Synthesis and Characterizations of Bioactive Glass Nanoparticle-Incorporated Triblock Copolymeric Injectable Hydrogel for Bone Tissue Engineering.

Recently, injectable hydrogels have attracted much interest in tissue engineering (TE) applications because of their controlled flowability, adaptability, and easy handling properties. This work emphasizes the synthesis and characterizations of bioactive glass (BAG) nanoparticle-reinforced poly(ethylene glycol) (PEG)- and poly(N-vinylcarbazole) (pNVC)-based minimally invasive composite injectable hydrogel suitable for bone regeneration. First, the copolymer was synthesized from a combination of PEG and pNVC through reversible addition-fragmentation chain-transfer (RAFT) polymerization and nanocomposite hydrogel constructs were subsequently prepared by conjugating BAG particles at varying loading concentrations. Gel permeation chromatography (GPC) analysis confirmed the controlled nature of the polymer. Various physicochemical characterization results confirmed the successful synthesis of copolymer and nanocomposite hydrogels that showed good gelling and injectability properties. Our optimal nanocomposite hydrogel formulation showed excellent swelling properties in comparison to the copolymeric hydrogel due to the presence of hydrophilic BAG particles. The bone cell proliferation rate was found to be evidently higher in the nanocomposite hydrogel than in the copolymeric hydrogel. Moreover, the enhanced level of ALP activity and apatite mineralization for the nanocomposite in comparison to that for the copolymeric hydrogel indicates accelerated in vitro osteogenesis. Overall, our study findings indicate BAG particle-conjugated nanocomposite hydrogels can be used as promising grafting materials in orthopedic reconstructive surgeries complementary to conventional bone graft substitutes in cancellous bone defects due to their 3D porous framework, minimal invasiveness, and ability to form any desired shape to match irregular bone defects.

Influence of Ultrasound and Magnetic Field Treatment Time on Carcinoma Cell Inhibition with Drug Carriers: An in Vitro Study.

The influence of exposing carcinoma cells to a static magnetic field (SMF) and low-intensity pulsed ultrasound (LIPUS), for different durations (15-45 min/d), in the presence of magnetic and non-magnetic drug carriers, on their in vitro inhibition is examined. Increasing the exposure time by 15 min/d decreased the culture duration by 24 h to achieve the same level of inhibition in colon (HCT116) and hepatocellular (HepG2) cells. Cell cycle analysis revealed enhanced cellular blockage in G1 and S phases with SMF + LIPUS exposure, and exposure for 45 min/d completely suppressed the S → G2 transition. Apoptosis of both types of cells increased with SMF + LIPUS treatment time, and HepG2 cells exhibited elevated necrosis with >30 min/d exposure. HepG2 cells also had higher amounts of reactive oxygen species (seven- to eightfold) than HCT116 cells (two- to sixfold), suggesting treatment effectiveness is cell and drug carrier dependent. The accelerated cellular activities are attributed to the enhanced internalization of drug carriers as a consequence of destabilized cellular membranes caused by the SMF + LIPUS-generated mechanical and electrical stimuli.

Mechanical behaviour of additively manufactured bioactive glass/high density polyethylene composites.

Bioactive glass (BAG) is a well-known biomaterial that can form a strong bond with hard and soft tissues and can also aid in bone regeneration. In this study, BAG is added to a polymer to induce bioactivity and to realize fused filament fabrication (FFF) based printing of polymer composites for potential orthopaedic implant applications. BAG (5, 10, and 20 wt%) is melt compounded with high density polyethylene (HDPE) and subsequently extruded into feedstock filament for FFF-printing. Tensile tests on developed filaments reveal that they are stiff enough to resist forces exerted during the printing process. Micrography of printed HDPE/BAG reveals perfect diffusion of raster interface indicating proper selection of printing parameters. Micrography of freeze fractured prints shows the homogeneous distribution and good dispersion of filler across the matrix. The tensile, flexural, and compressive modulus of FFF-printed HDPE/BAG parts increases with filler addition. BAG addition to the HDPE matrix enhances flexural and compressive strength. The tensile and flexural behaviour of FFF-prints is comparable to injection molded counterparts. Property maps exhibit the merits of present study over the existing literature pertaining to desired bone properties and polymer composites used in biomedical applications. It is envisioned that the development of HDPE/BAG composites for FFF-printing can lead to possible orthopaedic implants and scaffolds to mimic the bone properties in customised anatomical sites or injuries.

