Name: direct and indirect culture methods for studying biodegradable implant materials in vitro
Uploaded: 2022-04-15T13:00:00.000+00:00
Duration: 14 min 49 s
Description: Watch this Scientific Journal Video about Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro at JoVE.com

The authors appreciate the financial support from the U.S. National Science Foundation (NSF CBET award 1512764 and NSF PIRE 1545852), the National Institutes of Health (NIH NIDCR 1R03DE028631), the University of California (UC) Regents Faculty Development Fellowship, and Committee on Research Seed Grant (Huinan Liu), and UC-Riverside Dissertation Research Grant (Jiajia Lin). The authors appreciate the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) at the UC-Riverside for the use of SEM/EDS and Dr. Perry Cheung for the use of XRD instruments. The authors also appreciate Thanh Vy Nguyen and Queenie Xu for partial editing. The authors also would like to thank Cindy Lee for recording the narration for the video. Any opinions, findings, and conclusions, or recommendations expressed in this article are those of the authors and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health.

The authors have no conflicts of interest.

Different cell culture methods can be used to evaluate the in vitro cytocompatibility of biomaterials of interest for various aspects of applications in vivo. This article demonstrates three in vitro culture methods, i.e., direct culture, direct exposure culture, and exposure culture, to mimic different in vivo scenarios where biodegradable implant materials are used inside the human body. The direct culture method is mainly used to evaluate the behavior of newly seeded cells directly adherent to and surrounding the implant materials. The direct exposure culture method mimics the in vivo scenario where the implant materials come into direct contact with the established cells and tissues. The exposure culture method can be used to show how the degradation products from the implant materials and the changes in the local microenvironment can affect the established cells and tissues that are not directly in contact with the implant materials.
In the direct culture, the newly seeded cells under both direct and indirect contact conditions are evaluated. In the direct exposure culture, established cells can be evaluated under both direct and indirect contact conditions. In the exposure culture, only established cells under indirect contact conditions can be evaluated. The newly seeded cells under direct contact conditions in the direct culture are affected by material properties and material-induced changes in medium such as the changes in ion concentration and pH value.
The abovementioned material properties may include surface morphology, hydrophilicity, surface free energy, stiffness, and composition. The newly seeded cells under indirect contact conditions in the direct culture method and all the established cells in the direct exposure culture and exposure culture methods are mainly affected by material-induced changes in the medium. The three different methods described in this article are closer to the practical scenario of the in vivo environment than conventional methods such as the medium extract method. The medium extract method only evaluates the cytotoxicity of the degradation products of the material and does not reflect the dynamic process of sample degradation. In the culture methods described in this article, as the cells are cultured with the implant materials, the dynamic change of the biodegradable materials and medium environment can affect the cells in situ.
Although no in vitro studies can completely replace in vivo studies, in vitro studies are complementary and can provide valuable data in a low-cost and efficient manner. In vivo studies usually include all multiple variables in a model, whereas in vitro cell culture can study the effects of a single factor on cell-material interactions. The methods introduced in this article can mimic different scenarios of the relevant in vivo studies. We can create the linkages between different variables to provide supplements for in vivo studies. An in vivo model usually only includes the same tissue in an animal type. However, in vitro studies can include different cell types in one culture, which can study the combined effects of different variables on cell-material interactions. Moreover, it is relatively difficult to study the effects of dynamic environmental changes on cell-material interactions in in vivo models. The methods described in this article can investigate the effects of dynamic changes such as ion concentrations in the medium on cell behavior38.
