Manufacturing and validation of small-diameter vascular grafts: A mini review.

The field of small-diameter vascular grafts remains a challenge for biomaterials scientists. While decades of research have brought us much closer to developing biomimetic materials for regenerating tissues and organs, the physiological challenges involved in manufacturing small conduits that can transport blood while not inducing an immune response or promoting blood clots continue to limit progress in this area. In this short review, we present some of the most recent methods and advancements made by researchers working in the field of small-diameter vascular grafts. We also discuss some of the most critical aspects biomaterials scientists should consider when developing lab-made small-diameter vascular grafts.

BEaTS-ß: an open-source electromechanical bioreactor for simulating human cardiac disease conditions.

Heart disease remains the leading cause of worldwide mortality. Although the last decades have broadened our understanding of the biology behind the pathologies of heart disease, ex vivo systems capable of mimicking disease progression and abnormal heart function using human cells remain elusive. In this contribution, an open-access electromechanical system (BEaTS-ß) capable of mimicking the environment of cardiac disease is reported. BEaTS-ß was designed using computer-aided modeling to combine tunable electrical stimulation and mechanical deformation of cells cultured on a flexible elastomer. To recapitulate the clinical scenario of a heart attack more closely, in designing BEaTS-ß we considered a device capable to operate under hypoxic conditions. We tested human induced pluripotent stem cell-derived cardiomyocytes, fibroblasts, and coronary artery endothelial cells in our simulated myocardial infarction environment. Our results indicate that, under simulated myocardium infarction, there was a decrease in maturation of cardiomyocytes, and reduced survival of fibroblasts and coronary artery endothelial cells. The open access nature of BEaTS-ß will allow for other investigators to use this platform to investigate cardiac cell biology or drug therapeutic efficacy in vitro under conditions that simulate arrhythmia and/or myocardial infarction.

Development of in situ bioprinting: A mini review.

Bioprinting has rapidly progressed over the past decade. One branch of bioprinting known as in situ bioprinting has benefitted considerably from innovations in biofabrication. Unlike ex situ bioprinting, in situ bioprinting allows for biomaterials to be printed directly into or onto the target tissue/organ, eliminating the need to transfer pre-made three-dimensional constructs. In this mini-review, recent progress on in situ bioprinting, including bioink composition, in situ crosslinking strategies, and bioprinter functionality are examined. Future directions of in situ bioprinting are also discussed including the use of minimally invasive bioprinters to print tissues within the body.

Characterization of the Monocyte Response to Biomaterial Therapy for Cardiac Repair.

Biomaterials are scaffolds designed to mimic the extracellular matrix and stimulate tissue repair. Biomaterial therapies have shown promise for improving wound healing in cardiac tissue after ischemic injury. An unintentional consequence of biomaterial delivery may be the stimulation of inflammation through recruitment of circulating monocytes into the tissue. Monocytes are a type of leukocyte (white blood cell) that play a critical role in pathogen recognition, phagocytosis of foreign material, and presentation of antigens to initiate an adaptive immune response. An increase in the pro-inflammatory subset of monocytes, marked by Ly6C antigen expression, in response to biomaterials can lead to rapid material degradation, ineffective treatment, and worsening of tissue injury. Flow cytometry is a leading method for screening the recruitment of monocytes to the heart in response to biomaterial injection. Here, we describe the isolation of leukocytes from the heart, blood, and spleen of mice treated with a biomaterial post-myocardial infarction and describe a flow cytometry protocol used to quantify the levels of major leukocyte subtypes, including Ly6C+ inflammatory monocytes.

Nanoengineered Sprayable Therapy for Treating Myocardial Infarction.

