Proanthocyanidin surface preconditioning of dental pulp stem cell spheroids enhances dimensional stability and biomineralization in vitro.

AIM: Lack of adequate mechanical strength and progressive shrinkage over time remain challenges in scaffold-free microtissue-based dental pulp regeneration. Surface collagen cross-linking holds the promise to enhance the mechanical stability of microtissue constructs and trigger biological regulations. In this study, we proposed a novel strategy for surface preconditioning microtissues using a natural collagen cross-linker, proanthocyanidin (PA). We evaluated its effects on cell viability, tissue integrity, and biomineralization of dental pulp stem cell (DPSCs)-derived 3D cell spheroids. METHODOLOGY: Microtissue and macrotissue spheroids were fabricated from DPSCs and incubated with PA solution for surface collagen cross-linking. Microtissue viability was examined by live/dead staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with transverse dimension change monitored. Microtissue surface stiffness was measured by an atomic force microscope (AFM). PA-preconditioned microtissues and macrotissues were cultured under basal or osteogenic conditions. Immunofluorescence staining of PA-preconditioned microtissues was performed to detect dentin sialophosphoprotein (DSPP) and F-actin expressions. PA-preconditioned macrotissues were subjected to histological analysis, including haematoxylin-eosin (HE), alizarin red, and Masson trichrome staining. Immunohistochemistry staining was used to detect alkaline phosphatase (ALP) and dentin matrix acidic phosphoprotein 1 (DMP-1) expressions. RESULTS: PA preconditioning had no adverse effects on microtissue spheroid viability and increased surface stiffness. It reduced dimensional shrinkage for over 7 days in microtissues and induced a larger transverse-section area in the macrotissue. PA preconditioning enhanced collagen formation, mineralized nodule formation, and elevated ALP and DMP-1 expressions in macrotissues. Additionally, PA preconditioning induced higher F-actin and DSPP expression in microtissues, while inhibition of F-actin activity by cytochalasin B attenuated PA-induced dimensional change and DSPP upregulation. CONCLUSION: PA surface preconditioning of DPSCs spheroids demonstrates excellent biocompatibility while effectively enhancing tissue structure stability and promoting biomineralization. This strategy strengthens tissue integrity in DPSC-derived spheroids and amplifies osteogenic differentiation potential, advancing scaffold-free tissue engineering applications in regenerative dentistry.

Applications and sensory utilizations of magnetic levitation in 3D cell culture for tissue Engineering.

3D cell culture approaches are cell culture methods that provide good visualization of interactions between cells while preserving the natural growth pattern. In recent years, several studies have managed to implement magnetic levitation technology on 3D cell culture applications by either combining cells with magnetic nanoparticles (positive magnetophoresis) or applying a magnetic field directly to the cells in a high-intensity medium (negative magnetophoresis). The positive magnetophoresis technique consists of integrating magnetic nanoparticles into the cells, while the negative magnetophoresis technique consists of levitating the cells without labelling them with magnetic nanoparticles. Magnetic levitation methods can be used to manipulate 3D culture, provide more complex habitats and custom control, or display density data as a sensor.The present review aims to show the advantages, limitations, and promises of magnetic 3D cell culture, along with its application methods, tools, and capabilities as a density sensor. In this context, the promising magnetic levitation technique on 3D cell cultures could be fully utilized in further studies with precise control.

Spheroids and organoids as humanized 3D scaffold-free engineered tissues for SARS-CoV-2 viral infection and drug screening.

The new coronavirus (2019-nCoV) or the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was officially declared by the World Health Organization (WHO) as a pandemic in March 2020. To date, there are no specific antiviral drugs proven to be effective in treating SARS-CoV-2, requiring joint efforts from different research fronts to discover the best route of treatment. The first decisions in drug discovery are based on 2D cell culture using high-throughput screening. In this context, spheroids and organoids emerge as a reliable alternative. Both are scaffold-free 3D engineered constructs that recapitulate key cellular and molecular events of tissue physiology. Different studies have already shown their advantages as a model for different infectious diseases, including SARS-CoV-2 and for drug screening. The use of these 3D engineered tissues as an in vitro model can fill the gap between 2D cell culture and in vivo preclinical assays (animal models) as they could recapitulate the entire viral life cycle. The main objective of this review is to understand spheroid and organoid biology, highlighting their advantages and disadvantages, and how these scaffold-free engineered tissues can contribute to a better comprehension of viral infection by SARS-CoV-2 and to the development of in vitro high-throughput models for drug screening.

Recent Tissue Engineering Advances for the Treatment of Temporomandibular Joint Disorders.

Temporomandibular disorders (TMDs) are among the most common maxillofacial complaints and a major cause of orofacial pain. Although current treatments provide short- and long-term relief, alternative tissue engineering solutions are in great demand. Particularly, the development of strategies, providing long-term resolution of TMD to help patients regain normal function, is a high priority. An absolute prerequisite of tissue engineering is to understand normal structure and function. The current knowledge of anatomical, mechanical, and biochemical characteristics of the temporomandibular joint (TMJ) and associated tissues will be discussed, followed by a brief description of current TMD treatments. The main focus is on recent tissue engineering developments for regenerating TMJ tissue components, with or without a scaffold. The expectation for effectively managing TMD is that tissue engineering will produce biomimetic TMJ tissues that recapitulate the normal structure and function of the TMJ.

3D bioprinting of biomimetic aortic vascular constructs with self-supporting cells.

