Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials <em>In Vitro</em>

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and ...

University of California, Riverside

A novel class of biodegradable metals, magnesium (Mg) and Mg-based alloys, has recently attracted much attention because of unique biodegradation and mechanical properties for medical applications. Ideally, Mg-based devices should degrade no faster than the degradation products can be eliminated efficiently from the body. Additionally, for orthopedic and maxillofacial applications, the implant integration with the surrounding bone is critical for its clinical success. Therefore, it is necessary to thoroughly characterize Mg surface and degradation and investigate how these characteristics influence its interactions with essential cells, for example, bone marrow derived mesenchymal stem cells. The objectives of this study were to investigate (1) the effects of two surface conditions (the presence vs. absence of surface oxides) on Mg degradation and mesenchymal stem cell adhesion, and (2) the effects of two essential aqueous environments (the presence vs. absence of physiological ions and proteins) on Mg degradation. In an effort towards standardizing testing methods for Mg alloys, consistent and well-controlled experimental methods were designed to characterize the surface and degradation of Mg and its interactions with cells. The results demonstrated that original surface (oxidized vs. polished) conditions had a less pronounced effect on regulating initial cell adhesion, but did affect surface morphology and composition of the Mg samples after 24 h of cell culture. The presence versus absence of biological ions and proteins had a significant effect on Mg degradation mode and rate. In conclusion, the material surface and anatomical sites of implantation dependent on the intended applications must be carefully considered while assessing Mg alloys in vitro or in vivo for medical applications. Standardized testing procedures and methods are critically needed for developing more effective medical-grade Mg alloys.

The effects of surface and biomolecules on magnesium degradation and mesenchymal stem cell adhesion.

Crystalline Mg-Zn-Ca ternary alloys have recently attracted significant interest for biomedical implant applications due to their promising biocompatibility, bioactivity, biodegradability and mechanical properties. The objective of this study was to characterize as-cast Mg-xZn-0.5Ca (x=0.5, 1.0, 2.0, 4.0wt.%) alloys, and determine the adhesion and morphology of bone marrow derived mesenchymal stem cells (BMSCs) at the interface with the Mg-xZn-0.5Ca alloys. The direct culture method (i.e. seeding cells directly onto the surface of the sample) was established in this study to probe the highly dynamic cell-substrate interface and thus to elucidate the mechanisms of BMSC responses to dynamic alloy degradation. The results showed that the BMSC adhesion density on these alloys was similar to the cell-only positive control and the BMSC morphology appeared more anisotropic on the rapidly degrading alloy surfaces in comparison with the cell-only positive control. Importantly, neither culture media supplemented with up to 27.6mM Mg(2+) ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on BMSC responses. We speculated that degradation-induced dynamic surface topography played an important role in modulating cell morphology at the interface. This study presents a clinically relevant in vitro model for screening bioresorbable alloys, and provides useful design guidelines for determining the degradation rate of implants made of Mg-Zn-Ca alloys. 

Investigation of magnesium-zinc-calcium alloys and bone marrow derived mesenchymal stem cell response in direct culture.

This article reports the quantitative relationship between the concentration of magnesium oxide (MgO) nanoparticles and its distinct biological activities towards mammalian cells and infectious bacteria for the first time. The effects of MgO nanoparticles on the viability of bone marrow derived mesenchymal stem cells (BMSCs) and infectious bacteria (both gram-negative Escherichia coli and gram-positive Staphylococcus epidermidis) showed a concentration-dependent behavior in vitro. The critical concentrations of MgO nanoparticles identified in this study provided valuable guidelines for biomaterial design toward potential clinical translation. BMSCs density increased significantly when cultured in 200μg/mL of MgO in comparison to the Cells Only control without MgO. The density of BMSCs decreased significantly after culture in the media with 500μg/mL or more of MgO. Concentrations at or above 1000μg/mL of MgO resulted in complete BMSCs death. Quantification of colony forming units (CFU) revealed that the minimum bactericidal concentration (MBC) of MgO for E. coli and S. epidermidis was 1200μg/mL. The addition of MgO nanoparticles into the cultures increased the pH and Mg(2+) ion concentration in the respective culture media, which might have played a role in the observed cell responses but not the main factors. E. coli and S. epidermidis still proliferated significantly at alkaline pH up to 10 or with supplemental Mg(2+) dosages up to 50mM, indicating bactericidal properties of MgO are beyond the effects of increased media pH and Mg(2+) ion concentrations. MgO nanoparticles at a concentration of 200μg/mL provided dual benefits of promoting BMSC proliferation while reducing bacterial adhesion, which should be further studied for potential medical implant applications. The use of free MgO nanoparticles yielded detrimental effects to BMSCs in concentrations above 300μg/mL. We recommend further study into MgO nanoparticle as a coating material or as a part of a composite.

