Brandon T. Smith

Hyperthermia-triggered intracellular delivery of anticancer agent to HER2+ cells by HER2-specific Affibody (ZHER2-GS-Cys)-Conjugated Thermosensitive Liposomes (HER2+ Affisomes)

We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2(+)affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2(+)affisomes). HER2(+)affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2(+) affisomes showed at least 10-fold increase in binding to HER2(+) cells (SK-BR-3) when compared to HER2(-) cells (MDA-MB-468) at 37°C. A competition experiment using free ZHER2-GS-Cys blocked HER2(+) affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2(+) affisomes accumulated in the cytosol of SK-BR-3 cells at 37°C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2(+) affisome/SK-BR-3 cell complexes with a 45°C (±1°C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20min at 45°C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2(+)affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2(+)affisomes. Brief exposure of liposome-cell complexes at 45°C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2(+)affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.

3D printing for the design and fabrication of polymer-based gradient scaffolds

To accurately mimic the native tissue environment, tissue engineered scaffolds often need to have a highly controlled and varied display of three-dimensional (3D) architecture and geometrical cues. Additive manufacturing in tissue engineering has made possible the development of complex scaffolds that mimic the native tissue architectures. As such, architectural details that were previously unattainable or irreproducible can now be incorporated in an ordered and organized approach, further advancing the structural and chemical cues delivered to cells interacting with the scaffold. This control over the environment has given engineers the ability to unlock cellular machinery that is highly dependent upon the intricate heterogeneous environment of native tissue. Recent research into the incorporation of physical and chemical gradients within scaffolds indicates that integrating these features improves the function of a tissue engineered construct. This review covers recent advances on techniques to incorporate gradients into polymer scaffolds through additive manufacturing and evaluate the success of these techniques. As covered here, to best replicate different tissue types, one must be cognizant of the vastly different types of manufacturing techniques available to create these gradient scaffolds. We review the various types of additive manufacturing techniques that can be leveraged to fabricate scaffolds with heterogeneous properties and discuss methods to successfully characterize them. STATEMENT OF SIGNIFICANCE Additive manufacturing techniques have given tissue engineers the ability to precisely recapitulate the native architecture present within tissue. In addition, these techniques can be leveraged to create scaffolds with both physical and chemical gradients. This work offers insight into several techniques that can be used to generate graded scaffolds, depending on the desired gradient. Furthermore, it outlines methods to determine if the designed gradient was achieved. This review will help to condense the abundance of information that has been published on the creation and characterization of gradient scaffolds and to provide a single review discussing both methods for manufacturing gradient scaffolds and evaluating the establishment of a gradient.

Immunomodulatory properties of stem cells and bioactive molecules for tissue engineering.

The immune system plays a crucial role in the success of tissue engineering strategies. Failure to consider the interactions between implantable scaffolds, usually containing cells and/or bioactive molecules, and the immune system can result in rejection of the implant and devastating clinical consequences. However, recent research into mesenchymal stem cells, which are commonly used in many tissue engineering applications, indicates that they may play a beneficial role modulating the immune system. Likewise, direct delivery of bioactive molecules involved in the inflammatory process can promote the success of tissue engineering constructs. In this article, we will review the various mechanisms in which modulation of the immune system is achieved through delivered bioactive molecules and cells and contextualize this information for future strategies in tissue engineering.

Extrusion-based 3D printing of poly(propylene fumarate) scaffolds with hydroxyapatite gradients

Abstract The primary focus of this work is to present the current challenges of printing scaffolds with concentration gradients of nanoparticles with an aim to improve the processing of these scaffolds. Furthermore, we address how print fidelity is related to material composition and emphasize the importance of considering this relationship when developing complex scaffolds for bone implants. The ability to create complex tissues is becoming increasingly relevant in the tissue engineering community. For bone tissue engineering applications, this work demonstrates the ability to use extrusion-based printing techniques to control the spatial deposition of hydroxyapatite (HA) nanoparticles in a 3D composite scaffold. In doing so, we combined the benefits of synthetic, degradable polymers, such as poly(propylene fumarate) (PPF), with osteoconductive HA nanoparticles that provide robust compressive mechanical properties. Furthermore, the final 3D printed scaffolds consisted of well-defined layers with interconnected pores, two critical features for a successful bone implant. To demonstrate a controlled gradient of HA, thermogravimetric analysis was carried out to quantify HA on a per-layer basis. Moreover, we non-destructively evaluated the tendency of HA particles to aggregate within PPF using micro-computed tomography (μCT). This work provides insight for proper fabrication and characterization of composite scaffolds containing particle gradients and has broad applicability for future efforts in fabricating complex scaffolds for tissue engineering applications.

