Effects of a bioengineered allogeneic cellular construct on burn-related macrophage phenotype.

Bioengineered allogeneic cellularised constructs (BACC) exert pro-healing effects in burn wounds and skew macrophage phenotype towards a predominately reparative phenotype. However, whether BACC can modulate the phenotype of dysregulated macrophages, like those present in burn wounds, is not known. To better understand the macrophage modulatory characteristics of the BACC, primary human macrophages were polarised to the M2b phenotype, an immunosuppressive phenotype relevant to burn wounds, by simultaneously exposing macrophages to polystyrene plate-coated immunoglobulin G and the endotoxin lipopolysaccharide (LPS). The resulting macrophage phenotype upregulated both inflammatory and reparative genes, and increased secretion of the M2b marker CCL1 compared to five different in vitro macrophage phenotypes. M2b macrophages were cultured with the BACC in the presence or absence of LPS to mimic infection, which is a common occurrence in burn wounds. The BACC caused up-regulation of reparative gene sets and down-regulation of pro-inflammatory gene sets, even when LPS was present in the cell culture media. Co-cultures were maintained for 1, 3, or 5 days in the presence of LPS, and by day 1 both non-activated macrophages and M2b macrophages exhibited signs of endotoxin tolerance, as demonstrated by a reduced secretion of tumour necrosis factor α (TNFα) in response to fresh LPS stimulus. The BACC was not able to prevent endotoxin tolerance, but reparative genes were upregulated in macrophages chronically exposed to LPS. These results suggest that the BACC can promote a reparative phenotype in dysregulated macrophages relevant to the pathophysiology of burns.

Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks.

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-ß signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

Biopolymer-Based Microcarriers for Three-Dimensional Cell Culture and Engineered Tissue Formation.

The concept of three-dimensional (3D) cell culture has been proposed to maintain cellular morphology and function as in vivo. Among different approaches for 3D cell culture, microcarrier technology provides a promising tool for cell adhesion, proliferation, and cellular interactions in 3D space mimicking the in vivo microenvironment. In particular, microcarriers based on biopolymers have been widely investigated because of their superior biocompatibility and biodegradability. Moreover, through bottom-up assembly, microcarriers have opened a bright door for fabricating engineered tissues, which is one of the cutting-edge topics in tissue engineering and regeneration medicine. This review takes an in-depth look into the recent advancements of microcarriers based on biopolymers-especially polysaccharides such as chitosan, chitin, cellulose, hyaluronic acid, alginate, and laminarin-for 3D cell culture and the fabrication of engineered tissues based on them. The current limitations and potential strategies were also discussed to shed some light on future directions.

Morphogenesis of 3D vascular networks is regulated by tensile forces.

Understanding the forces controlling vascular network properties and morphology can enhance in vitro tissue vascularization and graft integration prospects. This work assessed the effect of uniaxial cell-induced and externally applied tensile forces on the morphology of vascular networks formed within fibroblast and endothelial cell-embedded 3D polymeric constructs. Force intensity correlated with network quality, as verified by inhibition of force and of angiogenesis-related regulators. Tensile forces during vessel formation resulted in parallel vessel orientation under static stretching and diagonal orientation under cyclic stretching, supported by angiogenic factors secreted in response to each stretch protocol. Implantation of scaffolds bearing network orientations matching those of host abdominal muscle tissue improved graft integration and the mechanical properties of the implantation site, a critical factor in repair of defects in this area. This study demonstrates the regulatory role of forces in angiogenesis and their capacities in vessel structure manipulation, which can be exploited to improve scaffolds for tissue repair.

Bioprinting Approaches to Engineering Vascularized 3D Cardiac Tissues.