The methods presented in this article are applicable for understanding in vitro cytocompatibility of all types of materials, including polymers, metals, ceramics, composites, and nanoparticles, and determining their interactions with different cells, bacteria, or fungi based on the intended applications. For example, Xu et al. evaluated the in vitro cytocompatibility of HyA-based hydrogels with BMSCs via the direct exposure culture method33. Cell adhesion densities and cell morphologies were analyzed under direct and indirect contact conditions. The cytotoxicity of HyA-based hydrogel composites might be related to the concentrations of Fe3+ and H+ ions released from the crosslinked HyA hydrogels during the cell culture experiment. Tian et al. cultured human urothelial cells (HUCs) with four different Mg alloys for 24 h and 48 h using the exposure culture method and their insoluble degradation products of MgO and Mg(OH)2 for 24 h using direct exposure culture to investigate cytocompatibility and degradation behaviors of Mg alloys containing zinc (Zn) and strontium (Sr) for potential ureteral stent application39. In this study, ZSr41_B containing 4 wt% Zn and 0.5 wt% Sr was found to have better cytocompatibility with HUCs among all the other Mg-4Zn-xSr alloys in both 24 h and 48 h exposure cultures. The results also showed that no visible adherent cells were found on the well plate when the concentrations of magnesium oxide (MgO) and magnesium hydroxide (Mg(OH)2) exceeded 1.0 mg/mL after 24 h of direct exposure culture. Therefore, Tian et al. concluded that reducing the degradation rates of Mg alloys is necessary to control the possible side effects toward future clinical translation. Wetteland et al. created a polymer-based nanocomposite by dispersing hydroxyapatite (nHA) and nMgO nanoparticles in a biodegradable PLGA polymer40. This nanocomposite was studied by culturing BMSCs with different samples using the direct culture method. The results demonstrated that improved dispersion of nanoparticles in the polymer could improve BMSC adhesion on nHA/PLGA but decrease the cell viability on nMgO/PLGA. Based on the results of in vitro cell studies, Wetteland et al. reported valuable insight for engineering optimal ceramic/polymer nanocomposites for different biomedical applications.
Cell morphologies and cell numbers can be observed and quantified in fluorescence images using software for quantitative image analysis such as ImageJ. We can investigate the effects of different materials on cell adhesion and morphology by quantifying the cell adhesion densities, cell aspect ratios, and cell spreading areas for different sample groups. The morphology of cells from the blank control group, where only the cells are cultured in medium, could serve as a standard of reference without any influence from sample materials. We can determine whether the sample materials would affect cell adhesion and morphology in vitro by comparing the cell adhesion densities and cell morphologies of the sample groups with those of the blank control. Cell spreading area reveals the preference of cell adhesion to the sample surface, showing how the cells interact with the sample materials. In this article, we reduced the reaction time for DAPI staining to be less than the vendor-recommended optimal time because biodegradable samples, such as pure magnesium, degrade rapidly in aqueous solutions. The morphology of cells adhered to the biodegradable materials may change if the staining process takes too long and the water exposure time is too long for the samples. Moreover, for the cells adherent to biodegradable materials, cell images should be taken promptly to reduce any possible changes in cell adhesion and morphology because of sample degradation.
Besides collecting results of cells, postculture medium and sample analyses are important because they will provide valuable data for analyzing the degradation mechanism, products, and rates of the implant materials. For example, biodegradable polymers such as PLGA may generate acidic degradation byproducts such as monomeric or oligomeric hydroxyl-carboxylic acids during the cell culture32, which may influence cell growth and proliferation. In contrast, biodegradable metals, such as magnesium and its alloys, produce hydroxide ions and hydrogen gas during their degradation31, which can significantly increase the local pH, and severe alkalinity may have adverse effects on local cell functions. Various biodegradable ceramics may also increase the pH of the medium41. In general, cells require a specific pH range in culture medium to function properly, and it is known that increased or decreased pH values in body fluids are harmful to life42. Measuring the pH of postculture medium is valuable for understanding any potential harm that biodegradable sample materials may cause in cell culture. Therefore, it is necessary to measure the pH value of the postculture medium to understand the potential mechanisms of how these biodegradable materials affect cells.