Incorporation of fast dissolving glucose porogens into an injectable calcium phosphate cement for bone tissue engineering

Calcium phosphate cements (CPCs) have been extensively investigated as scaffolds in bone tissue engineering in light of their chemical composition closely resembling the mineral component of bone extracellular matrix. Yet, the degradation kinetics of many CPCs is slow compared to de novo bone formation. In order to overcome this shortcoming, the use of porogens within CPCs has been suggested as a potential strategy to increase scaffold porosity and promote surface degradation. This study explored the usage of glucose microparticles (GMPs) as porogens for the introduction of macroporosity within CPCs, and characterized the handling properties and physicochemical characteristics of CPCs containing GMPs. Samples were fabricated with four different weight fractions of GMPs (10, 20, 30, and 40%) and two different size ranges (100-150μm and 150-300μm), and were assayed for porosity, pore size distribution, morphology, and compressive mechanical properties. Samples were further tested for their handling properties - specifically, setting time and cohesiveness. Additionally, these same analyses were conducted on samples exposed to a physiological solution in order to estimate the dissolution kinetics of GMPs and its effect on the properties of the composite. GMPs were efficiently encapsulated and homogeneously dispersed in the resulting composite. Although setting times increased for GMP/CPC formulations compared to control CPC material, increasing the Na2HPO4 concentration in the liquid phase decreased the initial setting time to clinically acceptable values (i.e. <15min). Incorporation of GMPs led to the formation of instant macroporosity upon cement setting, and encapsulated GMPs completely dissolved in three days, resulting in a further increase in scaffold porosity. However, the dissolution of GMPs decreased scaffold compressive strength. Overall, the introduction of GMPs into CPC resulted in macroporous scaffolds with good handling properties, as well as designer porosity and pore size distribution via selection of the appropriate size/weight fraction of GMPs. The data demonstrate that GMPs are promising porogens for the production of highly tunable porous CPC scaffolds. STATEMENT OF SIGNIFICANCE Calcium phosphate cements have shown great promise for the regeneration of bone. However, macropores (>100μm) are required for promoting bone ingrowth. Several studies have investigated methods to generate macroporosity within calcium phosphate cements but many of these methods either affect the cement setting or take weeks or months to generate the maximum porosity. This work offers a new method for generating macroporosity within calcium phosphate cements by utilizing glucose microparticles. The microparticles dissolve in less then 72h, thereby generating scaffolds with maximum porosity in short period of time. The results will offer a new method for generating macroporosity within calcium phosphate cements.

Bone Tissue Engineering Challenges in Oral & Maxillofacial Surgery.

Over the past decades, there has been a substantial amount of innovation and research into tissue engineering and regenerative approaches for the craniofacial region. This highly complex area presents many unique challenges for tissue engineers. Recent research indicates that various forms of implantable biodegradable scaffolds may play a beneficial role in the clinical treatment of craniofacial pathological conditions. Additionally, the direct delivery of bioactive molecules may further increase de novo bone formation. While these strategies offer an exciting glimpse into potential future treatments, there are several challenges that still must be overcome. In this chapter, we will highlight both current surgical approaches for craniofacial reconstruction and recent advances within the field of bone tissue engineering. The clinical challenges and limitations of these strategies will help contextualize and inform future craniofacial tissue engineering strategies.

Multimodal pore formation in calcium phosphate cements

Calcium phosphate cements (CPCs) are commonly used as bone substitute materials. However, their slow degradation rate and lack of macroporosity hinders new bone formation. Poly(dl-lactic-co-glycolic acid) (PLGA) incorporation is of great interest as, upon degradation, produces acidic by-products that enhance CPC degradation. Yet, new bone formation is delayed until PLGA degradation occurs a few weeks after implantation. Therefore, the aim of this study was to accelerate the early stage pore formation within CPCs in vitro. With that purpose, we incorporated the water-soluble porogen sucrose at different weight percentages (10 or 20 wt %) to CPC and CPC/PLGA composites. The results revealed that incorporation of sucrose porogens increased mass loss within the first week of in vitro degradation in groups containing sucrose compared to control groups. After week 1, a further mass loss was observed related to PLGA and CPC degradation. Macroporosity analysis confirmed that macroporosity formation is influenced by the dissolution of sucrose at an early stage and by the degradation of PLGA and CPC at a later stage. We concluded that the combination of sucrose and PLGA porogens in CPC is a promising approach to promote early stage bone tissue ingrowth and complete replacement of CPC through multimodal pore formation.