Probing the Influence of γ-Sterilization on the Oxidation, Crystallization, Sliding Wear Resistance, and Cytocompatibility of Chemically Modified Graphene-Oxide-Reinforced HDPE/UHMWPE Nanocomposites and Wear Debris.

Osteolysis and aseptic loosening due to wear at the articulating interfaces of prosthetic joints are considered to be the key concerns for implant failure in load-bearing orthopedic applications. In an effort to reduce the wear and processing difficulties of ultrahigh-molecular-weight polyethylene (UHMWPE), our research group recently developed high-density polyethylene (HDPE)/UHMWPE nanocomposites with chemically modified graphene oxide (mGO). Considering the importance of sterilization, this work explores the influence of γ-ray dosage of 25 kGy on the clinically relevant performance-limiting properties of these newly developed hybrid nanocomposites in vitro. Importantly, this work also probes into the cytotoxic effects of the wear debris of different compositions and sizes on MC3T3 murine osteoblasts and human mesenchymal stem cells (hMSCs). In particular, γ-ray-sterilized 1 wt % mGO-reinforced HDPE/UHMWPE nanocomposites exhibit an improvement in the oxidation index (16%), free energy of immersion (-12.1 mN/m), surface polarity (5.0%), and hardness (42%). Consequently, such enhancements result in better tribological properties, especially coefficient of friction (+13%) and wear resistance, when compared with UHMWPE. A spectrum of analyses using transmission electron microscopy (TEM) and in vitro cytocompatibility assessment demonstrate that phagocytosable (0.5-4.5 µm) sterilized 1 mGO wear particles, when present in culture media at 5 mg/mL concentration, induce neither significant reduction in MC3T3 murine osteoblast and hMSC growth nor cell morphology phenotype, during 24, 48, and 72 h of incubation. Taken together, this study suggests that γ-ray-sterilized HDPE/UHMWPE/mGO nanocomposites can be utilized as promising articulating surfaces for total joint replacements.

Combinatorial cassettes to systematically evaluate tissue-engineered constructs in recipient mice.

Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (µCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.

A Bioinformatics 3D Cellular Morphotyping Strategy for Assessing Biomaterial Scaffold Niches.

Many biomaterial scaffolds have been advanced to provide synthetic cell niches for tissue engineering and drug screening applications; however, current methods for comparing scaffold niches focus on cell functional outcomes or attempt to normalize materials properties between different scaffold formats. We demonstrate a three-dimensional (3D) cellular morphotyping strategy for comparing biomaterial scaffold cell niches between different biomaterial scaffold formats. Primary human bone marrow stromal cells (hBMSCs) were cultured on 8 different biomaterial scaffolds, including fibrous scaffolds, hydrogels, and porous sponges, in 10 treatment groups to compare a variety of biomaterial scaffolds and cell morphologies. A bioinformatics approach was used to determine the 3D cellular morphotype for each treatment group by using 82 shape metrics to analyze approximately 1000 cells. We found that hBMSCs cultured on planar substrates yielded planar cell morphotypes, while those cultured in 3D scaffolds had elongated or equiaxial cellular morphotypes with greater height. Multivariate analysis was effective at distinguishing mean shapes of cells in flat substrates from cells in scaffolds, as was the metric L1-depth (the cell height along its shortest axis after aligning cells with a characteristic ellipsoid). The 3D cellular morphotyping technique enables direct comparison of cellular microenvironments between widely different types of scaffolds and design of scaffolds based on cell structure-function relationships.

Machine learning based methodology to identify cell shape phenotypes associated with microenvironmental cues.

