Vascular tissues bioprinted with smooth muscle cell-only bioinks in support baths mimic features of native coronary arteries.

This study explores the bioprinting of a smooth muscle cell-only bioink into ionically crosslinked oxidized methacrylated alginate (OMA) microgel baths to create self-supporting vascular tissues. The impact of OMA microgel support bath methacrylation degree and cell-only bioink dispensing parameters on tissue formation, remodeling, structure and strength was investigated. We hypothesized that reducing dispensing tip diameter from 27 G (210m) to 30 G (159m) for cell-only bioink dispensing would reduce tissue wall thickness and improve the consistency of tissue dimensions while maintaining cell viability.

Endothelial Cells Increase Mesenchymal Stem Cell Differentiation in Scaffold-Free 3D Vascular Tissue.

In this study, we present a versatile, scaffold-free approach to create ring-shaped engineered vascular tissue segments using human mesenchymal stem cell-derived smooth muscle cells (hMSC-SMCs) and endothelial cells (ECs). We hypothesized that incorporation of ECs would increase hMSC-SMC differentiation without compromising tissue ring strength or fusion to form tissue tubes. Undifferentiated hMSCs and ECs were co-seeded into custom ring-shaped agarose wells using four different concentrations of ECs: 0%, 10%, 20%, and 30%.

Upskilling the cell therapy manufacturing workforce: design, implementation, and evaluation of a massive open online course.

Cell therapies have gained prominence as a promising therapeutic modality for treating a range of diseases. Despite the recent clinical successes of cell therapy products, very few formal training programs exist for cell therapy manufacturing. To meet the demand for a well-trained workforce, we assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing.

Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw.

Characterization of LL37 Binding to Collagen through Peptide Modification with a Collagen-Binding Domain.

Collagen-based biomaterials loaded with antimicrobial peptides (AMPs) present a promising approach for promoting wound healing while providing protection against infections. In our previous work, we modified the AMP LL37 by incorporating a collagen-binding domain (cCBD) as an anchoring unit for collagen-based wound dressings. We demonstrated that cCBD-modified LL37 (cCBD-LL37) exhibited improved retention on collagen after washing with PBS.

Manufacturing the multiscale vascular hierarchy: progress toward solving the grand challenge of tissue engineering.

In human vascular anatomy, blood flows from the heart to organs and tissues through a hierarchical vascular tree, comprising large arteries that branch into arterioles and further into capillaries, where gas and nutrient exchange occur. Engineering a complete, integrated vascular hierarchy with vessels large enough to suture, strong enough to withstand hemodynamic forces, and a branching structure to permit immediate perfusion of a fluidic circuit across scales would be transformative for regenerative medicine (RM), enabling the translation of engineered tissues of clinically relevant size, and perhaps whole organs. How close are we to solving this biological plumbing problem? In this review, we highlight advances in engineered vasculature at individual scales and focus on recent strategies to integrate across scales.

LL37 and collagen-binding domain-mediated LL37 binding with type I collagen: Quantification via QCM-D.

Antimicrobial peptide (AMP)-loaded biomaterials may represent a viable alternative for stimulating wound healing while protecting against infections. Previously, to develop an efficient delivery system for the cathelicidin antimicrobial peptide, LL37, our lab modified LL37 with a collagen-binding domain derived from collagenase (cCBD) as an anchoring unit to collagen-based wound dressings. However, a direct quantification of unmodified LL37 and cCBD-LL37 binding with collagen has not been performed.

Scaffold-free human mesenchymal stem cell construct geometry regulates long bone regeneration.

Biomimetic bone tissue engineering strategies partially recapitulate development. We recently showed functional restoration of femoral defects using scaffold-free human mesenchymal stem cell (hMSC) condensates featuring localized morphogen presentation with delayed in vivo mechanical loading. Possible effects of construct geometry on healing outcome remain unclear.

Alginate Affects Bioactivity of Chimeric Collagen-Binding LL37 Antimicrobial Peptides Adsorbed to Collagen-Alginate Wound Dressings.

Chronic infected wounds cause more than 23,000 deaths annually. Antibiotics and antiseptics are conventionally used to treat infected wounds; however, they can be toxic to mammalian cells, and their use can contribute to antimicrobial resistance. Antimicrobial peptides (AMPs) have been utilized to address the limitations of antiseptics and antibiotics.

Mechanistic predictions of the influence of collagen-binding domain sequences on human LL37 interactions with model lipids using quartz crystal microbalance with dissipation.

Modifications of human-derived antimicrobial peptide LL37 with collagen binding domains (CBD-LL37) hold promise as alternatives to antibiotics due to their wider therapeutic ratio than unmodified LL37 when interacting with collagen substrates such as commercial wound dressings. However, CBD-LL37 lipid membrane interaction mechanisms (against both mammalian and bacterial lipids) are not well understood. Our goal was to develop a mechanistic explanation of how CBDs modulate peptide-lipid interactions leading to their observed bioactivities, in order to better understand their potential for clinical applications.

Non-invasive functional molecular phenotyping of human smooth muscle cells utilized in cardiovascular tissue engineering.

Smooth muscle cell (SMC) diversity and plasticity are limiting factors in their characterization and application in cardiovascular tissue engineering. This work aimed to evaluate the potential of Raman microspectroscopy and Raman imaging to distinguish SMCs of different tissue origins and phenotypes. Cultured human SMCs isolated from different vascular and non-vascular tissues as well as fixed human SMC-containing tissues were analyzed.

A Method for High-Throughput Robotic Assembly of Three-Dimensional Vascular Tissue.