PURPOSE OF REVIEW: 3D bioprinting technologies hold significant promise for the generation of engineered cardiac tissue and translational applications in medicine. To generate a clinically relevant sized tissue, the provisioning of a perfusable vascular network that provides nutrients to cells in the tissue is a major challenge. This review summarizes the recent vascularization strategies for engineering 3D cardiac tissues. RECENT FINDINGS: Considerable steps towards the generation of macroscopic sizes for engineered cardiac tissue with efficient vascular networks have been made within the past few years. Achieving a compact tissue with enough cardiomyocytes to provide functionality remains a challenging task. Achieving perfusion in engineered constructs with media that contain oxygen and nutrients at a clinically relevant tissue sizes remains the next frontier in tissue engineering. The provisioning of a functional vasculature is necessary for maintaining a high cell viability and functionality in engineered cardiac tissues. Several recent studies have shown the ability to generate tissues up to a centimeter scale with a perfusable vascular network. Future challenges include improving cell density and tissue size. This requires the close collaboration of a multidisciplinary teams of investigators to overcome complex challenges in order to achieve success.

Growing Human Parathyroids in a Microphysiological System: A Novel Approach to Understanding and Developing New Treatments for Hyperparathyroidism.

We developed a novel model for studying hyperparathyroidism by growing ex vivo 3-dimensional human parathyroids as part of a microphysiological system (MPS) that mimics human physiology. The purpose of this study was to validate the parathyroid portion of the MPS. We prospectively collected parathyroid tissue from 46 patients with hyperparathyroidism for growth into pseudoglands. We evaluated pseudogland architecture and calcium responsiveness. Following 2 weeks in culture, dispersed cells successfully coalesced into pseudoglands ∼500-700 µm in diameter that mimicked the appearance of normal parathyroid glands. Functionally, they also appeared similar to intact parathyroids in terms of organization and calcium-sensing receptor expression. Immunohistochemical staining for calcium-sensing receptor revealed 240-450/cell units of mean fluorescence intensity within the pseudoglands. Finally, the pseudoglands showed varying levels of calcium responsiveness, indicated by changes in parathyroid hormone (PTH) levels. In summary, we successfully piloted the development of a novel MPS for studying the effects of hyperparathyroidism on human organ systems. We are currently evaluating the effect of PTH on adverse remodeling of tissue engineered cardiac, skeletal, and bone tissue within the MPS.

Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts.

The generation of an off-the-shelf in vitro engineered living tissue graft will likely require cryopreservation. However, the efficient addition and removal of cryoprotective agents (CPA) to cells throughout the volume of a three-dimensional (3D) tissue graft remains a significant challenge. In this work, we assessed whether a perfusion bioreactor-based method could be used to improve the viability of cryopreserved mesenchymal stromal cell- (MSC) based tissue constructs as compared to using conventional diffusion-based methods. The bioreactor was first used to saturate 3D constructs with CPA under perfused flow. Following cryopreservation, the bioreactor was then also used for the efficient removal of the CPA from the 3D tissues. We demonstrate that addition and removal of CPA under perfused flow significantly increased the viability of MSC within cryopreserved 3D tissue constructs as compared to conventional diffusion-based methods.

Femtosecond laser-induced cell-cell surgical attachment.

BACKGROUND AND OBJECTIVE: Laser-induced cell-cell surgical attachment using femtosecond laser pulses is reported. STUDY DESIGN/MATERIALS AND METHODS: We have demonstrated the ability to attach single cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength delivered from a Ti:Sapphire laser. To check that the cells did not go through a cell-fusion process, a fluorescent dye Calcein AM was used to verify that the fluorescent dye did not migrate from a dyed cell to a non-dyed cell. The mechanical integrity of the attached joint was assessed using an optical tweezer. RESULTS: Attachment of cells was performed without the induction of cell-cell fusion, with attachment efficiency of 95%, and while preserving the cells' viability. Cell-cell attachment was achieved by delivery of one to two trains of femtosecond laser pulses lasting 15 ms each. CONCLUSIONS: Laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane. The inner cell membrane remained intact during the attachment procedure, and isolation of the cells' cytoplasm from the surrounding medium was obtained. A strong physical attachment between the cells was obtained due to the bonding of the membranes' ionized phospholipid molecules and the formation of a joint cellular membrane at the connection point. The cellular attachment technique, femtosecond laser-induced cell-cell surgical attachment, can potentially provide a platform for the creation of engineered tissue and cell cultures.