Cell morphology has been identified as a potential indicator of stem cell response to biomaterials. However, determination of cell shape phenotype in biomaterials is complicated by heterogeneous cell populations, microenvironment heterogeneity, and multi-parametric definitions of cell morphology. To associate cell morphology with cell-material interactions, we developed a shape phenotyping framework based on support vector machines. A feature selection procedure was implemented to select the most significant combination of cell shape metrics to build classifiers with both accuracy and stability to identify and predict microenvironment-driven morphological differences in heterogeneous cell populations. The analysis was conducted at a multi-cell level, where a "supercell" method used average shape measurements of small groups of single cells to account for heterogeneous populations and microenvironment. A subsampling validation algorithm revealed the range of supercell sizes and sample sizes needed for classifier stability and generalization capability. As an example, the responses of human bone marrow stromal cells (hBMSCs) to fibrous vs flat microenvironments were compared on day 1. Our analysis showed that 57 cells (grouped into supercells of size 4) are the minimum needed for phenotyping. The analysis identified that a combination of minor axis length, solidity, and mean negative curvature were the strongest early shape-based indicator of hBMSCs response to fibrous microenvironment.

Investigation of In Vitro Bone Cell Adhesion and Proliferation on Ti Using Direct Current Stimulation.

Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 µA, were used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell-materials interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 µA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 µA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell-materials interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model.

In vitro biological and tribological properties of transparent magnesium aluminate (Spinel) and aluminum oxynitride (ALON®).

The purpose of this first generation investigation is to evaluate the in vitro cytotoxicity, cell-materials interactions and tribological performance of Spinel and ALON® transparent ceramics for potential wear resistant load bearing implant applications. Besides their non-toxicity, the high surface energy of these ceramics significantly enhanced in vitro cell-materials interactions compared to bioinert commercially pure Ti as control. These transparent ceramics with high hardness in the range of 1334 and 1543 HV showed in vitro wear rate of the order of 10⁻6 mm³ Nm⁻¹ against Al2O3 ball at a normal load of 20 N.

Effect of electrical polarization and composition of biphasic calcium phosphates on early stage osteoblast interactions.

In spite of having excellent biocompatibility and osteogenic property of hydroxyapatite (HAp) and ß-tricalcium phosphate (ß-TCP), concerns have been raised regarding their degradation kinetics. Complete in vivo degradation of HAp takes years because of its slow degradation rate, and fast degradation rate of ß-TCP limits its application. Biphasic calcium phosphates (BCPs) composed of both HAp and ß-TCP have controlled degradation to some extent. Here, we have prepared three different BCPs composed of ß-TCP and HAp. These BCP composites are successfully electrically polarized to generate surfaces with positive (P-poled) and negative (N-poled) charges. Thermally stimulated depolarized current measurement (TSDC) exhibits increased stored charge density with the increase in HAp percentage in the composites. Our study focuses on understanding the effect of composition variation as well as electrical polarization of these composites on early stage osteoblast-cell adhesion, proliferation, and extracellular matrix (ECM) formation capability. No matter what the composition is, N-poled (Negatively poled) surfaces show early stage osteoblast-cell adhesion, proliferation, and ECM formation when compared with U-poled (unpoled) and P-poled (positively poled) surfaces. Irrespective of the surface charge, an improved cell-material interactions are observed as the percentage of HAp content in the composites is increased. These electrically polarized BCP composites can have potential use in the area of orthopedics and dentistry.

Porous tantalum structures for bone implants: fabrication, mechanical and in vitro biological properties.

The relatively high cost of manufacturing and the inability to produce modular implants have limited the acceptance of tantalum, in spite of its excellent in vitro and in vivo biocompatibility. In this article, we report how to process Ta to create net-shape porous structures with varying porosity using Laser Engineered Net Shaping (LENS) for the first time. Porous Ta samples with relative densities between 45% and 73% have been successfully fabricated and characterized for their mechanical properties. In vitro cell materials interactions, using a human fetal osteoblast cell line, have been assessed on these porous Ta structures and compared with porous Ti control samples. The results show that the Young's modulus of porous Ta can be tailored between 1.5 and 20 GPa by changing the pore volume fraction between 27% and 55%. In vitro biocompatibility in terms of MTT assay and immunochemistry study showed excellent cellular adherence, growth and differentiation with abundant extracellular matrix formation on porous Ta structures compared to porous Ti control. These results indicate that porous Ta structures can promote enhanced/early biological fixation. The enhanced in vitro cell-material interactions on the porous Ta surface are attributed to its chemistry, its high wettability and its greater surface energy relative to porous Ti. Our results show that these laser-processed porous Ta structures can find numerous applications, particularly among older patients, for metallic implants because of their excellent bioactivity.

