Huinan Liu

Nanomedicine for implants: A review of studies and necessary experimental tools

The response of host organisms (including at the protein and cellular level) to nanomaterials is different than that observed to conventional materials. Nanomaterials are those materials which possess constituents less than 100 nm in at least one direction. This review will first introduce the use of nanomaterials in a variety of implant applications highlighting their promise towards regenerating tissues. Such reviewed studies will emphasize interactions of nanomaterials with various proteins and subsequently cells. Moreover, such advances in the use of nanomaterials as novel implants have been largely, to date, determined by conventional methods. However, the novel structure–property relationships unique for nanosized materials reside at the nanoscale. That is, the novelty of a nanomaterial can only be fully appreciated by characterizing their interactions with biological systems (such as proteins) with nanoscale resolution analytical tools. This characterization of nanomaterials at the nanoscale is critical to understanding and, hence, further promoting increased tissue growth on nanomaterials. For this reason, while more tools are needed for this emerging field, this review will also cover currently available surface characterization techniques that emphasize nanoscale resolution pertinent for characterizing biological interactions with nanomaterials, including attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (SIMS), colorimetric biological assays, circular dichroism (CD), and atomic force microscopy (AFM). Only through the coordination of nanoscale analytical tools with studies that highlight mechanisms of increased tissue growth on nanomaterials will we be able to design better implant materials.

Mimicking the nanofeatures of bone increases bone-forming cell adhesion and proliferation

There is a great need to design better orthopaedic implant devices by modifying their surface properties. In this respect, one approach that has received much attention of late is the simulation of the surface roughness of bone in synthetic orthopaedic implant materials. Bone has numerous nanometre features due to the presence of nanostructured entities such as collagen and hydroxyapatite. Despite this fact, current orthopaedic implant materials are smooth at the nanoscale. Previous studies have measured increased osteoblast (bone-forming cell) functions on biologically inspired nanophase titania compared to conventional titania formulations. In fact, in vitro calcium deposition by osteoblasts was up to three times higher on nanostructured compared to conventional titania. However, it was unclear in those studies what underlying surface properties (roughness, crystallinity, crystal phase, chemistry, etc) promoted enhanced functions of osteoblasts on nanophase titania. For that reason, the objective of the present in vitro study was to specifically determine the role nanostructured surface roughness of titania had on increasing functions of osteoblasts. To achieve this, the surface roughness of nanophase and conventional titania was transferred to a model tissue engineering polymer: poly-lactic-co-glycolic acid (PLGA). Results of the present study demonstrated greater osteoblast adhesion and proliferation for up to 5 days of culture on PLGA moulds of nanophase compared to conventional titania. In this manner, this study elucidated that the property of nanophase titania which increased osteoblast function was a large degree of nanometre surface features that mimicked bone. For this reason, nanophase materials deserve more attention in improving orthopaedic implant applications.

An in vitro evaluation of the Ca/P ratio for the cytocompatibility of nano-to-micron particulate calcium phosphates for bone regeneration

Calcium phosphate based bioceramics have been widely used for orthopedic applications due to their chemical similarity to natural bone. The Ca/P stoichiometry of calcium phosphates strongly influences their performance under biological conditions, which have not yet been fully elucidated to date. For this reason, the objective of this in vitro study was to understand the relationship between the Ca/P ratio of nano-to-micron particulate calcium phosphate substrates and their biological properties, such as osteoblast (bone-forming cell) viability, collagen production, alkaline phosphatase activity and nitric oxide (NO) production. A group of calcium phosphates with Ca/P ratios between 0.5 and 2.5 were obtained by intentionally adjusting the Ca/P stoichiometry of the initial reactants necessary for calcium phosphate precipitation. For samples with 0.5 and 0.75 Ca/P ratios, tricalcium phosphate (TCP) and Ca(2)P(2)O(7) phases were observed. In contrast, for samples with 1.0 and 1.33 Ca/P ratios, the only stable phase was TCP. For samples with a 1.5 Ca/P ratio, the TCP phase was dominant; however, small amounts of the hydroxyapatite (HA) phase started to appear. For samples with a 1.6 Ca/P ratio, the HA phase was dominant. Lastly, for samples with 2.0 and 2.5 Ca/P ratios, the CaO phase started to appear in the HA phase which was the dominant phase. Moreover, the average grain size and the average pore size decreased from micron-scale (e.g. 1370nm for a 0.5 Ca/P ratio) to nano-scale (e.g. 262nm for a 2.5 Ca/P ratio) with increasing Ca/P ratios. The porosity (%) of calcium phosphate substrates also decreased with increasing Ca/P ratios. Previous in vitro results demonstrated increased osteoblast adhesion on calcium phosphates with higher Ca/P ratios (up to 2.5). The present study showed that the collagen production by osteoblasts was similar between all the calcium phosphates but slightly lower with a 1.6 Ca/P ratio. Greater alkaline phosphatase activity by osteoblasts was observed in all the cultures with various calcium phosphates (0.5-2.5 Ca/P ratios) than in the control (only cells in culture). Ca/P ratios of <2 and 1 optimized osteoblast viability and promoted alkaline phosphatase activity in osteoblasts, respectively. However, the presence of the CaO phase in Ca/P ratios 2.0 increased osteoblast NO production and decreased osteoblast viability. In summary, this study provided evidence that the Ca/P ratio of calcium phosphate is a very important factor that should be considered when selecting nano-to-micron particulate calcium phosphates for various orthopedic applications.