Hierarchically Vascularized and Suturable Tissue Constructs created through Angiogenesis from Tissue-Engineered Vascular Grafts.

A major roadblock in implementing engineered tissues clinically lies in their limited vascularization. After implantation, such tissues do not integrate with the host's circulation as quickly as needed, commonly resulting in loss of viability and functionality. This study presents a solution to the vascularization problem that could enable the survival and function of large, transplantable, and vascularized engineered tissues. The technique allows vascularization of a cell laden hydrogel through angiogenesis from a suturable tissue-engineered vascular graft (TEVG) constructed from electrospun polycaprolactone with macropores. The graft is surrounded by a layer of cell-laden gelatin-methacryloyl hydrogel. The constructs are suturable and possess mechanical properties like native vessels. Angiogenesis occurs through the pores in the graft, resulting in a hydrogel containing an extensive vascular network that is connected to an implantable TEVG. The size of the engineered tissue and the degree of vascularization can be increased by adding multiple TEVGs into a single construct. The engineered tissue has the potential to be immediately perfused by the patient's blood upon surgical anastomosis to host vessels, enabling survival of implanted cells. These findings provide a meaningful step to address the longstanding problem of fabricating suturable pre-vascularized tissues which could survive upon implantation in vivo. STATEMENT OF SIGNIFICANCE: Creating vascularized engineered tissues that can be transplanted and rapidly perfused by the host blood supply is a major challenge which has limited the clinical impact of tissue engineering. In this study we demonstrate a technique to fabricate vascularized tissue constructs via angiogenesis from a suturable tissue-engineered vascular graft. The macroporous graft is surrounded with hydrogel, allowing endothelial cells to migrate from the lumen and vascularize the hydrogel layer with capillary-like structures connected to the macrovessel. The graft has comparable mechanical properties to native blood vessels and larger constructs can be fabricated by incorporating multiple grafts. These constructs could potentially be connected surgically to the circulation at an implantation site to support their immediate perfusion and survival.

Vascular units as advanced living materials for bottom-up engineering of perfusable 3D microvascular networks.

The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regeneration, remaining a central challenge in tissue engineering. In this study, we present a novel (micro)vascularization strategy that explores the use of specialized "vascular units" (VUs) as building blocks to initiate blood vessel formation and create perfusable, stroma-embedded 3D microvascular networks from the bottom-up. We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels. This leads to the formation of VUs-derived capillaries, which fuse with adjacent capillaries to form stable microvascular beds within a supportive, extracellular matrix-rich fibroblastic microenvironment. Using a custom-designed biomimetic fibrin-based vessel-on-chip (VoC), we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused. Moreover, VUs can establish capillary bridges between channels, extending the microvascular network throughout the entire device. When VUs and intestinal organoids (IOs) are combined within the VoC, the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs. This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device. These findings underscore the remarkable potential of VUs as building blocks for engineering microvascular networks, with versatile applications spanning from regenerative medicine to advanced in vitro models.

Implantation of a double allogeneic human engineered tissue graft on damaged heart: insights from the PERISCOPE phase I clinical trial.