It is important to measure the crucial ion concentrations in the postculture medium for biodegradable materials. For example, Cortez Alcaraz et al. measured the Mg2+ and Ca2+ ion concentrations of the postculture medium when they studied magnesium oxide nanoparticle-coated magnesium samples using direct culture with BMSCs34. The concentrations of magnesium ions indicate the degradation properties of different samples in vitro during cell culture, and the concentrations of calcium ions can provide information about calcium deposition during cell proliferation. Xu et al. measured Fe3+ ion concentrations of the postculture medium when they studied HyA hydrogels using direct exposure culture with BMSCs. They utilized Fe3+ ions to adjust the crosslinking densities of HyA33. Fe3+ ions may decrease the pH value of the culture medium, and high concentrations of Fe3+ ions might be toxic to the cells. Therefore, it is important to measure the concentrations of the ions of interest to improve the cytocompatibility of biodegradable materials and their associated degradation products.
We may collect different data to analyze the cell-material interactions for different materials. For example, as shown in Supplemental Table 1, Mg alloys degrade by reacting with water, and the degradation products may include Mg2+ and OH- ions, H2 gas, and some other insoluble degradation products such as Mg(OH)2. XRD, SEM, and EDX, which could be used to determine the mineral deposition formed on the material. We may study the effects of concentration of Mg2+ ions and pH values in medium on the cell behaviors. Moreover, we may use these results to study the gas evolution during metal degradation. In vitro studies have reported the critical tolerance level of H2 gas to be &#60;0.01 mL/cm2/day, and this has been widely used to screen magnesium alloys for temporary implant applications. Essentially, the amount of gas evolution is dependent on the degradation rate of the magnesium alloys.&#160;In another example, PLGA degrades due to the hydrolysis of its ester linkages in the presence of water. The degradation products of lactic acid and glycolic acid, as well as the pH values in the medium, could be studied to analyze cell-material interactions.&#160;The methods described in this article include the measurement of released ions and the pH values in the cell culture media and the mass change of the materials, which can be used to estimate the degradation rate of the materials.
Different materials usually behave differently in vitro and in vivo, and the methods for cytocompatibility studies should be selected based on the application environment and material type. For orthopedic applications, it is desirable to evaluate the interactions between the relevant bone cells and implants when they are in direct contact with each other. The direct culture method could be utilized to investigate the interactions between the newly seeded cells and the implant. In cardiovascular applications, as the established cells will directly or indirectly come into contact with the implanted stent materials, direct exposure culture and exposure culture methods may be used to evaluate the cytocompatibility of biodegradable metals for cardiovascular applications. We believe that the in vitro methods described in this article are feasible to provide initial evidence for the cytocompatibility of biodegradable implant materials. The culture methods need to be modified for different materials with varied degradation mechanisms, products, and rates. For instance, the culturing time for different materials can be modified based on varied degradation rates of different material types. Different results may be collected based on different degradation mechanisms and products of the materials.
In summary, it is important to analyze the cells, medium, and sample materials qualitatively and quantitatively, before and after in vitro cell culture, to understand the effects of biodegradable implant materials and their degradation products on cytocompatibility. The three culture methods presented in this article can be used for studying a wide range of biodegradable materials, including biodegradable polymers, ceramics, and metals for medical implant and tissue engineering applications. These in vitro cell studies are valuable for screening biodegradable materials, optimizing the design of implantable devices and scaffolds at the early stage of product development, and reducing potential toxicity to cells.

