface structure and chemistry was perfomed by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis, atomic force microscopy (AFM), and ζ-potential measurement, either on implants or disks or plates subjected to the same surface-engineering process. Results show that hydrophilicity and ensuing capillary rise of the hyaluronan-coated implant surface is unaffected by aging and dry storage. Chemical analysis of the implant surface by XPS and evaluation of the ζ potential indicate that hyaluronan chemistry and not that of titanium dictates interfacial properties. Comparison between XPS versus EDX and SEM versus AFM data confirm that the thickness of the hyaluronan surface layer is within the nanometer range. Data show that nanoengineering of the implant surface by linking of the hydrophilic hyaluronan molecule endows tested titanium implants by permanent wettability, without need of wet storage as presently performed to keep long-term hydrophilic implant surfaces. From an analytical point of view, the introduction in routine clinical practice of nanoengineered implant surfaces requires upgrading of analytical methods to the nanoscale.

Effects of Er:YAG laser and ultrasonic treatment on fibroblast attachment to root surfaces: an in vitro study.

BACKGROUND: The aim of this study was to analyze the effects of erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser and ultrasonic treatment on fibroblast attachment to periodontally diseased root surfaces. METHODS: Thirty single-rooted human periodontally involved teeth were included in this study. A total of 60 specimens were obtained from all selected teeth and were randomly assigned to the following three groups: group A, untreated control group; group B, ultrasonic group; and group C, Er:YAG laser at 160 mJ/pulse at 10 Hz group. All of the specimens were incubated in petri dishes with fibroblast suspension and observed by scanning electron microscopy. RESULTS: Laser-treated specimens showed a significantly higher cell density number, with a mean+/-SD of 3,720+/-316 cells/mm2. The ultrasonically treated group showed a lower cell density number, with a mean+/-SD of 658+/-140 cells/mm2. The untreated control group showed the lowest cell density number, with a mean+/-SD of 130+/-80 cells/mm2. Differences between all groups were significant (P<0.0001). CONCLUSION: The results of the study indicate that untreated control surfaces and ultrasonically treated surfaces exhibited a significantly lower number of attached cells compared to laser-treated specimens, which showed a significantly higher cell density number.

Bacterial Biofilm Morphology on a Failing Implant with an Oxidized Surface: A Scanning Electron Microscope Study.

This case report provided a unique opportunity to investigate the extent of microbiota infiltration on the oxidized implant surface that has been compromised by peri-implantitis. Scanning electron microscopic analysis confirmed the etiologic role of the bacteria on the loss of supporting structure and the difficulty in complete removal of bacterial infiltration on the implant surface. This case report emphasizes the need to perform definitive surface decontamination on failing dental implants prior to a regeneration procedure.

Novel bioceramic-reinforced hydrogel for alveolar bone regeneration.

UNLABELLED: The osseointegration of dental implants and their consequent long-term success is guaranteed by the presence, in the extraction site, of healthy and sufficient alveolar bone. Bone deficiencies may be the result of extraction traumas, periodontal disease and infection. In these cases, placement of titanium implants is contraindicated until a vertical bone augmentation is obtained. This goal is achieved using bone graft materials, which should simulate extracellular matrix (ECM), in order to promote osteoblast proliferation and fill the void, maintaining the space without collapsing until the new bone is formed. In this work, we design, develop and characterize a novel, moldable chitosan-pectin hydrogel reinforced by biphasic calcium phosphate particles with size in the range of 100-300µm. The polysaccharide nature of the hydrogel mimics the ECM of natural bone, and the ceramic particles promote high osteoblast proliferation, assessed by Scanning Electron Microscopy analysis. Swelling properties allow significant adsorption of water solution (up to 200% of solution content) so that the bone defect space can be filled by the material in an in vivo scenario. The incorporation of ceramic particles makes the material stable at different pH and increases the compressive elastic modulus, toughness and ultimate tensile strength. Furthermore, cell studies with SAOS-2 human osteoblastic cell line show high cell proliferation and adhesion already after 72h, and the presence of ceramic particles increases the expression of alkaline phosphatase activity after 1week. These results suggest a great potential of the developed moldable biomaterials for the regeneration of the alveolar bone. STATEMENT OF SIGNIFICANCE: The positive fate of a surgical procedure involving the insertion of a titanium screw still depends on the quality and quantity of alveolar bone which is present in the extraction site. Available materials are basically hard scaffold materials with un-predictable behavior in different condition and difficult shaping properties. In this work we developed a novel pectin-chitosan hydrogel reinforced with ceramic particles. Polysaccharides simulate the extracellular matrix of natural bone and the extensive in vitro cells culture study allows to assess that the incorporation of the ceramic particles promote a pro-osteogenic response. Shape control, easy adaption of the extraction site, predictable behavior in different environment condition, swelling properties and an anti-inflammatory response are the significant characteristics of the developed biomaterial.