BACKGROUND: In preclinical studies, the use of double allogeneic grafts has shown promising results in promoting tissue revascularization, reducing infarct size, preventing adverse remodelling and fibrosis, and ultimately enhancing cardiac function. Building upon these findings, the safety of PeriCord, an engineered tissue graft consisting of a decellularised pericardial matrix and umbilical cord Wharton's jelly mesenchymal stromal cells, was evaluated in the PERISCOPE Phase I clinical trial (NCT03798353), marking its first application in human subjects. METHODS: This was a double-blind, single-centre trial that enrolled patients with non-acute myocardial infarction eligible for surgical revascularization. Seven patients were implanted with PeriCord while five served as controls. FINDINGS: Patients who received PeriCord showed no adverse effects during post-operative phase and one-year follow-up. No significant changes in secondary outcomes, such as quality of life or cardiac function, were found in patients who received PeriCord. However, PeriCord did modulate the kinetics of circulating monocytes involved in post-infarction myocardial repair towards non-classical inflammation-resolving macrophages, as well as levels of monocyte chemoattractants and the prognostic marker Meteorin-like in plasma following treatment. INTERPRETATION: In summary, the PeriCord graft has exhibited a safe profile and notable immunomodulatory properties. Nevertheless, further research is required to fully unlock its potential as a platform for managing inflammatory-related pathologies. FUNDING: This work was supported in part by grants from MICINN (SAF2017-84324-C2-1-R); Instituto de Salud Carlos III (ICI19/00039 and Red RICORS-TERAV RD21/0017/0022, and CIBER Cardiovascular CB16/11/00403) as a part of the Plan Nacional de I + D + I, and co-funded by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) and AGAUR (2021-SGR-01437).

Nanomaterials-incorporated hydrogels for 3D bioprinting technology.

In the field of tissue engineering and regenerative medicine, various hydrogels derived from the extracellular matrix have been utilized for creating engineered tissues and implantable scaffolds. While these hydrogels hold immense promise in the healthcare landscape, conventional bioinks based on ECM hydrogels face several challenges, particularly in terms of lacking the necessary mechanical properties required for 3D bioprinting process. To address these limitations, researchers are actively exploring novel nanomaterial-reinforced ECM hydrogels for both mechanical and functional aspects. In this review, we focused on discussing recent advancements in the fabrication of engineered tissues and monitoring systems using nanobioinks and nanomaterials via 3D bioprinting technology. We highlighted the synergistic benefits of combining numerous nanomaterials into ECM hydrogels and imposing geometrical effects by 3D bioprinting technology. Furthermore, we also elaborated on critical issues remaining at the moment, such as the inhomogeneous dispersion of nanomaterials and consequent technical and practical issues, in the fabrication of complex 3D structures with nanobioinks and nanomaterials. Finally, we elaborated on plausible outlooks for facilitating the use of nanomaterials in biofabrication and advancing the function of engineered tissues.

A review of the evidence to support electrical stimulation-induced vascularization in engineered tissue.

Tissue engineering presents a promising solution for regenerative medicine and the success depends on the supply of oxygen/nutrients to the cells by rapid vascularization. More and more technologies are being developed to facilitate vascularization of engineered tissues. In this review, we indicated that a regulatory system which influences all angiogenesis associated cells to achieve their desired functional state is ideal for the construction of vascularized engineered tissues in vitro. We presented the evidence that electrical stimulation (ES) enhances the synergistic promotion of co-cultured angiogenesis associated cells and its potential regulatory mechanisms, highlighted the potential advantages of a combination of mesenchymal stem cells (MSCs), endothelial cells (ECs) and ES to achieve tissue vascularization, with particular emphasis on the different biological pathways of ES-regulated ECs. Finally, we proposed the future direction of using ES to reconstruct engineered tissue blood vessels, pointed out the potential advantages and disadvantages of ES application on tissue vascularization.

Hydrogel-Assisted Double Molding Enables Rapid Replication of Stereolithographic 3D Prints for Engineered Tissue Design.