For decades, biodegradable materials have been extensively studied and used in biomedical applications such as orthopedic1,2, dental3,4, and craniomaxillofacial5 applications. Unlike permanent implants and materials, biodegradable metals, ceramics, polymers, and their composites gradually degrade in the body over time via different chemical reactions in the physiological environment. For example, biodegradable metals such as magnesium (Mg) alloys1,6,7 and zinc (Zn) alloys8,9 are promising materials for bone fixation devices. Their biodegradability could eliminate the necessity for secondary surgeries to remove the implants after bone healing. Biodegradable ceramics such as calcium phosphate cements (CPCs) have shown exciting potential for the treatment of osteoporotic vertebral compression fractures in percutaneous kyphoplasty10. The CPCs provide mechanical support for the fractured vertebral body and gradually degrade after the fracture has healed.
Biodegradable polymers, such as some polysaccharides and polyesters, have also been widely explored for biomedical applications. For instance, chitosan hydrogel as a biodegradable polysaccharide has exhibited its capabilities for preventing infection and regenerating skin tissue11. Poly-L-lactic acid (PLLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA) are widely studied polyesters for fabricating 2D or 3D porous scaffolds for tissue engineering applications12,13,14. Moreover, composite materials integrate two or more phases of metals, ceramics, and polymers to provide advanced functions for a wide range of biomedical applications15,16,17. For example, PLGA and calcium phosphate composites can be used to fabricate biodegradable scaffolds for applications such as repairing skull bone defects18. These biodegradable scaffolds and implants could support and promote the growth of cells and tissues and then gradually degrade in the body over time.
As shown in Supplemental&#160;Table 1, different biodegradable materials may have varied degradation mechanisms, products, and rates. For example, magnesium alloys, such as Mg-2 wt % Zn-0.5 wt % Ca (ZC21)1, Mg-4 wt% Zn-1 wt% Sr (ZSr41)19, and Mg-9 wt% Al-1 wt% Zinc (AZ91)20, degrade by reacting with water, and their degradation products mainly include Mg2+ ions, OH- ions, H2 gas, and mineral depositions. The degradation rate for biodegradable metals varies depending on their different compositions, geometries, and degradation environments. For example, Cipriano et al.19 reported that ZSr41 wires (&#216;1.1 &#215; 15 mm) lost 85% mass while pure Mg wires with the same geometry lost 40% mass after being implanted in the rat tibiae for 47 days. Biodegradable ceramic materials such as hydroxyapatite (HA) and &#946;-tricalcium phosphate (&#946;-TCP) can degrade via solution-driven extracellular liquid dissolution or break down into small particles and then degrade via both extracellular liquid dissolution and cell-mediated resorption processes. The degradation products of these calcium phosphate-based ceramics may include Ca2+ ions, (PO4)3- ions, OH- ions, and mineral depositions21. The degradation rate for calcium phosphate ceramics is significantly affected by their crystal structures. For instance, Van Blitterswijk et al.22 reported that HA with 40 vol.% micropores did not lose any mass while &#946;-TCP with 40 vol.% micropores lost 30 &#177; 4% mass after being implanted in the tibiae of rabbits for 3 months. Polymers such as PLGA14,23 may degrade due to hydrolysis of the ester linkages in the presence of water, and the degradation products mainly include lactic and glycolic acids. It may take one month for PLGA 50/50 and several months for PLGA 95/5 to achieve complete degradation24.
Cell response and cytocompatibility testing are critical to evaluate and screen these biodegradable implant materials for biomedical applications. However, current standards from the International Organization for Standardization (ISO), such as ISO 10993-5:2009 &#34;Biological evaluation of medical devices-Part 5 Tests for in vitro cytotoxicity&#34;, were initially designed to assess the cytotoxicity of nondegradable biomaterials such as Ti alloys and Cr-Co alloys in vitro25. Specifically, ISO 10993-5:2009 only covers the in vitro cytotoxicity tests of the extract, direct contact, and indirect contact tests. In the extract test, the extract is prepared by immersing samples in extraction fluids such as culture media with serum and physiological saline solutions under one of the standard time and temperature conditions. The collected extract or dilution is then added into the cell culture to study cytotoxicity. For the direct contact test, direct contact between sample and cells is achieved by placing the test sample on the established (adhered) cell layer. In the indirect contact test, the culture media containing serum and melted agar is pipetted to cover the established cells. The sample is then placed onto the solidified agar layer with or without a filter.
The ISO standards have shown some limitations when applied to evaluate biodegradable materials in vitro. Unlike nondegradable materials, the degradation behaviors of biodegradable materials are dynamic and may change at a different time or in varied environmental conditions (e.g., temperature, humidity, media composition, and cell type). The extract test only evaluates the cytotoxicity of the degradation products of the material and does not reflect the dynamic process of sample degradation. Both direct and indirect contact tests of the ISO standard only characterize the interactions between the established cells and samples. Moreover, in the indirect contact test, the materials and cells are in different microenvironments that do not reflect the in vivo environment and do not capture the dynamic degradation of biodegradable materials.
The objective of this article is to introduce and discuss the cytocompatibility testing methods for various biodegradable implant materials to address the abovementioned limitations of the methods described in the current ISO standards. The methods presented in this article consider the dynamic degradation behavior of implant materials and the different circumstances of cell-material interactions in vivo. Specifically, this article provides three cytocompatibility testing methods, namely direct culture, direct exposure culture, and exposure culture for various biodegradable materials, including biodegradable polymers, ceramics, metals, and their composites for medical implant applications.
In the direct culture method, cells suspended in the culture media are directly seeded on the samples, thus evaluating the interactions between newly seeded cells and the implants. In the direct exposure culture, the samples are placed directly on the established cell layer to mimic the interactions of implants with established host cells in the body. In the exposure culture, the samples are placed in their respective well inserts and then introduced to the culture wells with established cells, which characterizes the responses of established cells to the changes in the local environment induced by implant degradation when they have no direct contact with implants. The direct culture and direct exposure culture methods evaluate the cells directly or indirectly in contact with the implant materials in the same culture well. The exposure culture characterizes the cells indirectly in contact with the implant materials within a prescribed distance in the same culture well.
This article presents a detailed description of the cytocompatibility testing for different biodegradable materials and their interactions with model cells, that is, bone marrow-derived mesenchymal stem cells (BMSCs). The protocols include the harvesting, culturing, seeding, fixing, staining, and imaging of the cells, along with analyses of postculture materials and media, which apply to a variety of biodegradable implant materials and a wide range of cell types. These methods are useful for screening biodegradable materials for different biomedical applications in terms of cell responses and cytocompatibility in vitro.