Concentration-dependent behaviors of bone marrow derived mesenchymal stem cells and infectious bacteria toward magnesium oxide nanoparticles.

Nanocomposite coatings offer multiple functions simultaneously to improve the interfacial properties of magnesium (Mg) alloys for skeletal implant applications, e.g., controlling the degradation rate of Mg substrates, improving bone cell functions, and providing drug delivery capability. However, the effective service time of nanocomposite coatings may be limited due to their early delamination from the Mg-based substrates. Therefore, the objective of this study was to address the delamination issue of nanocomposite coatings, improve the coating properties for reducing the degradation of Mg-based substrates, and thus improve their cytocompatibility with bone marrow derived mesenchymal stem cells (BMSCs). The surface conditions of the substrates, polymer component type of the nanocomposite coatings, and post-deposition processing are the key parameters that contribute to the efficacy of the nanocomposite coatings in regulating substrate degradation and bone cell responses. Specifically, the effects of metallic surface versus alkaline heat-treated hydroxide surface of the substrates on coating quality were investigated. For the nanocomposite coatings, nanophase hydroxyapatite (nHA) was dispersed in three types of biodegradable polymers, i.e., poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid) (PLLA), or poly(caprolactone) (PCL) to determine which polymer component could provide integrated properties for slowest Mg degradation. The nanocomposite coatings with or without post-deposition processing, i.e., melting, annealing, were compared to determine which processing route improved the properties of the nanocomposite coatings most significantly. The results showed that optimizing the coating processes addressed the delamination issue. The melted then annealed nHA/PCL coating on the metallic Mg substrates showed the slowest degradation and the best coating adhesion, among all the combinations of conditions studied; and, it improved the adhesion density of BMSCs. This study elucidated the key parameters for optimizing nanocomposite coatings on Mg-based substrates for skeletal implant applications, and provided rational design guidelines for the nanocomposite coatings on Mg alloys for potential clinical translation of biodegradable Mg-based implants.

A systemic study on key parameters affecting nanocomposite coatings on magnesium substrates.

Crystalline Mg-Zinc (Zn)-Strontium (Sr) ternary alloys consist of elements naturally present in the human body and provide attractive mechanical and biodegradable properties for a variety of biomedical applications. The first objective of this study was to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x=0.15, 0.5, 1.0, 1.5wt%; designated as ZSr41A, B, C, and D respectively) in the direct culture with human umbilical vein endothelial cells (HUVEC) in vitro. The second objective was to investigate, for the first time, the early-stage inflammatory response in cultured HUVECs as indicated by the induction of vascular cellular adhesion molecule-1 (VCAM-1). The results showed that the 24-h in vitro degradation of the ZSr41 alloys containing a β-phase with a Zn/Sr at% ratio ∼1.5 was significantly faster than the ZSr41 alloys with Zn/Sr at% ∼1. Additionally, the adhesion density of HUVECs in the direct culture but not in direct contact with the ZSr41 alloys for up to 24h was not adversely affected by the degradation of the alloys. Importantly, neither culture media supplemented with up to 27.6mM Mg ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on HUVEC responses. In contrast, the significantly higher, yet non-cytotoxic, Zn ion concentration from the degradation of ZSr41D alloy was likely the cause for the initially higher VCAM-1 expression on cultured HUVECs. Lastly, analysis of the HUVEC-ZSr41 interface showed near-complete absence of cell adhesion directly on the sample surface, most likely caused by either a high local alkalinity, change in surface topography, and/or surface composition. The direct culture method used in this study was proposed as a valuable tool for studying the design aspects of Zn-containing Mg-based biomaterials in vitro, in order to engineer solutions to address current shortcomings of Mg alloys for vascular device applications.

Cytocompatibility and early inflammatory response of human endothelial cells in direct culture with Mg-Zn-Sr alloys.

