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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 sp...
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 ...
The authors have no conflicts of interest.
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.
Name | Company | Catalog Number | Comments |
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 |
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