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

Summary

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.

Abstract

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.

Introduction

For decades, biodegradable materials have been extensively studied and used in biomedical applications such as orthopedic^1,2, dental^3,4, 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) alloys^1,6,

Protocol

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 a....

Results

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.......

Discussion

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 .......

Materials

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