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.
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.
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 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 (Ø1.1 × 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 β-tricalcium phosphate (β-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 β-TCP with 40 vol.% micropores lost 30 ± 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 "Biological evaluation of medical devices-Part 5 Tests for in vitro cytotoxicity", 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.
This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California at Riverside (UCR) for cell and tissue harvesting. A 12-week-old female Sprague-Dawley (SD) rat is shown as an example in the video. Younger female and male rats are preferred.
1. Cell culture preparation
NOTE: The three culture methods described in this article are generally applicable for different cell types that are adherent. Here, BMSCs harvested from rat weanlings will be introduced as an example for cell culture preparation. Depending on their relevance for specific medical applications, different cell types may be utilized, including primary cells harvested from animals or human donors and cell lines from a cell/tissue bank.
2. Sample preparation and sterilization
3. Cell culture methods
4. Postculture characterization of cells
NOTE: For direct culture and direct exposure culture, fix, stain, image, and analyze the cells adherent on both well plates and samples. For exposure culture, analyze the cells adhered to the well-plates.
5. Postculture analyses of media and samples
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 (ф = 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∙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∙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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. Reproduced from 34. Abbreviations: BMSCs = bone marrow-derived mesenchymal stem cells; SEM = scanning electron microscopy; EDX = energy dispersive X-Ray spectroscopy. Please click here to view a larger version of this figure.
Supplemental Table 1: Degradation mechanisms, products, and rates for different types of materials, and the results collected for the postculture sample and medium analysis. Please click here to download this Table.
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 <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. 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. 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.
The authors have nothing to disclose.
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.
10 mL serological pipette | VWR | 490019-704 | |
12-well tissue-culture-treated plates | Thermo Fisher Scientific | 353043 | |
15 mL conical tube (Polypropylene) | VWR | 89039-666 | |
18 G needle | BD | 305196 | |
25½ G needle | BD | 305122 | |
4′,6-diamidino-2- phenylindole dilactate (DAPI) | Invitrogen | D3571 | |
50 mL conical tube (Polypropylene) | VWR | 89039-658 | |
70 μm nylon strainer | Fisher Scientific | 50-105-0135 | |
Alexa Flour 488-phalloidin | Life technologies | A12379 | |
Biological safety cabinet | LABCONCO | Class II, Type A2 | |
Centrifuge | Eppendorf | Rotor F-35-6-30, Centrifuge5430 | |
Clear Fused Quartz Round Dish | AdValue Technology | FQ-4085 | |
CO2 incubator | SANYO | MCO-19AIC | |
CoolCell Freezer Container | Corning | 432000 | foam container designed to regulate temperature decrease |
Cryovial | Thermo Fisher Scientific | 5000-1020 | |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | 472301 | |
Dulbecco’s modified Eagle’s medium (DMEM) | Sigma-Aldrich | D5648 | |
EDX analysis software | Oxford Instruments | AztecSynergy | |
Energy dispersive X-ray spectroscopy (EDX) | FEI | 50mm2 X-Max50 SDD | |
Fetal bovine serum (FBS) | Thermo Fisher Scientific Inc. | SH30910 | |
Fluorescence microscope | Nikon | Eclipse Ti | |
Formaldehyde | VWR | 100496-496 | |
Hemacytometer | Hausser Scientific | 3520 | |
ImageJ software | National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin) | ||
Inductively coupled plasma optical emission spectrometry (ICP-OES) |
PerkinElmer | Optima 8000 | |
Optical microscope | VWR | VistaVision | |
Penicillin/streptomycin (P/S) | Thermo Fisher Scientific, Inc., | 15070063 | |
pH meter | VWR | model SB70P | |
Phosphate Buffered Saline (PBS) | VWR | 97062-730 | |
Scanning electronic microscope (SEM) | FEI | Nova NanoSEM 450 | |
surgical blade | VWR | 76353-728 | |
Tissue Culture Flasks | VWR | T-75, MSPP-90076 | |
Transwell inserts | Corning | 3460 | |
Trypsin-ethylenediaminetetraacetic acid solution (Trypsin-EDTA) | Sigma-Aldrich | T4049 | |
X-ray diffraction instrument (XRD) | PANalytical | Empyrean Series 2 |