Jelena Rnjak-Kovacina

Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering

We obtained low and high porosity synthetic human elastin scaffolds by adapting low (1 mL/h) and high (3 mL/h) flow rates respectively during electrospinning. Physical, mechanical and biological properties of these scaffolds were screened to identify the best candidates for the bioengineering of dermal tissue. SHE scaffolds that were electrospun at the higher flow rate presented increased fiber diameter and greater average pore size and over doubling of overall scaffold porosity. Both types of scaffold displayed Youngs moduli comparable to that of native elastin, but the high porosity scaffolds possessed higher tensile strength. Low and high porosity scaffolds supported early attachment, spreading and proliferation of primary dermal fibroblasts, but only high porosity scaffolds supported active cell migration and infiltration into the scaffold. High porosity SHE scaffolds promoted cell persistence and scaffold remodeling in vitro with only moderate scaffold contraction. The scaffolds persisted for at least 6 weeks in a mouse subcutaneous implantation study with fibroblasts on the exterior and infiltrating, evidence of scaffold remodeling including de novo collagen synthesis and early stage angiogenesis.

Increasing the Pore Size of Electrospun Scaffolds

Electrospinning has gained much attention in the past decade as an effective means of generating nano- to micro-scale polymer fibers that resemble native extracellular matrix. High porosity, pore interconnectivity, and large surface area to volume ratio of electrospun scaffolds make them highly conducive to cellular adhesion and growth. However, inherently small pores of electrospun scaffolds do not promote adequate cellular infiltration and tissue ingrowth. Cellular infiltration into the scaffold is essential for a range of tissue engineering applications and is particularly important in skin and musculoskeletal engineering. Pore size, porosity, and pore interconnectivity dictate the extent of cellular infiltration and tissue ingrowth into the scaffold; influence a range of cellular processes; and are crucial for diffusion of nutrients, metabolites, and waste products. A number of electrospinning techniques and postelectrospinning modifications have, therefore, been developed in order to increase the pore size of electrospun scaffolds. Diverse techniques ranging from simple variations in the electrospinning parameters to complex methodologies requiring highly specialized equipment have been explored and are described in this article.

Electrospun synthetic human elastin:collagen composite scaffolds for dermal tissue engineering

We present an electrospun synthetic human elastin:collagen composite scaffold aimed at dermal tissue engineering. The panel of electrospun human tropoelastin and ovine type I collagen blends comprised 80% tropoelastin+20% collagen, 60% tropoelastin+40% collagen and 50% tropoelastin+50% collagen. Electrospinning efficiency decreased with increasing collagen content under the conditions used. Physical and mechanical characterization encompassed fiber morphology, porosity, pore size and modulus, which were prioritized to identify the optimal candidate for dermal tissue regeneration. Scaffolds containing 80% tropoelastin and 20% collagen (80T20C) were selected on this basis for further cell interaction and animal implantation studies. 80T20C enhanced proliferation and migration rates of dermal fibroblasts in vitro and were well tolerated in a mouse subcutaneous implantation study where they persisted over 6 weeks. The 80T20C scaffolds supported fibroblast infiltration, de novo collagen deposition and new capillary formation.

pH-Dependent anticancer drug release from silk nanoparticles

Silk has traditionally been used as a suture material because of its excellent mechanical properties and biocompatibility. These properties have led to the development of different silk-based material formats for tissue engineering and regenerative medicine. Although there have been a small number of studies about the use of silk particles for drug delivery, none of these studies have assessed the potential of silk to act as a stimulus-responsive anticancer nanomedicine. This report demonstrates that an acetone precipitation of silk allows the formation of uniform silk nanoparticles (98 nm diameter, polydispersity index 0.109), with an overall negative surface charge (-33.6 ± 5.8 mV), in a single step. Silk nanoparticles are readily loaded with doxorubicin (40 ng doxorubicin/μg silk) and show pH-dependent release (pH 4.5≫ 6.0 > 7.4). In vitro studies with human breast cancer cell lines demonstrates that the silk nanoparticles are not cytotoxic (IC50 > 120 μg mL(-1) ) and that doxorubicin-loaded silk nanoparticles are able to overcome drug resistance mechanisms. Live cell fluorescence microscopy studies show endocytic uptake and lysosomal accumulation of silk nanoparticles. In summary, the pH-dependent drug release and lysosomal accumulation of silk nanoparticles demonstrate the ability of drug-loaded silk nanoparticles to serve as a lysosomotropic anticancer nanomedicine.

Robust bioengineered 3D functional human intestinal epithelium

Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

Tropoelastin - a versatile, bioactive assembly module

Elastin provides structural integrity, biological cues and persistent elasticity to a range of important tissues, including the vasculature and lungs. Its critical importance to normal physiology makes it a desirable component of biomaterials that seek to repair or replace these tissues. The recent availability of large quantities of the highly purified elastin monomer, tropoelastin, has allowed for a thorough characterization of the mechanical and biological mechanisms underpinning the benefits of mature elastin. While tropoelastin is a flexible molecule, a combination of optical and structural analyses has defined key regions of the molecule that directly contribute to the elastomeric properties and control the cell interactions of the protein. Insights into the structure and behavior of tropoelastin have translated into increasingly sophisticated elastin-like biomaterials, evolving from classically manufactured hydrogels and fibers to new forms, stabilized in the absence of incorporated cross-linkers. Tropoelastin is also compatible with synthetic and natural co-polymers, expanding the applications of its potential use beyond traditional elastin-rich tissues and facilitating finer control of biomaterial properties and the design of next-generation tailored bioactive materials.

