Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.     

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.     

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO₂. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.     

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides¹. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously¹. This combinatorial platform has been validated with conventional methods² and the polyanhydride film and nanoparticle libraries have been characterized with ¹H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion^1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.     

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions ¹. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA ² and PLGA ³ or a number of naturally occurring proteins such as collagen ⁴, hyaluronic acid ⁵ or fibrin ^6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body''s natural wound healing processes ⁸. Fibrin is cell-degradable and potentially autologous ⁹, making it an ideal temporary scaffold for tissue engineering.Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering ⁴. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers ¹⁰. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries ^11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture ¹³.After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions ⁶. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.     

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−ene and thiol−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−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−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.     

丙烯酸酯-PEG400 复合薄膜对胰岛素的体外控释研究

目的：在胰岛素非注射给药研究中,经皮给药系统凭借其独特的优势,已成为近年来医药领域的研发重点。控释膜的研究是 经皮给药系统中一个重要组成部分,然而涉及胰岛素通过控释膜释放的研究报道不多。本实验室通过紫外光催化技术合成出一 种丙烯酸酯-PEG复合薄膜作为胰岛素控释膜。本实验目的在于考察该复合薄膜在24 小时内对胰岛素的体外控释作用,从而为 胰岛素经皮给药制剂的基础研究作出贡献。方法：通过紫外光固化方法合成丙烯酸酯-PEG400 复合薄膜,通过HPLC的方法考察 丙烯酸酯-PEG400 复合薄膜对不同浓度胰岛素溶液的控释作用,通过比较薄膜对不同浓度胰岛素溶液的累积渗透量及渗透速率 等参数,研究薄膜对胰岛素的控释规律。结果：实验数据显示：丙烯酸酯-PEG 复合薄膜对3.0 mg/mL,6.0 mg/mL,9.0 mg/mL 这三 种不同浓度胰岛素控释曲线的相关因子分别为：0.9921,0.9950,0.9964。相关因子均大于0.99,表明该薄膜能很好的控制胰岛素溶 液实现线性释放。经计算,薄膜对3.0 mg/mL,6.0 mg/mL,9.0 mg/mL 这三种浓度胰岛素的累积渗透量分别为：266.69 ug/cm2,343.65 ug/cm2,460.10 ug/cm2。渗透速率分别为：9.24 ug·cm-2·h-1,13.40 ug·cm-2·h-1,19.04 ug·cm-2·h-1。以上两组数据表明, 薄膜对胰岛素的累积渗透量及渗透速率随胰岛素浓度的增加而增大。结论：通过实验结果我们可以看出,丙烯酸酯-PEG复合薄 膜能控制不同浓度的胰岛素溶液以恒定速率释放,通过对比薄膜对各浓度胰岛素的累积渗透量及渗透速率等参数,发现该薄膜 对胰岛素的释放速率受胰岛素浓度调节,具体表现为随胰岛素浓度的增加而增加。因此该薄膜不仅可以稳定控制胰岛素实现零 级释放,而且可以通过调节胰岛素浓度实现调节胰岛素释放速率的目的。由此可以看出,该薄膜是一种理想的胰岛素控释膜。同 时本实验作为胰岛素控释膜的基础研究,也为日后以该薄膜为控释膜的胰岛素经皮给药制剂的研发打下了坚实的基础。     

丙烯酸酯-PEG400复合薄膜对胰岛素的体外控释研究

目的：在胰岛索非注射给药研究中,经皮给药系统凭借其独特的优势,已成为近年来医药领域的研发重点。控释膜的研究是经皮给药系统中一个重要组成部分,然而涉及胰岛素通过控释膜释放的研究报道不多。本实验室通过紫外光催化技术合成出一种丙烯酸酯-PEG复合薄膜作为胰岛素控释膜。本实验目的在于考察该复合薄膜在24小时内对胰岛素的体外控释作用,从而为胰岛素经皮给药制剂的基础研究作出贡献。方法：通过紫外光固化方法合成丙烯酸酯-PEG400复合薄膜,通过HPLC的方法考察丙烯酸酯-PEG400复合薄膜对不同浓度胰岛素溶液的控释作用,通过比较薄膜对不同浓度胰岛素溶液的累积渗透量及渗透速率等参数,研究薄膜对胰岛索的控释规律。结果：实验数据显示：丙烯酸酯-PEG复合薄膜对3．0mg／mL,6．0mg／mL,9．0mg／mL这三种不同浓度胰岛素控释曲线的相关因子分别为：0．9921,0．9950,0．9964。相关因子均大于0．99,表明该薄膜能很好的控制胰岛素溶液实现线性释放。经计算,薄膜对3．0mg／mL,6．0mg／mL,9．0mg／mL这三种浓度胰岛素的累积渗透量分别为：266．69μg／cm-2,343．65μg／cm-2,460．10μg／cm2。渗透速率分别为：9．24μg／cm-2·h-1,13．40μg／cm-2·h-1,19．04μg／cm-2·h-1。以上两组数据表明,薄膜对胰岛素的累积渗透量及渗透速率随胰岛素浓度的增加而增大。结论：通过实验结果我们可以看出,丙烯酸酯一PEG复合薄膜能控制不同浓度的胰岛素溶液以恒定速率释放,通过对比薄膜对各浓度胰岛素的累积渗透量及渗透速率等参数,发现该薄膜对胰岛素的释放速率受胰岛素浓度调节,具体表现为随胰岛素浓度的增加而增加。因此该薄膜不仅可以稳定控制胰岛素实现零级释放,而且可以通过调节胰岛素浓度实现调节胰岛素释放速率的目的。由此可以看出,该薄膜是一种理想的胰岛素控释膜。同时本实验作为胰岛素控释膜的基础研究,也为日后以该薄膜为控释膜的胰岛素经皮给药制剂的研发打下了坚实的基础。     

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications^1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings^5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain^7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties^10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo¹⁵, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control^13,16,17.To this end, a custom microtensile tester was designed to accommodate microscale samples^13,17 with widely-varying Young''s moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling¹⁷. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young''s modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.