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 ...

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.

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	<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&#189; G needle</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,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 &#956;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>CO2 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&#8217;s modified Eagle&#8217;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>50mm2 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 
			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 sp...

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 ...

direct-indirect-culture-methods-for-studying-biodegradable-implant

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Bioengineering

4.9K Views.  University of California, Riverside. 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.

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 ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant adhesion strength than commercial tissue adhesives. Medical adhesives have attracted great attention as potential alternative tools to sutures and staples due to their convenience in usage with minimal invasiveness. Although there are several protocols for developing tissue adhesives including those commercially available such as fibrin glues and cyanoacrylate-based materials, mostly they require a series of chemical syntheses of organic molecules, or complicated protein-purification methods, in the case of bio-driven materials (i.e., fibrin glue). Also, the development of surgical glues exhibiting high adhesive properties while maintaining biodegradability is still a challenge due to difficulties in achieving good performance in the wet environment of the body. We illustrate a new method to prepare a medical glue, known as TAPE, by the weight-based separation of a water-immiscible supramolecular aggregate formed after a physical mixing of a plant-derived, wet-resistant adhesive molecule,&#160;Tannic Acid (TA), and a well-known biopolymer, Poly(Ethylene) glycol (PEG). With our approach, TAPE shows high adhesion strength, which is 2.5-fold more than commercial fibrin glue in the presence of water. Furthermore, TAPE is biodegradable in physiological conditions and can be used as a potent hemostatic glue against tissue bleeding. We expect the widespread use of TAPE in a variety of medical settings and drug delivery applications, such as polymers for muco-adhesion, drug depots, and others.

We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue.

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...