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. The course covers the basic cell and systems physiology underlying cell therapy products, in addition to explaining end-to-end manufacturing from cell acquisition through to patient treatment, industrialization, and regulatory processes. As of September 2023, >10,000 learners have enrolled in the course, and >90% of respondents to the course exit survey indicated that they were "very likely" or "likely" to recommend the course to a peer. In this article, we discuss our experience in the collaborative design and implementation of the online course as well as lessons learned from quantitative and qualitative student feedback. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.NEW & NOTEWORTHY We assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.

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/cm2 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. This release rate could be modulated by adjusting the adsorption concentration, decreasing to as low as 2 ng/cm2 total release over 7 days using a 12.5 µg/mL FGF2 solution. The surface energy and roughness could also be regulated using the adsorbed PEM. These properties were found to be substrate- and first-layer-dependent, supporting current theories of PEM assembly. When released, FGF2 from the PEMs was found to significantly enhance fibroblast proliferation as compared to culture conditions without FGF2. The results showed that growth factor release profiles and surface properties are easily controllable through modification of the PEM assembly steps and that these strategies can be effectively applied to common cell culture surfaces to control the cell fate.

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. In this study, we sought to determine the sequence-, structure-, and concentration-dependent properties underlying these differences in bioactivity. Molecular dynamics (MD) simulations allowed visualization of the structure of each peptide and calculation of residue-level helicity, revealing that residues within the CBD domains were not helical. Circular dichroism (CD) spectroscopy affirmed that the overall structures of LL37 and each CBD-LL37 peptide was primarily helical (greater than 67%) in a membrane-like solvent. Quartz crystal microbalance with dissipation (QCM-D) and imaging of fluorescent bilayers revealed unique, concentration-dependent interactions of each peptide with bilayers of different lipid compositions. Specifically, fCBD-LL37, which is less cytotoxic than LL37 and cCBD-LL37, demonstrated higher affinity toward anionic bilayers (model bacterial cell membranes) than zwitterionic bilayers (model mammalian cell membranes). In contrast, cCBD-LL37 and LL37 demonstrated similar affinities to both types of bilayers. This study demonstrates that the combination of MD, CD, and QCM-D may enable predictive modeling of the effects of primary sequence alterations on peptide secondary structure and membrane interactions. Understanding the structural and mechanistic properties of AMPs and their interactions with specific lipid bilayer compositions may enable the engineering of less cytotoxic AMPs with improved therapeutic indexes for human wound healing applications.

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 (210µm) to 30 G (159µm) for cell-only bioink dispensing would reduce tissue wall thickness and improve the consistency of tissue dimensions while maintaining cell viability. Printing with 30 G tips resulted in decreased mean wall thickness (318.6µm) without compromising mean cell viability (94.8%). Histological analysis of cell-only smooth muscle tissues cultured for 14 d in OMA support baths exhibited decreased wall thickness using 30 G dispensing tips, which correlated with increased collagen deposition and alignment. In addition, a TUNEL assay indicated a decrease in cell death in tissues printed with thinner (30 G) dispensing tips. Mechanical testing demonstrated that tissues printed with a 30 G dispensing tip exhibit an increase in ultimate tensile strength compared to those printed with a 27 G dispensing tip. Overall, these findings highlight the importance of precise control over bioprinting parameters to generate mechanically robust tissues when using cell-only bioinks dispensed and cultured within hydrogel support baths. The ability to control print dimensions using cell-only bioinks may enable bioprinting of more complex soft tissue geometries to generatein vitrotissue models.

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%. Co-seeded EC and hMSC rings were cultured in SMC differentiation medium for a total of 22 days. Tissue rings were then harvested for histology, Western blotting, wire myography, and uniaxial tensile testing to examine their structural and functional properties. Differentiated hMSC tissue rings comprising 20% and 30% ECs exhibited significantly greater SMC contractile protein expression, endothelin-1 (ET-1)-meditated contraction, and force at failure compared with the 0% EC rings. On average, the 0%, 10%, 20%, and 30% EC rings exhibited a contractile force of 0.745 ± 0.117, 0.830 ± 0.358, 1.31 ± 0.353, and 1.67 ± 0.351 mN (mean ± standard deviation [SD]) in response to ET-1, respectively. Additionally, the mean maximum force at failure for the 0%, 10%, 20%, and 30% EC rings was 88.5 ± 36. , 121 ± 59.1, 147 ± 43.1, and 206 ± 0.8 mN (mean ± SD), respectively. Based on these results, 30% EC rings were fused together to form tissue-engineered blood vessels (TEBVs) and compared with 0% EC TEBV controls. The addition of 30% ECs in TEBVs did not affect ring fusion but did result in significantly greater SMC protein expression (calponin and smoothelin). In summary, co-seeding hMSCs with ECs to form tissue rings resulted in greater contraction, strength, and hMSC-SMC differentiation compared with hMSCs alone and indicates a method to create a functional 3D human vascular cell coculture model.