In Vitro Cytokine Expression and In Vivo Healing and Inflammatory Response to a Collagen-Coated Synthetic Bone Filler.

The goal of the present work was to investigate the relationship between in vivo healing and inflammatory response and in vitro cytokine expression by macrophages of a synthetic bone filler (25% hydroxylapatite-75% ß-tricalcium phosphate) bearing a surface nanolayer of collagen. A clinically accepted, state-of-the-art xenograft material was used as a "negative control," that is, as a material that provides the correct clinical response for the intended use. In vitro data show that both materials exert a very low stimulation of proinflammatory cytokines by macrophages, and this was confirmed by the very mild inflammatory response detected in in vivo tests of local response in a rabbit model. Also, in vitro findings suggest a different mechanism of healing for the test and the control material, with a higher regenerative activity for the synthetic, resorbable filler, as confirmed by in vivo observation and literature reports. Thus, the simple in vitro model adopted provides a reasonable forecast of in vivo results, suggesting that new product development can be guided by in vitro tuning of cell-materials interactions.

Multifunctional commercially pure titanium for the improvement of bone integration: Multiscale topography, wettability, corrosion resistance and biological functionalization.

The objects of this research are commercially pure titanium surfaces, with multifunctional behavior, obtained through a chemical treatment and biological functionalization. The explored surfaces are of interest for dental implants, in contact with bone, where several simultaneous and synergistic actions are needed, in order to get a fast and effective osseointegration. The here described modified surfaces present a layer of titanium oxide, thicker than the native one, with a multi-scale surface topography (a surface roughness on the nano scale, which can be overlapped to a micro or macro roughness of the substrate) and a high density of OH groups, that increase surface wettability, induce a bioactive behavior (hydroxyapatite precipitation in simulated body fluid) and make possible the grafting of biomolecules (alkaline phosphatase, ALP, in the present research). The surface oxide is an efficient barrier against corrosion, with passive behavior both with and without application of an external voltage.

Surface analysis and effects on interfacial bone microhardness of collagen-coated titanium implants: a rabbit model.

PURPOSE: The aim of this study was to evaluate the surface chemistry and the microhardness at the implant-bone interface using a recently developed collagen-coated titanium implant in a short-term rabbit model. MATERIALS AND METHODS: Surface chemistry was evaluated by x-ray photoelectron spectroscopy (XPS), while in vivo studies involved 4-week implants mid-diaphysis in the lateral femurs of adult male rabbits. After conventional embedding and evaluation of histologic sections, the resinembedded blocks containing the implanted screws were used to measure bone hardness by means of an indentation test. RESULTS: Decomposition of the C1s peak obtained by XPS analysis confirmed that surface-immobilized collagen retained all the molecular features of the control, nonimmobilized reference. As to microhardness measurement, newly formed bone at the collagen-coated-implant/bone interface was significantly harder than bone at the interface of the uncoated control implant and bone. DISCUSSION: These results suggested that collagen coating significantly improves bone maturation and mineralization at the interface in comparison with uncoated commercially pure titanium. Surface modification of titanium implants by collagen coating has recently been discussed as a promising approach to the biochemical modification of implant surfaces. The present results support previous histologic findings and demonstrated that the biomolecular layer linked over the titanium implant can increase the bone healing rate, at least in this animal model. CONCLUSIONS: The present microhardness measurement at the bone-implant interface showed that collagen coating can significantly improve bone maturation and mineralization at the interface in comparison with uncoated commercially pure titanium, confirming and substantiating previous findings by histomorphometric measurements from the same model.