We report the development, as well as the in vitro and in vivo testing, of a sprayable nanotherapeutic that uses surface engineered custom-designed multiarmed peptide grafted nanogold for on-the-spot coating of an infarcted myocardial surface. When applied to mouse hearts, 1 week after infarction, the spray-on treatment resulted in an increase in cardiac function (2.4-fold), muscle contractility, and myocardial electrical conductivity. The applied nanogold remained at the treatment site 28 days postapplication with no off-target organ infiltration. Further, the infarct size in the mice that received treatment was found to be <10% of the total left ventricle area, while the number of blood vessels, prohealing macrophages, and cardiomyocytes increased to levels comparable to that of a healthy animal. Our cumulative data suggest that the therapeutic action of our spray-on nanotherapeutic is highly effective, and in practice, its application is simpler than other regenerative approaches for treating an infarcted heart.

3D Bioprinted Cardiac Tissues and Devices for Tissue Maturation.

Cardiovascular diseases are the leading cause of mortality worldwide. Given the limited endogenous regenerative capabilities of cardiac tissue, patient-specific anatomy, challenges in treatment options, and shortage of donor tissues for transplantation, there is an urgent need for novel approaches in cardiac tissue repair. 3D bioprinting is a technology based on additive manufacturing which allows for the design of precisely controlled and spatially organized structures, which could possibly lead to solutions in cardiac tissue repair. In this review, we describe the basic morphological and physiological specifics of the heart and cardiac tissues and introduce the readers to the fundamental principles underlying 3D printing technology and some of the materials/approaches which have been used to date for cardiac repair. By summarizing recent progress in 3D printing of cardiac tissue and valves with respect to the key features of cardiovascular tissue (such as contractility, conductivity, and vascularization), we highlight how 3D printing can facilitate surgical planning and provide custom-fit implants and properties that match those from the native heart. Finally, we also discuss the suitability of this technology in the design and fabrication of custom-made devices intended for the maturation of the cardiac tissue, a process that has been shown to increase the viability of implants. Altogether this review shows that 3D printing and bioprinting are versatile and highly modulative technologies with wide applications in cardiac regeneration and beyond.

Evaluation of Therapeutic Collagen-Based Biomaterials in the Infarcted Mouse Heart by Extracellular Matrix Targeted MALDI Imaging Mass Spectrometry.

The goal of this study was to develop strategies to localize human collagen-based hydrogels within an infarcted mouse heart, as well as analyze its impact on endogenous extracellular matrix (ECM) remodeling. Collagen is a natural polymer that is abundantly used in bioengineered hydrogels because of its biocompatibility, cell permeability, and biodegradability. However, without the use of tagging techniques, collagen peptides derived from hydrogels can be difficult to differentiate from the endogenous ECM within tissues. Imaging mass spectrometry is a robust tool capable of visualizing synthetic and natural polymeric molecular structures yet is largely underutilized in the field of biomaterials outside of surface characterization. In this study, our group leveraged a recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) technique to enzymatically target collagen and other ECM peptides within the tissue microenvironment that are both endogenous and hydrogel-derived. Using a multimodal approach of fluorescence microscopy and ECM-IMS techniques, we were able to visualize and relatively quantify significantly abundant collagen peptides in an infarcted mouse heart that were localized to regions of therapeutic hydrogel injection sites. On-tissue MALDI MS/MS was used to putatively identify sites of collagen peptide hydroxyproline site occupancy, a post-translational modification that is critical in collagen triple helical stability. Additionally, the technique could putatively identify over 35 endogenously expressed ECM peptides that were expressed in hydrogel-injected mouse hearts. Our findings show evidence for the use of MALDI-IMS in assessing the therapeutic application of collagen-based biomaterials.

Cardiac Sympathetic Positron Emission Tomography Imaging with Meta-[18F]Fluorobenzylguanidine is Sensitive to Uptake-1 in Rats.