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. However, the binding mechanism of cCBD-LL37 to collagen remained to be elucidated. In this study, we found that cCBD-LL37 showed a slightly higher affinity for collagen compared to LL37. Our results indicated that cCBD inhibited cCBD-LL37 binding to collagen but did not fully eliminate the binding. This suggests that cCBD-LL37 binding to collagen may involve more than just one-site-specific binding through the collagen-binding domain, with non-specific interactions also playing a role. Electrostatic studies revealed that both LL37 and cCBD-LL37 interact with collagen via long-range electrostatic forces, initiating low-affinity binding that transitions to close-range or hydrophobic interactions. Circular dichroism analysis showed that cCBD-LL37 exhibited enhanced structural stability compared to LL37 under varying ionic strengths and pH conditions, implying potential improvements in antimicrobial activity. Moreover, we demonstrated that the release of LL37 and cCBD-LL37 into the surrounding medium was influenced by the electrostatic environment, but cCBD could enhance the retention of peptide on collagen scaffolds. Collectively, these results provide important insights into cCBD-modified AMP-binding mechanisms and suggest that the addition of cCBD may enhance peptide structural stability and retention under varying electrostatic conditions.

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. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

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. In this study, we used quartz crystal microbalance with dissipation monitoring (QCM-D), immunohistochemistry (IHC), and atomic force microscopy (AFM) to establish and characterize an adsorbed layer of type I collagen on the QCM-D sensor and quantify peptide-collagen binding. A collagen deposition protocol was successfully established by measuring concentration-dependent deposition of collagen in QCM-D, and collagen self-assembly was observed by IHC and AFM. Hydrophobicity is known to affect the behavior of collagen adsorption. Therefore, we compared the deposition of collagen on hydrophilic SiO2-coated sensors vs. hydrophobic polystyrene (PS)-coated sensors via QCM-D, and found that the hydrophobic surface yielded more collagen adsorption, which suggested that hydrophobic surfaces are preferable for collagen layer establishment. There was no significant difference between LL37 and cCBD-LL37 binding with collagen, but the cCBD-LL37 showed better retention on the collagen after washing with PBS, indicating that there is an advantage to using cCBD as an anchoring unit to collagen. Collectively, these results provide important information on cCBD-mediated AMP-binding mechanisms and establish an effective method for quantifying peptide-collagen binding.

Engineered vascular tissue fabricated from aggregated smooth muscle cells.

The goal of this study was to develop a system to rapidly generate engineered tissue constructs from aggregated cells and cell-derived extracellular matrix (ECM) to enable evaluation of cell-derived tissue structure and function. Rat aortic smooth muscle cells seeded into annular agarose wells (2, 4 or 6 mm inside diameter) aggregated and formed thick tissue rings within 2 weeks of static culture (0.76 mm at 8 days; 0.94 mm at 14 days). Overall, cells appeared healthy and surrounded by ECM comprised of glycosoaminoglycans and collagen, although signs of necrosis were observed near the centers of the thickest rings. Tissue ring strength and stiffness values were superior to those reported for engineered tissue constructs cultured for comparable times. The strength (100-500 kPa) and modulus (0.5-2 MPa) of tissue rings increased with ring size and decreased with culture duration. Finally, tissue rings cultured for 7 days on silicone mandrels fused to form tubular constructs. Ring margins were visible after 7 days, but tubes were cohesive and mechanically stable, and histological examination confirmed fusion between ring subunits. This unique system provides a versatile new tool for optimization and functional assessment of cell-derived tissue, and a new approach to creating tissue-engineered vascular grafts.

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. Here, we hypothesized that localized presentation of transforming growth factor (TGF)-ß1 and bone morphogenetic protein (BMP)-2 to engineered hMSC tubes mimicking femoral diaphyses induces endochondral ossification, and that TGF-ß1 + BMP-2-presenting hMSC tubes enhance defect healing with delayed in vivo loading vs. loosely packed hMSC sheets. Localized morphogen presentation stimulated chondrogenic priming/endochondral differentiation in vitro. Subcutaneously, hMSC tubes formed cartilage templates that underwent bony remodeling. Orthotopically, hMSC tubes stimulated more robust endochondral defect healing vs. hMSC sheets. Tissue resembling normal growth plate was observed with negligible ectopic bone. This study demonstrates interactions between hMSC condensation geometry, morphogen bioavailability, and mechanical cues to recapitulate development for biomimetic bone tissue engineering.

Differentiation of cardiomyocytes from human embryonic stem cells is accompanied by changes in the extracellular matrix production of versican and hyaluronan.

Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N- and O-linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2, and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS-1 and HAS-2 mRNA in cardiomyocyte cultures, with HAS-2 most abundant. Interestingly, HAS-3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process.

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. In previous work, we modified the human AMP LL37 with collagen-binding domains from collagenase (cCBD) or fibronectin (fCBD) to facilitate peptide tethering and delivery from collagen-based wound dressings. We found that cCBD-LL37 and fCBD-LL37 were retained and active when bound to 100% collagen scaffolds. Collagen wound dressings are commonly made as composites with other materials, such as alginate. The goal of this study was to investigate how the presence of alginate affects the tethering, release, and antimicrobial activity of LL37 and CBD-LL37 peptides adsorbed to commercially available collagen-alginate wound dressings (FIBRACOL Plus-a 90% collagen and 10% alginate wound dressing). We found that over 85% of the LL37, cCBD-LL37, and fCBD-LL37 was retained on FIBRACOL Plus over a 14-day release study (90.3, 85.8, and 98.6%, respectively). Additionally, FIBRACOL Plus samples loaded with peptides were bactericidal toward Pseudomonas aeruginosa, even after 14 days in release buffer but demonstrated no antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. The presence of alginate in solution induced conformational changes in the cCBD-LL37 and LL37 peptides, resulting in increased peptide helicity, and reduced antimicrobial activity against P. aeruginosa. Peptide-loaded FIBRACOL Plus scaffolds were not cytotoxic to human dermal fibroblasts. This study demonstrates that CBD-mediated LL37 tethering is a viable strategy to reduce LL37 toxicity, and how substrate composition plays a crucial role in modulating the antimicrobial activity of tethered AMPs.

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. The authors studied time- and concentration-dependent interactions of CBD-LL37 modified with collagenase (cCBD) and fibronectin (fCBD) CBDs, with zwitterionic and anionic supported lipid bilayers, in order to model mammalian erythrocytes and bacterial cells, respectively. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to characterize peptide-lipid interactions at concentrations in the immunomodulatory (0.5-1.0 µM), antimicrobial (1.0-5.0 µM), and cytotoxic (5.0-10.0 µM) ranges. Their prior work with zwitterionic membranes demonstrated that cCBD-LL37 formed transmembrane pores while fCBD-LL37 underwent surface adsorption. Our goal in this study is to better interpret these results, by investigating the data at a wider concentration range and for two types of lipids, and by applying the Voigt-Kelvin viscoelastic model to calculate thickness and density changes of the peptide-lipid films as a function of time and concentration, thus providing information to help build detailed mechanisms of peptide/bilayer interactions. For pore-forming cCBD-LL37 and unmodified LL37, they found that there was a relationship between layer thicknesses and pore formation, which was attributed to different peptide orientation changes influenced by bilayer charge prior to pore formation. Specifically, cCBD-LL37 at 0.5 and 1.0 µM demonstrated higher thicknesses on zwitterionic than anionic membranes, indicating that prior to insertion into zwitterionic membranes, it orients perpendicular to the surface, which was also consistent with the higher dissipation changes observed on zwitterionic membranes. fCBD-LL37 demonstrated a bilayer adsorption mechanism with a preference toward anionic lipids. Adsorption of fCBD-LL37 onto anionic lipids demonstrated a rapid first adsorption step that transitioned depending on the number of fCBD-LL37 molecules on the bilayer. For this peptide at higher concentrations, greater dissipation changes were observed than for fCBD-LL37 physically adsorbed onto surfaces without bilayers. This suggests that peptide-peptide interactions promoted by the fCBD domain dominated after saturation. The development of a structure-function relationship for cCBD-LL37 and fCBD-LL37 demonstrates promise for using QCM-D predictions to inform the rational design of novel, antimicrobial, and noncytotoxic CBD-LL37 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. In addition, Raman spectra and images of tissue-engineered SMC constructs were acquired. Routine techniques such as qPCR, histochemistry, histological and immunocytological staining were performed for comparative gene and protein expression analysis. We identified that SMCs of different tissue origins exhibited unique spectral information that allowed a separation of all groups of origin by multivariate data analysis (MVA). We were further able to non-invasively monitor phenotypic switching in cultured SMCs and assess the impact of different culture conditions on extracellular matrix remodeling in the tissue-engineered ring constructs. Interestingly, we identified that the Raman signature of the human SMC-based ring constructs was similar to the one obtained from native aortic tissue. We conclude that Raman microspectroscopic methods are promising tools to characterize cells and define cellular and extracellular matrix components on a molecular level. In this study, in situ measurements were marker-independent, fast, and identified cellular differences that were not detectable by established routine techniques. Perspectively, Raman microspectroscopy and MVA in combination with artificial intelligence can be suitable for automated quality monitoring of (stem) cell and cell-based tissue engineering products. STATEMENT OF SIGNIFICANCE: The accessibility of autologous blood vessels for surgery is limited. Tissue engineering (TE) aims to develop functional vascular replacements; however, no commercially available TE vascular graft (TEVG) exists to date. One limiting factor is the availability of a well-characterized and safe cell source. Smooth muscle cells (SMCs) are generally used for TEVGs. To engineer a TEVG, proliferating SMCs of the synthesizing phenotype are essential, whereas functional, sustainable TEVGs require SMCs of the contractile phenotype. SMC diversity and plasticity are therefore limiting factors, also for their quality monitoring and application in TE. In this study, Raman microspectroscopy and imaging combined with machine learning tools allowed the non-destructive, marker-independent characterization of SMCs, smooth muscle tissues and TE SMC-constructs. The spectral information was specific enough to distinguish for the first time the phenotypic switching in SMCs in real-time, and monitor the impact of culture conditions on ECM remodeling in the TE SMC-constructs.