Mechanical properties of dispersed ceramic nanoparticles in polymer composites for orthopedic applications.

Ceramic/polymer composites have been considered as third-generation orthopedic biomaterials due to their ability to closely match properties (such as surface, chemistry, biological, and mechanical) of natural bone. It has already been shown that the addition of nanophase compared with conventional (or micron-scale) ceramics to polymers enhances bone cell functions. However, in order to fully take advantage of the promising nanometer size effects that nanoceramics can provide when added to polymers, it is critical to uniformly disperse them in a polymer matrix. This is critical since ceramic nanoparticles inherently have a strong tendency to form larger agglomerates in a polymer matrix which may compromise their properties. Therefore, in this study, model ceramic nanoparticles, specifically titania and hydroxyapatite (HA), were dispersed in a model polymer (PLGA, poly-lactic-co-glycolic acid) using high-power ultrasonic energy. The mechanical properties of the resulting PLGA composites with well-dispersed ceramic (either titania or HA) nanoparticles were investigated and compared with composites with agglomerated ceramic nanoparticles. Results demonstrated that well-dispersed ceramic nanoparticles (titania or HA) in PLGA improved mechanical properties compared with agglomerated ceramic nanoparticles even though the weight percentage of the ceramics was the same. Specifically, well-dispersed nanoceramics in PLGA enhanced the tensile modulus, tensile strength at yield, ultimate tensile strength, and compressive modulus compared with the more agglomerated nanoceramics in PLGA. In summary, supplemented by previous studies that demonstrated greater osteoblast (bone-forming cell) functions on well-dispersed nanophase ceramics in polymers, the present study demonstrated that the combination of PLGA with well-dispersed nanoceramics enhanced mechanical properties necessary for load-bearing orthopedic/dental applications.

Nanomaterials enhance osteogenic differentiation of human mesenchymal stem cells similar to a short peptide of BMP-7.

Background Nanomaterials have unique advantages in controlling stem cell function due to their biomimetic characteristics and special biological and mechanical properties. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration. Methods This in vitro study investigated the effects of nano-hydroxyapatite, nano-hydroxyapatite-polylactide- co-glycolide (PLGA) composites, and a bone morphogenetic protein (BMP-7)- derived short peptide (DIF-7c) on osteogenic differentiation of human mesenchymal stem cells (MSC). The peptide was chemically functionalized onto nano-hydroxyapatite, incorporated into a nanophase hydroxyapatite-PLGA composite or PLGA control, or directly injected into culture media. Results Unlike the PLGA control, the nano-hydroxyapatite-PLGA composites promoted adhesion of human MSC. Importantly, nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites promoted osteogenic differentiation of human MSCs, comparable with direct injection of the DIF-7c peptide into culture media. Conclusion Nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites provide a promising alternative in directing the adhesion and differentiation of human MSC. These nanocomposites should be studied further to clarify their effects on MSC functions and bone remodeling in vivo, eventually translating to clinical applications.

Degradation and antibacterial properties of magnesium alloys in artificial urine for potential resorbable ureteral stent applications.

This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli viability and reduced the colony forming units over a 3-day incubation period in an artificial urine (AU) solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the AU solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.