Self-assembled tissues have potential to serve both as implantable grafts and as tools for disease modeling and drug screening. For these applications, tissue production must ultimately be scaled-up and automated. Limited technologies exist for precisely manipulating self-assembled tissues, which are fragile early in culture.

Targeted Delivery of Bioactive Molecules for Vascular Intervention and Tissue Engineering.

Cardiovascular diseases are the leading cause of death in the United States. Treatment often requires surgical interventions to re-open occluded vessels, bypass severe occlusions, or stabilize aneurysms. Despite the short-term success of such interventions, many ultimately fail due to thrombosis or restenosis (following stent placement), or incomplete healing (such as after aneurysm coil placement).

Concentration-Dependent, Membrane-Selective Activity of Human LL37 Peptides Modified with Collagen Binding Domain Sequences.

Antimicrobial peptides (AMPs) such as LL37 are promising alternatives to antibiotics to treat wound infections due to their broad activity, immunomodulatory functions, and low likelihood of antimicrobial resistance. To deliver LL37 to chronic wounds, we developed two chimeric LL37 peptides with C-terminal collagen binding domains (CBD) derived from collagenase ( cCBD-LL37) and fibronectin ( fCBD-LL37) as a strategy for noncovalent tethering of LL37 onto collagen-based, commercially available wound dressings. The addition of CBD sequences to LL37 resulted in differences in cytotoxicity against human fibroblasts and antimicrobial activity against common wound pathogens.

A Modular Strategy to Engineer Complex Tissues and Organs.

Currently, there are no synthetic or biologic materials suitable for long-term treatment of large tracheal defects. A successful tracheal replacement must (1) have radial rigidity to prevent airway collapse during respiration, (2) contain an immunoprotective respiratory epithelium, and (3) integrate with the host vasculature to support epithelium viability. Herein, biopolymer microspheres are used to deliver chondrogenic growth factors to human mesenchymal stem cells (hMSCs) seeded in a custom mold that self-assemble into cartilage rings, which can be fused into tubes.

Assembly of Tissue-Engineered Blood Vessels with Spatially Controlled Heterogeneities.

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue-engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of certain vascular diseases. We developed a system for generating self-assembled human smooth muscle cell (SMC) ring units, which were fused together into TEBVs.

A QCM-D study of the concentration- and time-dependent interactions of human LL37 with model mammalian lipid bilayers.

The human antimicrobial peptide LL37 is promising as an alternative to antibiotics due to its biophysical interactions with charged bacterial lipids. However, its clinical potential is limited due to its interactions with zwitterionic mammalian lipids leading to cytotoxicity. Mechanistic insight into the LL37 interactions with mammalian lipids may enable rational design of less toxic LL37-based therapeutics.

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold.

High-density human mesenchymal stem cell rings with spatiotemporally-controlled morphogen presentation as building blocks for engineering bone diaphyseal tissue.

Emerging biomimetic tissue engineering strategies aim to partially recapitulate fundamental events that transpire during embryonic skeletal development; namely, cellular self-organization and targeted morphogenetic pathway activation. Here, we describe self-assembled, scaffold-free human mesenchymal stem cell (hMSC) rings featuring microparticle-mediated presentation of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2). We tested the hypothesis that spatiotemporally-controlled dual presentation of TGF-β1 and BMP-2 is superior in modulating endochondral ossification of high-density cellular constructs compared to single morphogen delivery.

Growth-Factor-Releasing Polyelectrolyte Multilayer Films to Control the Cell Culture Environment.

Polyelectrolyte multilayers (PEMs) are of great interest as cell culture surfaces because of their ability to modify topography and surface energy and release biologically relevant molecules such as growth factors. In this work, fibroblast growth factor 2 (FGF2) was adsorbed directly onto polystyrene, plasma-treated polystyrene, and glass surfaces with a poly(methacrylic acid) and poly-l-histidine PEM assembled above it. Up to 14 ng/cm of FGF2 could be released from plasma-treated polystyrene surfaces over the course of 7 days with an FGF2 solution concentration of 100 μg/mL applied during the adsorption process.

Pro-myogenic and low-oxygen culture increases expression of contractile smooth muscle markers in human fibroblasts.

Smooth muscle cells (SMCs) are essential for tissue engineering strategies to fabricate organs such as blood vessels, the oesophagus and bladder, and to create disease models of these systems. In order for such therapies and models to be feasible, SMCs must be sourced effectively to enable production of large numbers of functional cells. In vitro, SMCs divide slowly and demonstrate short proliferative lifespans compared with other types of cells, including stem cells and fibroblasts, limiting the number of cells that can be derived from expansion in culture of a primary isolation.

Fabrication and characterization of electrospun polycaprolactone and gelatin composite cuffs for tissue engineered blood vessels.

Sewing cuffs incorporated within tissue-engineered blood vessels (TEBVs) enable graft anastomosis in vivo, and secure TEBVs to bioreactors in vitro. Alternative approaches to cuff design are required to achieve cuff integration with scaffold-free TEBVs during tissue maturation. To create porous materials that promote tissue integration, we used electrospinning to fabricate cuffs from polycaprolactone (PCL), PCL blended with gelatin, and PCL coated with gelatin, and evaluated cuff mechanical properties, porosity, and cellular attachment and infiltration.

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds.

Despite significant advances in the fabrication of bioengineered scaffolds for tissue engineering, delivery of nutrients in complex engineered human tissues remains a challenge. By taking advantage of the similarities in the vascular structure of plant and animal tissues, we developed decellularized plant tissue as a prevascularized scaffold for tissue engineering applications. Perfusion-based decellularization was modified for different plant species, providing different geometries of scaffolding.