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

IMPACT STATEMENT: 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. Here, we presented a method for automatically stacking self-assembled smooth muscle cell rings onto mandrels, using a custom-designed well plate and robotic punch system. Rings then fuse into tissue-engineered blood vessels (TEBVs). This is a critical step toward automating TEBV production that may be applied to other tubular tissues as well.

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. To this end, we studied concentration- and time-dependent interactions of LL37 with zwitterionic model phosphatidylcholine (PC) bilayers with quartz crystal microbalance with dissipation (QCM-D). LL37 mass adsorption and PC bilayer viscoelasticity changes were monitored by measuring changes in frequency (Δf) and dissipation (ΔD), respectively. The Voigt-Kelvin viscoelastic model was applied to Δf and ΔD to study changes in bilayer thickness and density with LL37 concentration. At low concentrations (0.10-1.00 µM), LL37 adsorbed onto bilayers in a concentration-dependent manner. Further analyses of Δf, ΔD and thickness revealed that peptide saturation on the bilayers was a threshold for interactions observed above 2.00 µM, interactions that were rapid, multi-step, and reached equilibrium in a concentration- and time-dependent manner. Based on these data, we proposed a model of stable transmembrane pore formation at 2.00-10.0 µM, or transition from a primarily lipid to a primarily protein film with a transmembrane pore formation intermediate state at concentrations of LL37 > 10 µM. The concentration-dependent interactions between LL37 and PC bilayers correlated with the observed concentration-dependent biological activities of LL37 (antimicrobial, immunomodulatory and non-cytotoxic at 0.1-1.0 µM, hemolytic and some cytotoxicity at 2.0-13 µM and cytotoxic at >13 µM).

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). Bioactive molecules capable of modulating host tissue responses and preventing these complications have been identified, but systemic delivery is often harmful or ineffective. This review discusses the use of localized bioactive molecule delivery methods to enhance the long-term success of vascular interventions, such as drug-eluting stents and aneurysm coils, as well as nanoparticles for targeted molecule delivery. Vascular grafts in particular have poor patency in small diameter, high flow applications, such as coronary artery bypass grafting (CABG). Grafts fabricated from a variety of approaches may benefit from bioactive molecule incorporation to improve patency. Tissue engineering is an especially promising approach for vascular graft fabrication that may be conducive to incorporation of drugs or growth factors. Overall, localized and targeted delivery of bioactive molecules has shown promise for improving the outcomes of vascular interventions, with technologies such as drug-eluting stents showing excellent clinical success. However, many targeted vascular drug delivery systems have yet to reach the clinic. There is still a need to better optimize bioactive molecule release kinetics and identify synergistic biomolecule combinations before the clinical impact of these technologies can be realized.

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. Gelatin blending significantly decreased cuff ultimate tensile stress and failure strain over PCL alone, but no significant differences were observed in elastic modulus or failure load. Interestingly, gelatin incorporation by blending or coating did not produce significant differences in cellular attachment or pore size. We then created tissue tubes by fusing self-assembled smooth muscle cell rings together with electrospun cuffs on either end. After 7 days, rings and cuffs fused seamlessly, and the resulting tubes were harvested for pull-to-failure tests to measure the strength of cuff-tissue integration. Tubes with gelatin-coated PCL cuffs failed more frequently at the cuff-tissue interface compared to PCL and PCL:gelatin blended groups. This work demonstrates that electrospun cuffs integrated successfully with scaffold-free TEBVs, and that the addition of gelatin did not significantly improve cuff integration over PCL alone for this application. Electrospun cuffs may aid cannulation for dynamic culture and testing of tubular constructs during engineered tissue maturation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 817-826, 2018.