<table><tbody><tr><td>10 mL serological pipette</td><td>VWR</td><td>490019-704</td><td></td></tr><tr><td>12-well tissue-culture-treated plates</td><td>Thermo Fisher Scientific</td><td>353043</td><td></td></tr><tr><td>15 mL conical tube (Polypropylene)</td><td>VWR</td><td>89039-666</td><td></td></tr><tr><td>18 G needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>25&amp;frac12; G needle</td><td>BD</td><td>305122</td><td></td></tr><tr><td>4&amp;prime;,6-diamidino-2- phenylindole dilactate (DAPI)</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>50 mL conical tube (Polypropylene)</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>70 &amp;mu;m nylon strainer</td><td>Fisher Scientific</td><td>50-105-0135</td><td></td></tr><tr><td>Alexa Flour 488-phalloidin</td><td>Life technologies</td><td>A12379</td><td></td></tr><tr><td>Biological safety cabinet</td><td>LABCONCO</td><td>Class II, Type A2</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>Rotor F-35-6-30, Centrifuge5430</td><td></td></tr><tr><td>Clear Fused Quartz Round Dish</td><td>AdValue Technology</td><td>FQ-4085</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>SANYO</td><td>MCO-19AIC</td><td></td></tr><tr><td>CoolCell Freezer Container</td><td>Corning</td><td>432000</td><td>foam container designed to regulate temperature decrease</td></tr><tr><td>Cryovial</td><td>Thermo Fisher Scientific</td><td>5000-1020</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>EDX analysis software</td><td>Oxford Instruments</td><td>AztecSynergy</td><td></td></tr><tr><td>Energy dispersive X-ray spectroscopy (EDX)</td><td>FEI</td><td>50mm&lt;sup&gt;2&lt;/sup&gt; X-Max50 SDD</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific Inc.</td><td>SH30910</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Formaldehyde</td><td>VWR</td><td>100496-496</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser Scientific</td><td>3520</td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin)</td><td></td><td></td></tr><tr><td>Inductively coupled plasma&lt;br/&gt; optical emission spectrometry (ICP-OES)</td><td>PerkinElmer</td><td>Optima 8000</td><td></td></tr><tr><td>Optical microscope</td><td>VWR</td><td>VistaVision</td><td></td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Thermo Fisher Scientific, Inc.,</td><td>15070063</td><td></td></tr><tr><td>pH meter</td><td>VWR</td><td>model SB70P</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>VWR</td><td>97062-730</td><td></td></tr><tr><td>Scanning electronic microscope (SEM)</td><td>FEI</td><td>Nova NanoSEM 450</td><td></td></tr><tr><td>surgical blade</td><td>VWR</td><td>76353-728</td><td></td></tr><tr><td>Tissue Culture Flasks</td><td>VWR</td><td>T-75, MSPP-90076</td><td></td></tr><tr><td>Transwell inserts</td><td>Corning</td><td>3460</td><td></td></tr><tr><td>Trypsin-ethylenediaminetetraacetic acid solution (Trypsin-EDTA)</td><td>Sigma-Aldrich</td><td>T4049</td><td></td></tr><tr><td>X-ray diffraction instrument (XRD)</td><td>PANalytical</td><td>Empyrean Series 2</td><td></td></tr></tbody></table>