Dysfunction of the cardiac sympathetic nervous system contributes to the development of cardiovascular diseases including ischemia, heart failure, and arrhythmias. Molecular imaging probes such as meta-[123I]iodobenzylguanidine have demonstrated the utility of assessing neuronal integrity by targeting norepinephrine transporter (NET, uptake-1). However, current radiotracers can report only on innervation due to suboptimal kinetics and lack sensitivity to NET in rodents, precluding mechanistic studies in these species. The objective of this work was to characterize myocardial sympathetic neuronal uptake mechanisms and kinetics of the positron emission tomography (PET) radiotracer meta-[18F]fluorobenzylguanidine ([18F]mFBG) in rats. Automated synthesis using spirocyclic iodonium(III) ylide radiofluorination produces [18F]mFBG in 24 ± 1% isolated radiochemical yield and 30-95 GBq/µmol molar activity. PET imaging in healthy rats delineated the left ventricle, with monoexponential washout kinetics (kmono = 0.027 ± 0.0026 min-1, Amono = 3.08 ± 0.33 SUV). Ex vivo biodistribution studies revealed tracer retention in the myocardium, while pharmacological treatment with selective NET inhibitor desipramine, nonselective neuronal and extraneuronal uptake-2 inhibitor phenoxybenzamine, and neuronal ablation with neurotoxin 6-hydroxydopamine reduced myocardial retention by 33, 76, and 36%, respectively. Clearance of [18F]mFBG from the myocardium was unaffected by treatment with uptake-1 and uptake-2 inhibitors following peak myocardial activity. These results suggest that myocardial distribution of [18F]mFBG in rats is dependent on both NET and extraneuronal transporters and that limited reuptake to the myocardium occurs. [18F]mFBG may therefore prove useful for imaging intraneuronal dysfunction in small animals.

Building new cardiac vasculature and myocardium: where are we at?

PURPOSE OF REVIEW: This review describes the latest advances in cell therapy, biomaterials and 3D bioprinting for the treatment of cardiovascular disease. RECENT FINDINGS: Cell therapies offer the greatest benefit for patients suffering from chronic ischemic and nonischemic cardiomyopathy. Rather than replacing lost cardiomyocytes, the effects of most cell therapies are mediated by paracrine signalling, mainly through the induction of angiogenesis and immunomodulation. Cell preconditioning, or genetic modifications are being studied to improve the outcomes. Biomaterials offer stand-alone benefits such as bioactive cues for cell survival, proliferation and differentiation, induction of vascularization or prevention of further cardiomyocyte death. They also provide mechanical support or electroconductivity, and can be used to deliver cells, growth factors or drugs to the injured site. Apart from classical biomaterial manufacturing techniques, 3D bioprinting offers greater spatial control over biomaterial deposition and higher resolution of the details, including hollow vessel-like structures. SUMMARY: Cell therapy induces mainly angiogenesis and immunomodulation. The ability to induce direct cardiomyocyte regeneration to replace the lost cardiomyocytes is, however, still missing until embryonic or induced pluripotent stem cell use becomes available. Cell therapy would benefit from combinatorial use with biomaterials, as these can prolong cell retention and survival, offer additional mechanical support and provide inherent bioactive cues. Biomaterials can also be used to deliver growth factors, drugs, and other molecules. 3D bioprinting is a high-resolution technique that has great potential in cardiac therapy.

Mimicking biofilm formation and development: Recent progress in in vitro and in vivo biofilm models.

Biofilm formation in living organisms is associated to tissue and implant infections, and it has also been linked to the contribution of antibiotic resistance. Thus, understanding biofilm development and being able to mimic such processes is vital for the successful development of antibiofilm treatments and therapies. Several decades of research have contributed to building the foundation for developing in vitro and in vivo biofilm models. However, no such thing as an "all fit" in vitro or in vivo biofilm models is currently available. In this review, in addition to presenting an updated overview of biofilm formation, we critically revise recent approaches for the improvement of in vitro and in vivo biofilm models.

A low cost and open access system for rapid synthesis of large volumes of gold and silver nanoparticles.