In vitro evaluation of the surface effects on magnesium-yttrium alloy degradation and mesenchymal stem cell adhesion.

Magnesium (Mg) alloys present many advantages over current materials used in medical implants and devices. However, the rapid degradation of Mg alloys can raise the local pH and create gas cavities. Fundamental understanding of their biodegradation processes is necessary for their success in clinical applications. This study investigated how the oxidized and polished surfaces of a Mg-yttrium (Y) alloy affected the degradation mode and rate in cell culture media versus deionized water. The interactions of the alloy surfaces with cells were examined in vitro using bone marrow derived mesenchymal stem cells, since they are critical cells for bone tissue regeneration. The polished surface was more stable than the oxidized surface in cell culture media, but less stable in water. When comparing polished and oxidized surfaces, their degradation modes were similar in water, but different in cell culture media. The microstructure, roughness, and oxygen content of the alloy surface contributed to these differences. The presence or absence of a stable degradation layer determined the rate of Y loss and the inhibiting or promoting behavior of Y on degradation. The initial alloy surfaces not only influenced the degradation, but also determined cell attachment, which is critical for tissue integration. The polished surface showed more cell adhesion than the oxidized surface, mainly because of its slower degradation rate and lesser effect on the local pH. In conclusion, this study demonstrated that both the Mg alloy surfaces and the immersion fluids played important roles in controlling the degradation and cellular interactions.

Increased osteoblast functions on nanophase titania dispersed in poly-lactic-co-glycolic acid composites

The design of nanophase titania/poly-lactic-co-glycolic acid (PLGA) composites offers an exciting approach to combine the advantages of a degradable polymer with nano-size ceramic grains to optimize physical and biological properties for bone regeneration. Importantly, nanophase titania mimics the size scale of constituent components of bone since it is a nanostructured composite composed of nanometre dimensioned hydroxyapatite well dispersed in a mostly collagen matrix. For these reasons, the objective of the present in vitro study was to investigate osteoblast (bone-forming cell) adhesion and long-term functions on nanophase titania/PLGA composites. Since nanophase titania tended to significantly agglomerate when added to polymers, different sonication output powers were applied in this study to improve titania dispersion. Results demonstrated that the dispersion of titania in PLGA was enhanced by increasing the intensity of sonication and that greater osteoblast adhesion correlated with improved nanophase titania dispersion in PLGA. Moreover, results correlated better osteoblast long-term functions, such as alkaline phosphatase activity and calcium-containing mineral deposition, on nanophase titania/PLGA composites compared to plain PLGA. In fact, the greatest collagen production by osteoblasts occurred when cultured on nanophase titania sonicated in PLGA at the highest powers. In this manner, the present study demonstrates that PLGA composites with well dispersed nanophase titania can enhance osteoblast functions necessary for improved bone tissue engineering applications.

The effects of nanostructured hydroxyapatite coating on the biodegradation and cytocompatibility of magnesium implants.

Magnesium (Mg) alloys, a novel class of degradable, metallic biomaterials, have attracted growing interest as a promising alternative for medical implant and device applications due to their advantageous mechanical and biological properties. Although its biodegradability is an attractive property, rapid degradation of Mg in the physiological environments imposes a major obstacle that limits the translation of Mg-based implants to clinical applications. Therefore, the objective of this study was to develop a nanostructured hydroxyapatite (nHA) coating on polished Mg substrates to mediate the rapid degradation of Mg while improving its integration with bone tissue for orthopedic applications. The nHA coatings were deposited on polished Mg using the patented transonic particle acceleration (Spire Biomedical) process. Surface morphology, elemental compositions, and crystal structures were characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction (XRD) analysis, respectively. The degradation of nHA-coated and non-coated Mg samples was investigated by incubating the samples in phosphate buffered saline and revised simulated body fluid, under standard cell culture conditions. Rat bone marrow stromal cells (BMSCs) were harvested and cultured with nHA-coated and non-coated Mg samples to determine cytocompatibility. The degradation results suggested that the nHA coatings decreased Mg degradation. Improved BMSC adhesion was observed on the surfaces of the nHA-coated and non-coated Mg samples, in comparison with the cells on the culture plate surrounding the Mg samples. In conclusion, nHA coatings showed promise for improving the biodegradation and cytocompatibility properties of Mg-based orthopedic implants and should be further studied.

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

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.