direct and indirect culture methods for studying biodegradable implant materials in vitro

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. 
This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. 
This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.

Figure 4 shows the representative fluorescence images of BMSCs under direct and indirect contact conditions using different culture methods. Figure 4A,B show the BMSCs under direct and indirect contact conditions after the same 24 h direct culture with ZC21 magnesium alloys1. The ZC21 alloys consist of 97.5 wt% Magnesium, 2 wt% Zinc, and 0.5 wt% calcium. The cells that have no direct contact with the ZC21 alloy samples spread better than those that have direct contact with the samples. As shown in Figure 4C,D, the cells under direct and indirect contact conditions all exhibit normal morphology after a 24 h direct exposure with hyaluronic acid (HyA) hydrogels crosslinked by Fe3+ ions. However, the number of cells under the indirect contact condition is lower than that under the direct contact condition33. Another study reported the effects of degradation of ZSr41 alloys (&#1092; = 1.1 mm) on BMSCs after a 24 h exposure culture19. The ZSr41 alloys consist of 95 wt% magnesium, 4 wt% zinc, and 1 wt% strontium. Figure 4E shows the representative fluorescence images of BMSCs adherent to the culture well at a location 3.5 mm away from the well center, after a 24 h exposure culture with the biodegradable pins19.
Figure 5 shows the example data for quantified cell adhesion density. As shown in Figure 5A, in the 24 h direct exposure (24h_DE) culture, BMSCs in direct contact with the ZC21 have significantly greater cell adhesion density than any other group. In the 24 h direct culture (24h_D), BMSCs in direct contact with the ZC21 show significantly higher cell adhesion density than the Mg group, significantly lower than the Glass reference group, but no statistical difference compared with the Ti alloy (T64) control. As shown in Figure 5B, in the indirect contact condition of direct exposure culture, BMSC adhesion density is significantly higher for the ZC21 group than the Mg group. However, it shows no significant difference compared with the T64 and cells-only control groups. In the indirect contact condition of direct culture, BMSC adhesion density is significantly higher for the ZC21 group than for the Mg group but shows no significant difference compared with the T64 and cells-only control groups1.
Figure 6A shows the pH value of postculture medium after the direct exposure culture and direct culture. For the direct exposure culture, the pH values of medium range from 8.3 to 8.4 for all samples. In the direct culture, the pH values of medium range from 7.9 to 8 across the groups. Figure 9B shows the Mg2+ ion concentration in the postculture medium. In both the direct exposure culture and the direct culture, the Mg2+ ion concentrations in the ZC21 and Mg groups are significantly higher than in any other control groups1. Figure 7 shows the XRD patterns for ZSr41 and pure Mg after a 3-day exposure culture. In Figure 7A, the crystalline phases of Mg, Ca(OH)2, ZnO, MgO&#8729;H2O, Ca(HPO4)(H2O)2, and Ca5(PO4)3(OH) (i.e., hydroxyapatite or HA), Mg17Sr2 are found on the surface of ZSr41. In Figure 7B, the crystalline phases of Mg, Ca(OH)2, Mg3(PO4)2, Mg7(PO4)2(OH)8, Ca2P2O7&#8729;5H2O are on the surface of pure Mg19. Figure 8A shows the overlay of SEM images and EDX maps of surface elemental composition for MgO-coated Mg and the control of Mg substrates and Glass after 24 h of direct culture with BMSCs. Figure 8B shows the quantitative surface elemental composition of the sample surfaces, indicating different depositions formed during cell culture34.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63065/63065fig01.jpg" /> 
Figure 1: Schematic diagram showing the steps to harvest BMSCs from rat weanlings. This figure is modified from 35. Abbreviation: BMSCs = bone marrow-derived mesenchymal stem cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63065/63065fig02.jpg" /> 
Figure 2: Schematic diagram showing the three cell culture methods. (A) Direct culture, (B) direct exposure culture, and (C) exposure culture. This figure is modified from 36. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63065/63065fig03.jpg" /> 
Figure 3: Schematic diagrams showing direct culture and direct exposure culture. (A) Cells under direct contact and indirect contact conditions in direct culture and direct exposure culture. (B) Utilization of an imaging guide to take pictures of the cells adhered to the well plate at different distances away from the center of the samples in exposure culture. Figure 3B is modified from 37. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63065/63065fig04.jpg" /> 
Figure 4: Representative fluorescence images of BMSCs. (A, B) Direct and indirect contact conditions after a 24 h direct culture with ZC21 alloys. (C, D) Direct exposure culture with HyA hydrogels. (E) On the culture plate after a 24 h exposure culture with ZSr41 alloys. Scale bars = 100 &#181;m. A and B are reproduced from 1; C and D are reproduced from 33; and E is reproduced from 19. Abbreviations: BMSCs = bone marrow-derived mesenchymal stem cells; HyA = hyaluronic acid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63065/63065fig05v2.jpg" /> 
Figure 5: Quantitative results for cell adhesion density of BMSCs. (A) Direct contact and (B) indirect contact conditions after the 24 h direct exposure culture (24 h_DE) and direct culture (24 h_D). This figure is reproduced from 1. Abbreviations: BMSCs = bone marrow-derived mesenchymal stem cells; DE = direct exposure culture; D = direct culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63065/63065fig06v2.jpg" /> 
Figure 6: Representative results for postculture analyses of medium after the 24 h direct exposure culture and direct culture. (A) pH values and (B) Mg2+ ion concentrations. This figure is reproduced from 1. Abbreviations: DE = direct exposure culture; D = direct culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63065/63065fig07.jpg" /> 
Figure 7: Representative postculture results for phase analyses of biodegradable metallic samples after 3 days of culture with BMSCs. (A) X-ray diffraction spectrum for ZSr41. (B) XRD spectrum for pure Mg. This figure is reproduced from 19. Abbreviations: BMSCs = bone marrow-derived mesenchymal stem cells; XRD = X-ray diffraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63065/63065fig08.jpg" /> 
Figure 8: Representative postculture results for surface analyses of samples after 24 h of direct culture with BMSCs, including surface microstructure, morphology, and composition. (A) Overlay of SEM images and EDX maps of surface elemental composition for MgO-coated Mg., non-coated Mg control, and Glass reference. (B) Surface elemental composition (at. %) quantified from EDX analyses. Scale bars = 200 &#181;m. Reproduced from 34. Abbreviations: BMSCs = bone marrow-derived mesenchymal stem cells; SEM = scanning electron microscopy; EDX = energy dispersive X-Ray spectroscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/63065fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table 1: Degradation mechanisms, products, and rates for different types of materials, and the results collected for the postculture sample and medium analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63065/Supplemental Table1_HL.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro at JoVE.com