Tissue-engineered in vitro models are an essential tool in biomedical research. Tissue geometry is a key determinant of function, but controlling the geometry of microscale tissues remains challenging. Additive manufacturing approaches have emerged as a promising means for rapid and iterative changes in the geometry of microdevices. However, it has been shown that poly(dimethylsiloxane) (PDMS) cross-linking is often inhibited at the interface of materials printed with stereolithography. While approaches to replica mold stereolithographic three-dimensional (3D) prints have been described, these methods are inconsistent and often lead to print destruction when unsuccessful. Additionally, 3D-printed materials often leach toxic chemicals into directly molded PDMS. Here, we developed a double molding approach that allows precise replication of high-resolution stereolithographic prints into poly(dimethylsiloxane) (PDMS) elastomer, facilitating rapid design iterations and highly parallelized sample production. Inspired by lost wax casting, we used hydrogels as intermediary molds to transfer high-resolution features from high-resolution 3D prints into PDMS, while previously published work focused on enabling direct molding of PDMS onto 3D prints through the use of coatings and post-cross-linking treatments of the 3D print itself. Hydrogel mechanical properties, including cross-link density, predict replication fidelity. We demonstrate the ability of this approach to replicate a variety of shapes that would be impossible to create using photolithography techniques traditionally used to create engineered tissue designs. This method also enabled the replication of 3D-printed features into PDMS that would not be possible with direct molding as the stiffness of these materials leads to material fracture when unmolding, while the increased toughness in the hydrogels can elastically deform around complex features and maintain replication fidelity. Finally, we highlight the ability of this method to minimize the potential for toxic materials to transfer from the original 3D print into the PDMS replica, enhancing its use for biological applications. This minimization of the transfer of toxic materials has not been reported in other previously reported methods describing replication of 3D prints into PDMS, and we demonstrate its use through the creation of stem cell-derived microheart muscles. This method can also be used in future studies to understand the effects of geometry on engineered tissues and their constitutive cells.

New Solutions for Old Problems: How Reproductive Tissue Engineering Has Been Revolutionizing Reproductive Medicine.

Acquired disorders and congenital defects of the male and female reproductive systems can have profound impacts on patients, causing sexual and endocrine dysfunction and infertility, as well as psychosocial consequences that affect their self-esteem, identity, sexuality, and relationships. Reproductive tissue engineering (REPROTEN) is a promising approach to restore fertility and improve the quality of life of patients with reproductive disorders by developing, replacing, or regenerating cells, tissues, and organs from the reproductive and urinary systems. In this review, we explore the latest advancements in REPROTEN techniques and their applications for addressing degenerative conditions in male and female reproductive organs. We discuss current research and clinical outcomes and highlight the potential of 3D constructs utilizing biomaterials such as scaffolds, cells, and biologically active molecules. Our review offers a comprehensive guide for researchers and clinicians, providing insights into how to reestablish reproductive tissue structure and function using innovative surgical approaches and biomaterials. We highlight the benefits of REPROTEN for patients, including preservation of fertility and hormonal production, reconstruction of uterine and cervical structures, and restoration of sexual and urinary functions. Despite significant progress, REPROTEN still faces ethical and technical challenges that need to be addressed. Our review underscores the importance of continued research in this field to advance the development of effective and safe REPROTEN approaches for patients with reproductive disorders.

3D engineered tissue models for studying human-specific infectious viral diseases.

Viral infections cause damage to various organ systems by inducing organ-specific symptoms or systemic multi-organ damage. Depending on the infection route and virus type, infectious diseases are classified as respiratory, nervous, immune, digestive, or skin infections. Since these infectious diseases can widely spread in the community and their catastrophic effects are severe, identification of their causative agent and mechanisms underlying their pathogenesis is an urgent necessity. Although infection-associated mechanisms have been studied in two-dimensional (2D) cell culture models and animal models, they have shown limitations in organ-specific or human-associated pathogenesis, and the development of a human-organ-mimetic system is required. Recently, three-dimensional (3D) engineered tissue models, which can present human organ-like physiology in terms of the 3D structure, utilization of human-originated cells, recapitulation of physiological stimuli, and tight cell-cell interactions, were developed. Furthermore, recent studies have shown that these models can recapitulate infection-associated pathologies. In this review, we summarized the recent advances in 3D engineered tissue models that mimic organ-specific viral infections. First, we briefly described the limitations of the current 2D and animal models in recapitulating human-specific viral infection pathology. Next, we provided an overview of recently reported viral infection models, focusing particularly on organ-specific infection pathologies. Finally, a future perspective that must be pursued to reconstitute more human-specific infectious diseases is presented.

Label-Free Quantification of Microscopic Alignment in Engineered Tissue Scaffolds by Polarized Raman Spectroscopy.