Rapid synthesis of nanomaterials in scalable quantities is critical for accelerating the discovery and commercial translation of nanoscale-based technologies. The synthesis of metal nanogold and silver in volumes larger than 100 mL is not automatized and might require of the use of harsh conditions that in most cases is detrimental for the production of nanoparticles with reproducible size distributions. In this work, we present the development and optimization of an open-access low-cost NanoParticle Flow Synthesis System (NPFloSS) that allows for the rapid preparation of volumes of up to 1 L of gold and silver nanoparticle aqueous solutions.

BEaTS-α an open access 3D printed device for in vitro electromechanical stimulation of human induced pluripotent stem cells.

3D printing was used to develop an open access device capable of simultaneous electrical and mechanical stimulation of human induced pluripotent stem cells in 6-well plates. The device was designed using Computer-Aided Design (CAD) and 3D printed with autoclavable, FDA-approved materials. The compact design of the device and materials selection allows for its use inside cell incubators working at high humidity without the risk of overheating or corrosion. Mechanical stimulation of cells was carried out through the cyclic deflection of flexible, translucent silicone membranes by means of a vacuum-controlled, open-access device. A rhythmic stimulation cycle was programmed to create a more physiologically relevant in vitro model. This mechanical stimulation was coupled and synchronized with in situ electrical stimuli. We assessed the capabilities of our device to support cardiac myocytes derived from human induced pluripotent stem cells, confirming that cells cultured under electromechanical stimulation presented a defined/mature cardiomyocyte phenotype. This 3D printed device provides a unique high-throughput in vitro system that combines both mechanical and electrical stimulation, and as such, we foresee it finding applications in the study of any electrically responsive tissue such as muscles and nerves.

Mybl2 rejuvenates heart explant-derived cells from aged donors after myocardial infarction.

While cell therapy is emerging as a promising option for patients with ischemic cardiomyopathy (ICM), the influence of advanced donor age and a history of ischemic injury on the reparative performance of these cells are not well defined. As such, intrinsic changes that result from advanced donor age and ischemia are explored in hopes of identifying a molecular candidate capable of restoring the lost reparative potency of heart explant-derived cells (EDCs) used in cell therapy. EDCs were cultured from myocardial biopsies obtained from young or old mice 4 weeks after randomization to experimental myocardial infarction or no intervention. Advanced donor age reduces cell yield while increasing cell senescence and the secretion of senescence-associated cytokines. A history of ischemic injury magnifies these effects as cells are more senescent and have lower antioxidant reserves. Consistent with these effects, intramyocardial injection of EDCs from aged ischemic donors provided less cell-mediated cardiac repair. A transcriptome comparison of ICM EDCs shows aging modifies many of the pathways responsible for effective cell cycle control and DNA damage/repair. Over-expression of the barely explored antisenescent transcription factor, Mybl2, in EDCs from aged ICM donors reduces cell senescence while conferring salutary effects on antioxidant activity and paracrine production. In vivo, we observed an increase in cell retention and vasculogenesis after treatment with Mybl2-over-expressing EDCs which improved heart function in infarcted recipient hearts. In conclusion, Mybl2 over-expression rejuvenates senescent EDCs sourced from aged ICM donors to confer cell-mediated effects comparable to cells from young nonischemic donors.

The Progression of Regenerative Medicine and its Impact on Therapy Translation.

Despite regenerative medicine (RM) being one of the hottest topics in biotechnology for the past 3 decades, it is generally acknowledged that the field's performance at the bedside has been somewhat disappointing. This may be linked to the novelty of these technologies and their disruptive nature, which has brought an increasing level of complexity to translation. Therefore, we look at how the historical development of the RM field has changed the translational strategy. Specifically, we explore how the pursuit of such novel regenerative therapies has changed the way experts aim to translate their ideas into clinical applications, and then identify areas that need to be corrected or reinforced in order for these therapies to eventually be incorporated into the standard-of-care. This is then linked to a discussion of the preclinical and postclinical challenges remaining today, which offer insights that can contribute to the future progression of RM.