Controlling the lateral distribution and alignment of human gingival fibroblasts by micropatterned polysaccharide surfaces.

Adhesion and alignment of primary human gingival fibroblast to Petri dishes were controlled by proper modification of the surface chemistry of the solid substrate. In particular, air plasma (glow-discharge) treatment, coupled to a masking technique, was used to pattern cell-adhesive areas on a cell-adhesion-resistant alginate coated surface. Fibroblasts were successfully confined to the air plasma-treated areas, which show a much greater growth rate and cell density as compared to the original alginate coated surface. Beside the implications for the study of the mechanisms of bio-adhesion at hydrophilic surfaces, these systems can be of interest for a number of applications in dental surgery and materials.

Surface chemistry effects of topographic modification of titanium dental implant surfaces: 2. In vitro experiments.

PURPOSE: To determine, in vitro, cytotoxicity and cell adhesion on 3 different implant surfaces. MATERIALS AND METHODS: All samples had machined surfaces, but they were subjected to different cleaning procedures, which produced 3 different surface chemistries. One of the samples was "as-produced" from the machining tools. The other samples were subjected to partial and total cleaning routines. Cytotoxicity was evaluated using mouse fibroblast cultures, and cell adhesion was evaluated with osteoblast-like SaOS-2 cells. RESULTS: The "as-produced" sample showed a pronounced surface contamination by lubricating oils. For partially and totally cleaned samples, an increasing amount of titanium and a decreasing carbon/titanium ratio was observed as cleaning became more complete. DISCUSSION: Differences in surface chemistry such as those normally found on titanium implant surfaces (see part 1 of this series) can lead to those same effects which, in in vitro experiments, are normally accounted for in terms of surface topography alone. CONCLUSION: Effects related to surface chemistry can operate over and above surface topography, making it impossible, without proper characterization, to make definite statements about the role of topography alone.

Surface chemistry effects of topographic modification of titanium dental implant surfaces: 1. Surface analysis.

PURPOSE: To analyze the surface composition of 34 different commercially available titanium dental implants. MATERIALS AND METHODS: Surface composition was evaluated by x-ray photoelectron spectroscopy (XPS). Samples were divided into 4 groups, depending on their surface topography (machined, sandblasted, acid etched, or plasma sprayed). RESULTS: Statistical analysis of the data showed a clear relationship between surface composition and topography, which can be easily accounted for by the chemical effects of the surface treatment performed. On average, acid-etched and plasma-sprayed surfaces had higher titanium and lower carbon concentration than machined surfaces. DISCUSSION AND CONCLUSION: Current studies aimed at the evaluation of implants with different topography should not implicitly assume that topography is the only variable controlling the biologic response. Rather, when comparing different topographies, it should be taken into account that surface chemistry may be a variable as well.

Plasma of argon accelerates murine fibroblast adhesion in early stages of titanium disk colonization.