This article reports anodization of Mg in KOH electrolyte and the associated surface, degradation, and biological properties for bioresorbable implant applications. The preparation procedures for electrodes and anodization setup significantly enhanced reproducibility of samples. The results of anodization performed at the applied potentials of 1.8, 1.9, or 2.0V showed that the sample anodized at 1.9V and annealed, referred to as the 1.9 AA sample, had homogenous surface microstructure and elemental composition, and a reduction in corrosion current density in the electrochemical testing. In comparison with Mg control, the 1.9 AA sample showed a distinct mode of degradation, e.g., continuous growth of a passivation layer enriched with Ca and P instead of typical localized pitting and undermining, and a greater release rate of Mg ions when immersed in physiologically relevant media. In the direct culture with bone marrow derived mesenchymal stem cells (BMSCs) in vitro, the 1.9 AA sample did not affect BMSC adhesion and morphology under indirect contact; however, the 1.9 AA sample showed a reduction in cell spreading under direct contact. The change in surface topography/composition at the dynamic interface of the anodized-annealed Mg sample might have contributed to the change in BMSC morphology. In summary, this study demonstrated the potential of anodic oxidation to modulate the degradation behaviors of Mg-based biomaterials and BMSC responses in vitro, and confirmed the value of direct culture method for studying cytocompatibility of Mg-based biomaterials for medical implant applications.

Anodization of magnesium for biomedical applications - Processing, characterization, degradation and cytocompatibility.

Magnesium (Mg) alloys are promising materials for biodegradable implants, but their clinical translation requires improved control over their degradation rates. Proteins may be a major contributing factor to Mg alloy degradation, but are not yet fully understood. This article reports the effects of fetal bovine serum (FBS), a physiologically relevant mixture of proteins, on Mg and Mg alloy degradation. FBS had little impact on mass loss of pure Mg during immersion degradation, regardless of whether or not a native oxide layer was present on the sample surface. FBS reduced the mass loss of Mg-Yttrium (MgY) alloy with an oxidized surface during immersion degradation, but increased the mass loss for the same alloy with a metallic surface (surface oxides were removed). FBS also influenced the mode of degradation by limiting the depth of pit formation during degradation processes on commercially pure Mg with metallic or oxidized surfaces and on MgY alloy with oxidized surfaces. The results demonstrated that serum proteins had significant interactions with Mg-based biodegradable metals, and these interactions may be modified by alloy composition and processing. Therefore, proteins should be taken into account when designing experiments to assess degradation of Mg-based implants.

The Effects of Serum Proteins on Magnesium Alloy Degradation in Vitro.

Magnesium (Mg) and its alloys have been widely investigated as the most promising biodegradable metals to replace conventional non-degradable metals for temporary medical implant applications. New Mg alloys have been developed for medical applications in recent years; and the concept of alloying Mg with less-toxic elements have aroused tremendous interests due to the promise to address the problems associated with rapid degradation of Mg without compromising its cytocompatibility and biocompatibility. Of particular interests for orthopedic/spinal implant applications are the additions of calcium (Ca) and strontium (Sr) into Mg matrix because of their beneficial properties for bone regeneration. In this study, degradation and cytocompatibility of four binary MgSr alloys (Mg-xSr, x = 0.2, 0.5, 1 and 2 wt%) and four ternary MgCaSr alloys (Mg-1Ca-xSr, x = 0.2, 0.5, 1 and 2 wt%) were investigated and compared via direct culture with bone marrow-derived mesenchymal stem cells (BMSCs). The influence of the alloy composition on the degradation rates were studied and compared. Moreover, the cellular responses to the binary MgSr alloys and the ternary MgCaSr alloys were comparatively evaluated; and the critical factors influencing BMSC behaviors were discussed. This study screened the degradability and in vitro cytocompatibility of the binary MgSr alloys and the ternary MgCaSr alloys. Mg-1Sr, Mg-1Ca-0.5Sr and Mg-1Ca-1Sr alloys are recommended for further in vivo studies toward clinical translation due to their best overall performances in terms of degradation and cytocompatibility among all the alloys studied in the present work.

In vitro evaluation of MgSr and MgCaSr alloys via direct culture with bone marrow derived mesenchymal stem cells.