Lyophilized Silk Sponges: A Versatile Biomaterial Platform for Soft Tissue Engineering

We present a silk biomaterial platform with highly tunable mechanical and degradation properties for engineering and regeneration of soft tissues such as, skin, adipose, and neural tissue, with elasticity properties in the kilopascal range. Lyophilized silk sponges were prepared under different process conditions and the effect of silk molecular weight, concentration and crystallinity on 3D scaffold formation, structural integrity, morphology, mechanical and degradation properties, and cell interactions in vitro and in vivo were studied. Tuning the molecular weight distribution (via degumming time) of silk allowed the formation of stable, highly porous, 3D scaffolds that held form with silk concentrations as low as 0.5% wt/v. Mechanical properties were a function of silk concentration and scaffold degradation was driven by beta-sheet content. Lyophilized silk sponges supported the adhesion of mesenchymal stem cells throughout 3D scaffolds, cell proliferation in vitro, and cell infiltration and scaffold remodeling when implanted subcutaneously in vivo.

Biocompatibility of silk-tropoelastin protein polymers

Blended polymers are used extensively in many critical medical conditions as components of permanently implanted devices. Hybrid protein polymers containing recombinant human tropoelastin and silk fibroin have favorable characteristics as implantable scaffolds in terms of mechanical and biological properties. A firefly luciferase transgenic mouse model was used to monitor real-time IL-1β production localized to the site of biomaterial implantation, to observe the acute immune response (up to 5 days) to these materials. Significantly reduced levels of IL-1β were observed in silk/tropoelastin implants compared to control silk only implants at 1, 2 and 3 days post-surgery. Subsequently, mice (n = 9) were euthanized at 10 days (10D) and 3 weeks (3W) post-surgery to assess inflammatory cell infiltration and collagen deposition, using histopathology and immunohistochemistry. Compared to control silk only implants, fewer total inflammatory cells were found in silk/tropoelastin (∼29% at 10D and ∼47% at 3W). Also fewer ingrowth cells (∼42% at 10D and ∼63% at 3W) were observed within the silk/tropoelastin implants compared to silk only. Lower IL-6 (∼52%) and MMP-2 (∼84%) (pro-inflammatory) were also detected for silk/tropoelastin at 10 days. After 3 weeks implantation, reduced neovascularization (vWF ∼43%), fewer proliferating cells (Ki67 ∼58% and PCNA ∼41%), macrophages (F4/80 ∼64%), lower IL-10 (∼47%) and MMP-9 (∼55%) were also observed in silk/tropoelastin materials compared to silk only. Together, these results suggest that incorporation of tropoelastin improves on the established biocompatibility of silk fibroin, uniquely measured here as a reduced foreign body inflammatory response.

Silk as a biocohesive sacrificial binder in the fabrication of hydroxyapatite load bearing scaffolds.

Limitations of current clinical methods for bone repair continue to fuel the demand for a high strength, bioactive bone replacement material. Recent attempts to produce porous scaffolds for bone regeneration have been limited by the intrinsic weakness associated with high porosity materials. In this study, ceramic scaffold fabrication techniques for potential use in load-bearing bone repairs have been developed using naturally derived silk from Bombyx mori. Silk was first employed for ceramic grain consolidation during green body formation, and later as a sacrificial polymer to impart porosity during sintering. These techniques allowed preparation of hydroxyapatite (HA) scaffolds that exhibited a wide range of mechanical and porosity profiles, with some displaying unusually high compressive strength up to 152.4 ± 9.1 MPa. Results showed that the scaffolds exhibited a wide range of compressive strengths and moduli (8.7 ± 2.7 MPa to 152.4 ± 9.1 MPa and 0.3 ± 0.1 GPa to 8.6 ± 0.3 GPa) with total porosities of up to 62.9 ± 2.7% depending on the parameters used for fabrication. Moreover, HA-silk scaffolds could be molded into large, complex shapes, and further machined post-sinter to generate specific three-dimensional geometries. Scaffolds supported bone marrow-derived mesenchymal stem cell attachment and proliferation, with no signs of cytotoxicity. Therefore, silk-fabricated HA scaffolds show promise for load bearing bone repair and regeneration needs.

The Effect of Sterilization on Silk Fibroin Biomaterial Properties

The effects of common sterilization techniques on the physical and biological properties of lyophilized silk fibroin sponges are described. Sterile silk fibroin sponges were cast using a pre-sterilized silk fibroin solution under aseptic conditions or post-sterilized via autoclaving, γ radiation, dry heat, exposure to ethylene oxide, or hydrogen peroxide gas plasma. Low average molecular weight and low concentration silk fibroin solutions could be sterilized via autoclaving or filtration without significant loses of protein. However, autoclaving reduced the molecular weight distribution of the silk fibroin protein solution, and silk fibroin sponges cast from autoclaved silk fibroin were significantly stiffer compared to sponges cast from unsterilized or filtered silk fibroin. When silk fibroin sponges were sterilized post-casting, autoclaving increased scaffold stiffness, while decreasing scaffold degradation rate in vitro. In contrast, γ irradiation accelerated scaffold degradation rate. Exposure to ethylene oxide significantly decreased cell proliferation rate on silk fibroin sponges, which was rescued by leaching ethylene oxide into PBS prior to cell seeding.