Improved in situ seeding of 3D printed scaffolds using cell-releasing hydrogels

The design of tissue engineered scaffolds based on polymerized high internal phase emulsions (polyHIPEs) has emerged as a promising bone grafting strategy. We previously reported the ability to 3D print emulsion inks to better mimic the structure and mechanical properties of native bone while precisely matching defect geometry. In the current study, redox-initiated hydrogel carriers were investigated for in situ delivery of human mesenchymal stem cells (hMSCs) utilizing the biodegradable macromer, poly(ethylene glycol)-dithiothreitol. Hydrogel carrier properties including network formation time, sol-gel fraction, and swelling ratio were modulated to achieve rapid cure without external stimuli and a target cell-release period of 5-7 days. These in situ carriers enabled improved distribution of hMSCs in 3D printed polyHIPE grafts over standard suspension seeding. Additionally, carrier-loaded polyHIPEs supported sustained cell viability and osteogenic differentiation of hMSCs post-release. In summary, these findings demonstrate the potential of this in situ curing hydrogel carrier to enhance the cell distribution and retention of hMSCs in bone grafts. Although initially focused on improving bone regeneration, the ability to encapsulate cells in a hydrogel carrier without relying on external stimuli that can be attenuated in large grafts or tissues is expected to have a wide range of applications in tissue engineering.

Incorporation of fast dissolving glucose porogens and poly(lactic-co-glycolic acid) microparticles within calcium phosphate cements for bone tissue regeneration

This study investigated the effects of incorporating glucose microparticles (GMPs) and poly(lactic-co-glycolic acid) microparticles (PLGA MPs) within a calcium phosphate cement on the cements handling, physicochemical properties, and the respective pore formation. Composites were fabricated with two different weight fractions of GMPs (10 and 20 wt%) and two different weight fractions of PLGA MPs (10 and 20 wt%). Samples were assayed for porosity, pore morphology, and compressive mechanical properties. An in vitro degradation study was also conducted. Samples were exposed to a physiological solution for 3 days, 4 wks, and 8 wks in order to understand how the inclusion of GMPs and PLGA MPs affects the composites porosity and mass loss over time. GMPs and PLGA MPs were both successfully incorporated within the composites and all formulations showed an initial setting time that is appropriate for clinical applications. Through a main effects analysis, we observed that the incorporation of GMPs had a significant effect on the overall porosity, mean pore size, mode pore size, and in vitro degradation rate of PLGA MPs as early as after 3 days (p < 0.05). After 4 wks and 8 wks, these same properties were affected by the inclusion of both types of MPs (p < 0.05). Advanced polymer chromatography confirmed that the degradation of PLGA MPs coincided with an increase in composite porosity, mean pore size, and mode pore size. Finally, it was observed that the inclusion of GMPs slowed the degradation of PLGA MPs in vitro and reduced the solution acidity due to PLGA degradation products. Our results suggest that the dual inclusion of GMPs and PLGA MPs is a valuable approach for the generation of early macropores, while also mitigating the effect of acidic degradation products from PLGA MPs on their degradation kinetics. STATEMENT OF SIGNIFICANCE A multitude of strategies and techniques have been investigated for the introduction of macropores with calcium phosphate cements (CPC). However, many of these strategies take several weeks to months to generate a maximal porosity or the degradation products of the porogen can trigger a localized inflammatory response in vivo. As such, it was hypothesized that the fast dissolution of glucose microparticles (GMPs) in a CPC composite also incorporating poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) will create an initial macroporosity and increase the surface area within the CPC, thus enhancing the diffusion of PLGA degradation products and preventing a significant decrease in pH. Furthermore, as PLGA degradation occurs over several weeks to months, additional macroporosity will be generated at later time points within CPCs. The results offer a new method for generating macroporosity in a multimodal fashion that also mitigates the effects of acidic degradation products.