Electrically polarized HAp-coated Ti: in vitro bone cell-material interactions.

Electrically polarized bulk sintered hydroxyapatite (HAp) compacts have been shown to accelerate mineralization and bone tissue ingrowth in vivo. In this work, a comprehensive study has been carried out to investigate the influence of surface charge and polarity on in vitro bone cell adhesion, proliferation and differentiation on electrically polarized HAp-coated Ti. Uniform and crack free sol-gel derived HAp coatings of 20+/-1.38microm thickness were polarized by application of an external d.c. field of 2.0kVcm(-1) at 400 degrees C for 1h. In vitro bioactivity of polarized HAp coatings was evaluated by soaking in simulated body fluid, and bone cell-material interactions were studied by culturing with human fetal osteoblast cells (hFOB) for a maximum period of 11 days. Scanning electron microscopic observation showed that accelerated mineralization on negatively charged surfaces favored rapid cell attachment and faster tissue ingrowth over non-polarized HAp coating surfaces, while positive charge on HAp coating surfaces restricted apatite nucleation with limited cellular response. Immunochemistry and confocal microscopy confirmed that the cell adhesion and early stage differentiation were more pronounced on negatively charged coating surfaces as hFOB cells expressed higher vinculin and alkaline phosphatase proteins on negatively charged surface compared to cells grown on all other surfaces. Our results in this study are process independent and potentially applicable to any other commercially available coating techniques.

Role of surface charge and wettability on early stage mineralization and bone cell-materials interactions of polarized hydroxyapatite.

Our objective was to determine the role of surface charge and wettability on early stage mineralization as well as bone cell adhesion and proliferation on polarized HAp surface. To estimate the surface wettability, contact angles were measured in water, simulated body fluid (SBF) and Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM). Experimental results show that HAp surface wettability and surface energy can be tailored by inducing surface charge without introducing any volumetric effects in the material. Increasing the surface charge increased the wettability and also the energy of HAp surfaces in all tested media. A maximum surface energy of 49.47+/-3.76mJ/m(2) was estimated for positively charged HAp surfaces polarized at 400(o)C. The in vitro bioactivity of polarized HAp samples was evaluated by soaking in SBF and DMEM (cell media). Cell-materials interaction was studied by culturing with human fetal osteoblast cells (hFOB). In vitro results show that tailoring the combined effect of wettability and charge polarity on the HAp surface enable differential binding of inorganic ions (e.g., Ca(2+), Cl(-), Na(+), HCO(3)(-) etc) and organic cell adhesive proteins (e.g., fibronectin, vitronectin etc) with different surface properties, which results in accelerated or decelerated mineralization as well as cell adhesion and proliferation on polarized HAp surface.

HDPE-Al2O3-HAp composites for biomedical applications: processing and characterizations.

The objective of this work is to demonstrate how the stiffness, hardness, as well as the biocompatibility property, of bioinert high-density polyethylene (HDPE) can be significantly improved by the combined addition of both bioinert and bioactive ceramic fillers. For this purpose, different volume fractions of hydroxyapatite and alumina, limited to a total of 40 vol %, have been incorporated in HDPE matrix. All the hybrid composites and monolithic HDPE were developed under optimized hot pressing condition (130 degrees C, 0.5 h, 92 MPa pressure). The results of the mechanical property characterization reveal that higher elastic modulus (6.2 GPa) and improved hardness (226.5 MPa) could be obtained in the developed HDPE-20 vol %-HAp-20 vol % Al(2)O(3) composite. Under the selected fretting conditions against various counterbody materials (steel, Al(2)O(3), and ZrO(2)), an extremely low COF of (0.07-0.11) and higher wear resistance (order of 10(-6) mm(3)/Nm) are obtained with the HDPE/20 vol % HAp/20 vol % Al(2)O(3) composite in both air and simulated body fluid environment. Importantly, in-vitro cell culture study using L929 fibroblast cells confirms favorable cell adhesion properties in the developed hybrid composite.