Magnesium oxide nanoparticle (nMgO) is a light metal based antimicrobial nanoparticle that can be metabolized and fully resorbed in the body. To take advantage of the antimicrobial properties of nMgO for medical use, it is necessary to determine the minimal inhibitory, bactericidal and fungicidal concentrations (MIC, MBC and MFC) of nMgO against prevalent infectious bacteria and yeasts. The objective of this study was to use consistent methods and conditions to reveal and directly compare the efficacy of nMgO against nine prevalent pathogenic microorganisms, including two gram-negative bacteria, three gram-positive bacteria with drug-resistant strains, and four yeasts with drug-resistant strains. The MIC of nMgO varied from 0.5 mg/mL to 1.2 mg/mL and the minimal lethal concentration (MLC) of nMgO at 90% killing varied from 0.7 mg/mL to 1.4 mg/mL against different pathogenic bacteria and yeasts. The most potent concentrations (MPC) of nMgO were 1.4 and/or 1.6 mg/mL, depending on the type of bacteria and yeasts tested. As the concentration of nMgO increased, the adhesion of bacteria and yeasts decreased. Moreover, S. epidermidis biofilm was disrupted at 1.6 mg/mL of nMgO. E. coli and some yeasts showed membrane damage after cultured with ≥0.5 mg/mL nMgO. Overall, nMgO killed both planktonic bacteria and disrupted nascent biofilms, suggesting new antimicrobial mechanisms of nMgO. Production of reactive oxygen species (ROS), Ca ion concentrations, and quorum sensing likely contribute to the action mechanisms of nMgO against planktonic bacteria, but transient alkaline pH of 7 to 10 or increased Mg ion concentrations from 1 to 50 mM showed no inhibitory or killing effects on bacteria such as S. epidermidis. Further studies are needed to determine if specific concentrations of nMgO at MIC, MLC or MPC level can be integrated into medical devices to evoke desired antimicrobial responses without harming host cells.

Antimicrobial Activities and Mechanisms of Magnesium Oxide Nanoparticles (nMgO) against Pathogenic Bacteria, Yeasts, and Biofilms.

Current urological devices such as ureteral stents and catheters still face serious problems, such as encrustation and biofilm formation. Magnesium (Mg) and its alloys showed great potentials as an alternative material for urological devices, due to their excellent biodegradability and antibacterial property. In this study, a serial of four promising Mg alloys which contain zinc (Zn) and strontium (Sr), i.e., Mg-4Zn-xSr (ZSr41) alloys, were investigated in vitro for potential ureteral stent application. Specifically, these four alloys have 4 wt% Zn in all and 0.15 wt% Sr in ZSr41_A, 0.5 wt% Sr in ZSr41_B, 1.0 wt% Sr in ZSr41_C and 1.5 wt% Sr in ZSr41_D. The cytocompatibility and degradation behaviors of Mg-4Zn-xSr alloys were studied by culturing with human urothelial cells (HUCs) for 24 h and 48 h using exposure culture method. ZSr41_B showed a better cytocompatibility with HUCs among all the Mg-4Zn-xSr alloys in both 24-hour and 48-hour cultures. Moreover, the cytocompatibility of insoluble degradation products of Mg, i.e., MgO and Mg(OH), was also investigated by culturing different concentrations of MgO and Mg(OH) nanoparticles with HUCs for 24 h and 48 h. The concentration of MgO and Mg(OH) particles at 0.5 mg/mL and above, showed a significant decrease of cell density and cell size after 24-hour and 48-hour cultures. The concentration of MgO and Mg(OH) at 1.0 mg/mL and above, showed no viable cells after 24-hour culture. Collectively, it is recommended to further reduce the degradation rates of Mg alloys in order to control possible side effects of the soluble and insoluble degradation products and to take the benefits of Mg-based biodegradable ureteral stents toward the future clinical translation.

Responses of human urothelial cells to magnesium-zinc-strontium alloys and associated insoluble degradation products for urological stent applications.

Magnesium (Mg) and its alloys have shown attractive biocompatibility and mechanical strength for medical applications, but low corrosion resistance of Mg in physiological environment limits its broad clinical translation. Hydroxyapatite (HA) nanoparticles (nHA) are promising coating materials for decreasing degradation rates and prolonging mechanical strength of Mg-based implants while enhancing bone healing due to their osteoconductivity and osteoinductivity. Conformal HA coatings with nano-to-submicron structures, namely nHA and mHA coatings, were deposited successfully on Mg plates and rods using a transonic particle acceleration (TPA) process under two different conditions, characterized, and investigated for their effects on Mg degradation in vitro. The nHA and mHA coatings enhanced corrosion resistance of Mg and retained 86-90% of ultimate compressive strength after in vitro immersion in rSBF for 6 weeks, much greater than non-coated Mg that only retained 66% of strength. Mg-based rods with or without coatings showed slower degradation than the respective Mg-based plates in rSBF after 6 weeks, likely because of the greater surface-to-volume ratio of Mg plates than Mg rods. This indicates that Mg-based plate and screw devices may undergo different degradation even when they have the same coatings and are implanted at the same or similar anatomical locations. Therefore, in addition to locations of implantation, the geometry, dimension, surface area, volume, and mass of Mg-based implants and devices should be carefully considered in their design and processing to ensure that they not only provide adequate structural and mechanical stability for bone fixation, but also support the functions of bone cells, as clinically required for craniomaxillofacial (CMF) and orthopedic implants. When the nHA and mHA coated Mg and non-coated Mg plates were cultured with bone marrow derived mesenchymal stem cells (BMSCs) using the in vitro direct culture method, greater cell adhesion densities were observed under indirect contact conditions than that under direct contact conditions for the nHA and mHA coated Mg. In comparison with non-coated Mg, the nHA and mHA coated Mg reduced BMSC adhesion densities directly on the surface, but increased the average BMSC adhesion densities under indirect contact. Further long-term studies in vitro and in vivo are necessary to elucidate the effects of nHA and mHA coatings on cell functions and tissue healing.