Delivering More of an Injectable Human Recombinant Collagen III Hydrogel Does Not Improve Its Therapeutic Efficacy for Treating Myocardial Infarction.

Injectable hydrogels are a promising method to enhance repair in the heart after myocardial infarction (MI). However, few studies have compared different strategies for the application of biomaterial treatments. In this study, we use a clinically relevant mouse MI model to assess the therapeutic efficacy of different treatment protocols for intramyocardial injection of a recombinant human collagen III (rHCIII) thermoresponsive hydrogel. Comparing a single hydrogel injection at an early time point (3 h) versus injections at multiple time points (3 h, 1 week, and 2 weeks) post-MI revealed that the single injection group led to superior cardiac function, reduced scar size and inflammation, and increased vascularization. Omitting the 3 h time point and delivering the hydrogel at 1 and 2 weeks post-MI led to poorer cardiac function. The positive effects of the single time point injection (3 h) on scar size and vascular density were lost when the hydrogel's collagen concentration was increased from 1% to 2%, and it did not confer any additional functional improvement. This study shows that early treatment with a rHCIII hydrogel can improve cardiac function post-MI but that injecting more rHCIII (by increased concentration or more over time) can reduce its efficacy, thus highlighting the importance of investigating optimal treatment strategies of biomaterial therapy for MI.

Collagen-Based Microcapsules As Therapeutic Materials for Stem Cell Therapies in Infarcted Myocardium.

As cell therapies emerged, it was quickly realized that pro-regenerative cells directly injected into injured tissue struggled within the inflammatory microenvironment. By using microencapsulation, i.e., encapsulating cells within polymeric biomaterials, they are henceforth protected from the harmful extracellular cues, while still being able to receive oxygen and nutrients and release secreted factors. Previous work showed that stem cells encapsulated within a biologically inert material (agarose) were able to significantly improve the function of the infarcted mouse heart. With the aim of using more bioresponsive microcapsules, we sought to develop an enzymatically degradable, type I collagen-based microcapsule for the intramyocardial delivery of bone marrow-derived mesenchymal stromal cells in a murine model of myocardial infarction.

Multifunctional Nano and Collagen-Based Therapeutic Materials for Skin Repair.

A novel strategy is needed for treating nonhealing wounds, which is able to simultaneously eradicate pathogenic bacteria and promote tissue regeneration. This would improve patient outcome and reduce the number of lower limb amputations. In this work, we present a multifunctional therapeutic approach able to control bacterial infections, provide a protective barrier to a full-thickness wound, and improve wound healing in a clinically relevant animal model. Our approach uses a nanoengineered antimicrobial nanoparticle for creating a sprayable layer onto the wound bed that prevents bacterial proliferation and also eradicates preformed biofilms. As a protective barrier for the wound, we developed a thermoresponsive collagen-based matrix that has prohealing properties and is able to fill wounds independent of their geometries. Our results indicate that using a combination of the matrix with full-thickness microscopic skin tissue columns synergistically contributed to faster and superior skin regeneration in a nonhealing wound model in diabetic mice.

Light-Activated Peptide-Based Materials for Sutureless Wound Closure.

Using chemically modified extracellular matrix proteins, such as collagen, in combination with light for tissue bonding reduces inflammation and minimizes scarring. However, full length animal or recombinant human collagen proteins are difficult to isolate/produce. Thus, short biomimetic collagen peptides with properties equivalent to collagen at both structural and functional levels may be ideal building blocks for the development of remotely triggered adhesives and fillers. In this work, the conjugation of self-assembling collagen-like peptides to acrylate functionalized polyethylene glycol units yielded adhesive filler materials activated by visible light through the incorporation of a photosensitizer. When tested in a murine skin wound model, the photoactivated adhesives showed reduced scar formation and promoted epithelial regeneration.

Injectable human recombinant collagen matrices limit adverse remodeling and improve cardiac function after myocardial infarction.

Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium's mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.