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

We introduce three methods of direct culture, direct exposure culture, and exposure culture for evaluating the in vitro cytocompatibility of biodegradable implant materials. These in vitro methods mimic different in vivo cell-implant interactions and can be applied to study various biodegradable materials.

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.0K Views.  University of California, Riverside. Directe en indirecte kweekmethoden voor het bestuderen van biologisch afbreekbare implantaatmaterialen in vitro. In de afgelopen decennia zijn biologisch afbreekbare materialen uitgebreid onderzocht voor biomedische toepassingen, zoals orthopedische, tandheelkundige en craniale maxillofaciale implantaten. om de biologisch afbreekbare materialen voor biomedische toepassingen te screenen, is het noodzakelijk om deze materialen te evalueren in termen van in vitro celresponsen, cytocompatibilitei...

Video: Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

Medicine

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

Effects of Mechanical Methods Used in Peri-implantitis Treatment on Implant Surface Decontamination and Roughness

Various mechanical methods have been proposed for decontaminating dental implant surfaces with varying success. This in vitro study evaluated the decontamination efficiency of an air abrasion (AA) system with erythritol powder, a polyether-ether-ketone (PEEK) ultrasonic tip, and titanium curettes (TIT) and their effects on implant surface topography using scanning electron microscopy (SEM). A total of 60 implants were stained with permanent red ink and placed in 3D-printed Class 1A and Class 1B peri-implantitis defects, forming six groups (n=10 per group) based on defect type and treatment protocol. Additionally, one positive and one negative control implant was used. Erythritol powder, PEEK ultrasonic tips, and titanium curettes were applied for 2 min in Class 1A defects and 3 minutes in Class 1B defects. Residual red ink areas were quantified with digital software, and implant surface changes were analyzed using SEM and EDS. None of the methods achieved complete decontamination. However, erythritol powder was significantly the most effective, leaving a residual ink rate of 24% &#177; 6% (p &#60; 0.001). PEEK ultrasonic tips resulted in 41% &#177; 4% residual ink, while titanium curettes left 55% &#177; 3%. Significant differences were observed among all methods. No significant difference in decontamination efficacy was found between Class 1A and Class 1B defects. SEM analysis showed minimal surface damage with erythritol powder and PEEK tips, whereas titanium curettes caused moderate to severe damage. Based on both decontamination efficiency and surface preservation, erythritol powder and PEEK tips are safe and effective options for peri-implantitis treatment, while titanium curettes are less effective and cause considerable surface damage. These findings may assist clinicians in peri-implantitis treatment planning.

The present protocol describes an experimental model based on ink-staining which can be used for in vitro implant surface decontamination and roughness research to contribute to clinical decision-making.

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

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