Nano-to-Submicron Hydroxyapatite Coatings for Magnesium-based Bioresorbable Implants - Deposition, Characterization, Degradation, Mechanical Properties, and Cytocompatibility.

Silver nanoparticles (AgNPs) and regenerated silk fibroin (RSF) have recently attracted significant interests for their potential applications in preventing wound-related infections and in tissue engineering. Indeed, nano-silver has long been recognized as one of the most effective antimicrobial agents, and silk fibroin is well known for its capability of stimulating cell activities and facilitating tissue regeneration. In this study, a green synthesis approach was used to create a composite hydrogel (CoHy) of RSF stabilized with CarboxymethylCellulose-Na (CMC-Na) and loaded with AgNPs. Their swelling ratios were up to 59 g/g when tested in different physiologically relevant fluids. Material characterizations by Scanning electron microscopy (SEM) with Energy Dispersive X-ray Spectroscopy (EDS), and X-Ray Diffraction (XRD) confirmed the presence of AgNPs on the surface. Antimicrobial properties of the CoHy samples were evaluated using agar diffusion tests. The results showed distinct inhibition zones against major microorganisms found in wound infections, including Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Methicillin Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), Candida albicans (C. albicans) and Fluconazole-resistant Candida albicans (FRCA). Cytocompatibility studies with rat bone marrow derived mesenchymal stem cells (BMSCs) in vitro showed that the adhesion density of BMScs on the CoHy loaded with 1 mg/mL was similar to the cell-only control group for the first 24 h of culture; moreover, higher cell proliferation was observed on the CoHy without AgNPs, indicating the regenerative potentials of the RSF/CMC composite hydrogels.

Photo-assisted green synthesis of silver doped silk fibroin/carboxymethyl cellulose nanocomposite hydrogels for biomedical applications.

This article reports fabrication, characterization, degradation and electrical properties of biodegradable magnesium (Mg) microwires coated with two functional polymers, and the first in vivo evidence on the feasibility of Mg-based biodegradable microelectrodes for neural recording. Conductive poly(3,4‑ethylenedioxythiophene) (PEDOT) coating was first electrochemically deposited onto Mg microwire surface, and insulating biodegradable poly(glycerol sebacate) (PGS) was then spray-coated onto PEDOT surface to improve the overall properties of microelectrode. The assembled PGS/PEDOT-coated Mg microelectrodes showed high homogeneity in coating thickness, surface morphology and composition before and after in vivo recording. The charge storage capacity (CSC) of PGS/PEDOT-coated Mg microwire (1.72 mC/cm) was nearly 5 times higher than the standard platinum (Pt) microwire widely used in implantable electrodes. The Mg-based microelectrode demonstrated excellent neural-recording capability and stability during in vivo multi-unit neural recordings in the auditory cortex of a mouse. Specifically, the Mg-based electrode showed clear and stable onset response, and excellent signal-to-noise ratio during spontaneous-activity recordings and three repeats of stimulus-evoked recordings at two different anatomical locations in the auditory cortex. During 10 days of immersion in artificial cerebrospinal fluid (aCSF) in vitro, PGS/PEDOT-coated Mg microelectrodes showed slower degradation and less change in impedance than PEDOT-coated Mg electrodes. The biodegradable PGS coating protected the PEDOT coating from delamination, and prolonged the mechanical integrity and electrical properties of Mg-based microelectrode. Mg-based novel microelectrodes should be further studied toward clinical translation because they can potentially eliminate the risks and costs associated with secondary surgeries for removal of failed or no longer needed electrodes.

Huinan H. Liu

Materials Science and Engineering Program,
Department of Bioengineering,
Stem Cell Center

University of California, Riverside

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

Huinan H. Liu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Materials Science and Engineering Program,Department of Bioengineering,Stem Cell Center

University of California, Riverside

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

Materials Science and Engineering Program,
Department of Bioengineering,
Stem Cell Center