PURPOSE: This study was conducted to analyze how a cleaning treatment using plasma of argon would affect fibroblast growth on titanium disks at different time points to determine whether this treatment could enhance soft tissue healing around titanium dental implant abutments. MATERIALS AND METHODS: Sixty sterile disks made of machined grade 5 titanium were divided into two groups; 30 disks were left untreated (control) and 30 were cleaned using plasma of argon (test). To simulate clinical conditions during soft tissue healing around titanium abutments, both groups were immersed in a culture of murine fibroblasts (L929) for 2, 8, or 48 hours. After preparation, they were stained using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) to label the cellular nuclei and fluorescent phalloidin to label the cellular bodies. The nuclei were counted, and cellular bodies were analyzed with fluorescent microscopy and imaging analysis software. Analysis was performed at the three different time points. RESULTS: Fibroblast adhesion for the test group was statistically significantly greater versus the control group at 2 and 8 hours but not at 48 hours. At 2 and 8 hours, the cellular bodies in the test group appeared flatter and more spread out, revealing more advanced cellular adhesion, compared to the cells observed in the control group. At 48 hours, the test and control specimens were nearly indistinguishable. CONCLUSION: The removal of organic and inorganic contaminants from the surfaces of titanium disks using plasma of argon accelerated fibroblast adhesion in the early stages of colonization (2 to 8 hours). This effect disappeared after 48 hours as a result of saturation. Clinically, abutment cleaning using plasma of argon might positively affect soft tissue healing in early stages.

Effect of titanium implant surface nanoroughness and calcium phosphate low impregnation on bone cell activity in vitro.

BACKGROUND AND OBJECTIVE: In the field of bone implant surfaces, the effects of nanoscale modifications have received significant attention. In the present study, bone cell activity on 2 implant surfaces with similar microtopography but distinct chemistry and nanotopography (sandblasted/acid-etched surface as control group, and calcium phosphate (CaP) low impregnated surface (Ossean) as test group, both from Intra-Lock, Boca Raton, FL) were evaluated. STUDY DESIGN: The 2 surfaces were characterized by X-ray photoelectronic spectroscopy (XPS) and scanning electron microscopy (SEM) up to x200,000 magnification. The micrometer level roughness profiles were evaluated by means of computer software. Cell adhesion, proliferation, and alkaline phosphatase activity were assessed with human SaOS-2 osteoblasts and bone mesenchymal stem cells in nonosteogenic culture conditions. RESULTS: The XPS and SEM results showed that the Ossean surface presented low levels of CaP impregnation within the titanium oxide layer and texturization at the nanometer scale (nanoroughness) compared with the control surface. Moreover Ossean surface induced significantly higher cell differentiation levels than the control (P < .01). CONCLUSION: This study showed that both homogeneous nanoroughness and CaP low impregnation differently affected in vitro bone cell behavior compared with the control moderately rough surface with less texturing in the nanometer scale. However, the relative importance of nanotopography and surface chemistry in cell reactions is yet to be determined.

Alkaline phosphatase grafting on bioactive glasses and glass ceramics.

Bone integration of orthopaedic or dental implants and regeneration of damaged bone at the surgical site are still unresolved problems in prosthetic surgery. For this reason, biomimetic surfaces (i.e. both inorganic and biological bioactive surfaces) represent a challenge for bone implantation. In this research work a hydrolase enzyme (alkaline phosphatase) was covalently grafted to inorganic bioactive glass and glass ceramic surfaces, in order to impart biological bioactivity. The functionalized samples were analysed by means of X-ray photoelectron spectroscopy in order to verify enzyme presence on the surface. Enzyme activity was measured by means of UV-visual spectroscopy after reaction with the natural substrate. Scanning electron microscopy-energy-dispersive spectroscopy observations allowed monitoring of the morphological and chemical modification of the materials during the different steps of functionalization. In vitro inorganic bioactivity was investigated by soaking samples in simulated body fluid. Enzymatic activity of the samples was tested and compared before and after soaking. Enzymatic activity of the solution was monitored at different experimental times. This study demonstrates that alkaline phosphatase could be successfully grafted onto different bioactive surfaces while maintaining its activity. Presence of the enzyme in vitro enhances the inorganic bioactivity of the materials tested.