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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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,

Protokół

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....

Wyniki

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.......

Dyskusje

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 .......

Ujawnienia

The authors have no conflicts of interest.

Podziękowania

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 ....

Materiały

NameCompanyCatalog NumberComments
10 mL serological pipetteVWR490019-704
12-well tissue-culture-treated platesThermo Fisher Scientific353043
15 mL conical tube (Polypropylene)VWR89039-666
18 G needleBD305196
25½ G needleBD305122
4′,6-diamidino-2- phenylindole dilactate (DAPI)InvitrogenD3571
50 mL conical tube (Polypropylene)VWR89039-658
70 μm nylon strainerFisher Scientific50-105-0135
Alexa Flour 488-phalloidinLife technologiesA12379
Biological safety cabinetLABCONCOClass II, Type A2
CentrifugeEppendorfRotor F-35-6-30, Centrifuge5430
Clear Fused Quartz Round DishAdValue TechnologyFQ-4085
CO2 incubatorSANYOMCO-19AIC
CoolCell Freezer ContainerCorning432000foam container designed to regulate temperature decrease
CryovialThermo Fisher Scientific5000-1020
Dimethyl Sulfoxide (DMSO)Sigma-Aldrich472301
Dulbecco’s modified Eagle’s medium (DMEM)Sigma-AldrichD5648
EDX analysis softwareOxford InstrumentsAztecSynergy
Energy dispersive X-ray spectroscopy (EDX)FEI50mm2 X-Max50 SDD
Fetal bovine serum (FBS)Thermo Fisher Scientific Inc.SH30910
Fluorescence microscopeNikonEclipse Ti
FormaldehydeVWR100496-496
HemacytometerHausser Scientific3520
ImageJ softwareNational Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin)
Inductively coupled plasma
optical emission spectrometry (ICP-OES)
PerkinElmerOptima 8000
Optical microscopeVWRVistaVision
Penicillin/streptomycin (P/S)Thermo Fisher Scientific, Inc.,15070063
pH meterVWRmodel SB70P
Phosphate Buffered Saline (PBS)VWR97062-730
Scanning electronic microscope (SEM)FEINova NanoSEM 450
surgical bladeVWR76353-728
Tissue Culture FlasksVWRT-75, MSPP-90076
Transwell insertsCorning3460
Trypsin-ethylenediaminetetraacetic acid solution (Trypsin-EDTA)Sigma-AldrichT4049
X-ray diffraction instrument (XRD)PANalyticalEmpyrean Series 2

Odniesienia

  1. Zhang, C., et al. Antimicrobial bioresorbable Mg-Zn-Ca alloy for bone repair in a comparison study with Mg-Zn-Sr Alloy and pure Mg. ACS Biomaterials Science & Engineering. 6 (1), 517-538 (2019).
  2. Xu, C., et al.

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Biodegradable ImplantsIn Vitro Cell CultureCytocompatibilityCytotoxicityDirect CultureDirect Exposure CultureExposure CultureMesenchymal Stem CellsBiomedical Applications

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