Friction and wear properties of novel HDPE--HAp--Al2O3 biocomposites against alumina counterface.

In an effort to enhance physical properties of biopolymers (high-density polyethylene, HDPE) in terms of elastic modulus and hardness, various ceramic fillers, like alumina (Al2O3) and hydroxyapatite (HAp) are added, and therefore it is essential to assess the friction and wear resistance properties of HDPE biocomposites. In this perspective, HDPE composites with varying ceramic filler content (upto 40 vol%) were fabricated under the optimal compression molding conditions and their friction and wear properties were evaluated against Al2O3 at fretting contacts. All the experiments were conducted at a load of 10 N for duration of 100,000 cycles in both dry as well as simulated body fluid (SBF). Such planned set of experiments has been designed to address three important issues: (a) whether the improvement in physical properties (hardness, E-modulus) will lead to corresponding improvement in friction and wear properties; (b) whether the fretting in SBF will provide sufficient lubrication in order to considerably enhance the tribological properties, as compared to that in ambient conditions; and (c) whether the generation of wear debris particles be reduced for various compositionally modified polymer composites, in comparison to unreinforced HDPE. The experimental results indicate the possibility of achieving extremely low coefficient of friction (COF approximately 0.047) as well as higher wear resistance (wear rate in the order of approximately 10(-7) mm3 N(-1) m(-1)) with the newly developed composites in SBF. A low wear depth of 3.5-4 microm is recorded, irrespective of fretting environment. Much effort has been put forward to correlate the friction and wear mechanisms with abrasion, adhesion, and wear debris formation.

Fretting wear properties of hydroxyapatite, alumina containing high density polyethylene biocomposites against zirconia.

Considering the importance of wear on the materials performance in biomedical applications, the major objective of the present work is to investigate the friction and fretting wear behavior of various HDPE-based composites against zirconia counterbody, both in air and simulated body fluid (SBF) environment. Both Al(2)O(3) and/or HAp fillers (upto 40 vol %) have been incorporated in HDPE to improve the hardness and elastic modulus of HDPE. The fretting wear study indicates that extremely low COF (approximately 0.055-0.075) as well as higher wear resistance (wear rate in the order of approximately 10(-6) mm(3)/N m) can be achieved with the newly developed composites in SBF. A low wear depth of 3-7 microm is recorded, irrespective of fretting environment. Besides reporting the phenomenological tribological data, major focus has been on to understand the underlying mechanism of material removal at fretting contacts. Such understanding has been established in discussing the wear mechanisms in terms of deformation of polymer matrix, tribolayer formation, and wear debris generation.

Tribological investigation of novel HDPE-HAp-Al2O3 hybrid biocomposites against steel under dry and simulated body fluid condition.

Among various biocompatible polymers, polyethylene based materials have received wider attention because of its excellent stability in body fluid, inertness, and easy formability. Attempts have been made to improve their physical properties (modulus/strength) to enable them to be used as load bearing hard tissue replacement applications. Among such attempts, high density polyethylene (HDPE)-hydroxyapatite (HAp) composite (HAPEX), has already been developed for total hip replacement (THR) acetabular cup and low load bearing bone tissue replacement. In the present work, alumina has been added as a partial replacement of HAp phase to improve the mechanical and tribological properties of the HAPEX composite. In an attempt to assess the suitability of the developed composite in THR application, the tribological properties against steel counterbody under both in air and simulated body fluid (SBF), have been investigated and efforts have been made to understand the wear mechanisms. The fretting wear study indicates the possibility of achieving extremely low COF (Coefficient of Friction approximately 0.09) as well as higher wear resistance (order of 10(-6) mm(3)/N m) with the newly developed composites in SBF. A low wear depth of approximately 4.6-5.3 microm is recorded, irrespective of fretting environment. The implication of the work is that optimal and combined addition of bioactive and bioinert ceramic filler to HDPE can provide a good opportunity to obtain hybrid biocomposites with better combination of physical properties (modulus, hardness) as well as low friction and high wear resistance.