Monitoring of extracellular matrix (ECM) microstructure is essential in studying structure-associated cellular processes, improving cellular function, and for ensuring sufficient mechanical integrity in engineered tissues. This paper describes a novel method to study the microscale alignment of the matrix in engineered tissue scaffolds (ETS) that are usually composed of a variety of biomacromolecules derived by cells. First, a trained loading function was derived from Raman spectra of highly aligned native tissue via principal component analysis (PCA), where prominent changes associated with specific Raman bands (e.g., 1444, 1465, 1605, 1627-1660, and 1665-1689 cm-1) were detected with respect to the polarization angle. These changes were mainly caused by the aligned matrix of many compounds within the tissue relative to the laser polarization, including proteins, lipids, and carbohydrates. Hence this trained function was applied to quantify the alignment within ETS of various matrix components derived by cells. Furthermore, a simple metric called Amplitude Alignment Metric (AAM) was derived to correlate the orientation dependence of polarized Raman spectra of ETS to the degree of matrix alignment. It was found that the AAM was significantly higher in anisotropic ETS than isotropic ones. The PRS method revealed a lower p-value for distinguishing the alignment between these two types of ETS as compared to the microscopic method for detecting fluorescent-labeled protein matrices at a similar microscopic scale. These results indicate that the anisotropy of a complex matrix in engineered tissue can be assessed at the microscopic scale using a PRS-based simple metric, which is superior to the traditional microscopic method. This PRS-based method can serve as a complementary tool for the design and assessment of engineered tissues that mimic the native matrix organizational microstructures.

Long-Term Culture Performance of a Polyelectrolyte Complex Microcapsule Platform for Hyaline Cartilage Repair.

Articular cartilage (AC) tissue repair and regeneration remains an ongoing challenge. One component of the challenge is the limited ability to scale an engineered cartilage graft to clinically relevant sizes while maintaining uniform properties. In this paper, we report on the evaluation of our polyelectrolyte complex microcapsule (PECM) platform technology as a technique for generating cartilage-like spherical modules. Bone marrow-derived mesenchymal stem cells (bMSCs) or primary articular chondrocytes were encapsulated within PECMs composed of methacrylated hyaluronan, collagen I, and chitosan. The formation of cartilage-like tissue in the PECMs over a 90-day culture was characterized. The results showed that chondrocytes exhibited superior growth and matrix deposition compared to either chondrogenically-induced bMSCs or a mixed PECM culture containing both chondrocytes and bMSCs. The chondrocyte-generated matrix filled the PECM and produced substantial increases in capsule compressive strength. The PECM system thus appears to support intracapsular cartilage tissue formation and the capsule approach promotes efficient culture and handling of these micro tissues. Since previous studies have proven the feasibility of fusing such capsules into large tissue constructs, the results suggest that encapsulating primary chondrocytes in PECM modules may be a viable route toward achieving a functional articular cartilage graft.

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle.

Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle ("myobundles"). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p < 0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo.

A macroscopic approach for stress-driven anisotropic growth in bioengineered soft tissues.

The simulation of growth processes within soft biological tissues is of utmost importance for many applications in the medical sector. Within this contribution, we propose a new macroscopic approach for modelling stress-driven volumetric growth occurring in soft tissues. Instead of using the standard approach of a-priori defining the structure of the growth tensor, we postulate the existence of a general growth potential. Such a potential describes all eligible homeostatic stress states that can ultimately be reached as a result of the growth process. Making use of well-established methods from visco-plasticity, the evolution of the growth-related right Cauchy-Green tensor is subsequently defined as a time-dependent associative evolution law with respect to the introduced potential. This approach naturally leads to a formulation that is able to cover both, isotropic and anisotropic growth-related changes in geometry. It furthermore allows the model to flexibly adapt to changing boundary and loading conditions. Besides the theoretical development, we also describe the algorithmic implementation and furthermore compare the newly derived model with a standard formulation of isotropic growth.