Cardiovascular diseases are the leading cause of deaths throughout the world. Vascular diseases are mostly treated with autografts and blood vessel transplantations. However, traditional grafting methods have several problems including lack of suitable harvest sites, additional surgical costs for harvesting procedure, pain, infection, lack of donors, and even no substitutes at all. Recently, tissue engineering and regenerative medicine approaches are used to regenerate damaged or diseased tissues. Most of the tissue engineering investigations have been based on the cell seeding into scaffolds by providing a suitable environment for cell attachment, proliferation, and differentiation. Because of the challenges such as difficulties in seeding cells spatially, rejection, and inflammation of biomaterials used, the recent tissue engineering studies focus on scaffold-free techniques. In this paper, the development of novel computer aided algorithms and methods are developed for 3D bioprinting of scaffold-free biomimetic macrovascular structures. Computer model mimicking a real human aorta is generated using imaging techniques and the proposed computational algorithms. An optimized three-dimensional bioprinting path planning are developed with the proposed self-supported model. Mouse embryonic fibroblast (MEF) cell aggregates and support structures (hydrogels) are 3D bioprinted layer-by-layer according to the proposed self-supported method to form an aortic tissue construct.

Synovium-Derived Mesenchymal Stem Cell-Based Scaffold-Free Fibrocartilage Engineering for Bone-Tendon Interface Healing in an Anterior Cruciate Ligament Reconstruction Model.

BACKGROUND: Current tendon and ligament reconstruction surgeries rely on scar tissue healing which differs from native bone-to-tendon interface (BTI) tissue. We aimed to engineer Synovium-derived mesenchymal stem cells (Sy-MSCs) based scaffold-free fibrocartilage constructs and investigate in vivo bone-tendon interface (BTI) healing efficacy in a rat anterior cruciate ligament (ACL) reconstruction model. METHODS: Sy-MSCs were isolated from knee joint of rats. Scaffold-free sy-MSC constructs were fabricated and cultured in differentiation media including  TGF-ß-only, CTGF-only, and TGF-ß + CTGF. Collagenase treatment on tendon grafts was optimized to improve cell-to-graft integration. The effects of fibrocartilage differentiation and collagenase treatment on BTI integration was assessed by conducting histological staining, cell adhesion assay, and tensile testing. Finally, histological and biomechanical analyses were used to evaluate in vivo efficacy of fibrocartilage construct in a rat ACL reconstruction model. RESULTS: Fibrocartilage-like features were observed with in the scaffold-free sy-MSC constructs when applying TGF-ß and CTGF concurrently. Fifteen minutes collagenase treatment increased cellular attachment 1.9-fold compared to the Control group without affecting tensile strength. The failure stress was highest in the Col + D + group (22.494 ± 13.74 Kpa) compared to other groups at integration analysis in vitro. The ACL Recon + FC group exhibited a significant 88% increase in estimated stiffness (p = 0.0102) compared to the ACL Recon group at the 4-week postoperative period. CONCLUSION: Scaffold-free, fibrocartilage engineering together with tendon collagenase treatment enhanced fibrocartilaginous BTI healing in ACL reconstruction.

Enhanced articular cartilage regeneration using costal chondrocyte-derived scaffold-free tissue engineered constructs with ascorbic acid treatment.

Background: Cartilage tissue engineering faces challenges related to the use of scaffolds and limited seed cells. This study aims to propose a cost-effective and straightforward approach using costal chondrocytes (CCs) as an alternative cell source to overcome these challenges, eliminating the need for special culture equipment or scaffolds. Methods: CCs were cultured at a high cell density with and without ascorbic acid treatment, serving as the experimental and control groups, respectively. Viability and tissue-engineered constructs (TEC) formation were evaluated until day 14. Slices of TEC samples were used for histological staining to evaluate the secretion of glycosaminoglycans and different types of collagen proteins within the extracellular matrix. mRNA sequencing and qPCR were performed to examine gene expression related to cartilage matrix secretion in the chondrocytes. In vivo experiments were conducted by implanting TECs from different groups into the defect site, followed by sample collection after 12 weeks for histological staining and scoring to evaluate the extent of cartilage regeneration. Hematoxylin-eosin (HE), Safranin-O-Fast Green, and Masson's trichrome stainings were used to examine the content of cartilage-related matrix components in the in vivo repair tissue. Immunohistochemical staining for type I and type II collagen, as well as aggrecan, was performed to assess the presence and distribution of these specific markers. Additionally, immunohistochemical staining for type X collagen was used to observe any hypertrophic changes in the repaired tissue. Results: Viability of the chondrocytes remained high throughout the culture period, and the TECs displayed an enriched extracellular matrix suitable for surgical procedures. In vitro study revealed glycosaminoglycan and type II collagen production in both groups of TEC, while the TEC matrix treated with ascorbic acid displayed greater abundance. The results of mRNA sequencing and qPCR showed that genes related to cartilage matrix secretion such as Sox9, Col2, and Acan were upregulated by ascorbic acid in costal chondrocytes. Although the addition of Asc-2P led to an increase in COL10 expression according to qPCR and RNA-seq results, the immunofluorescence staining results of the two groups of TECs exhibited similar distribution and fluorescence intensity. In vivo experiments showed that both groups of TEC could adhere to the defect sites and kept hyaline cartilage morphology until 12 weeks. TEC treated with ascorbic acid showed superior cartilage regeneration as evidenced by significantly higher ICRS and O'Driscoll scores and stronger Safranin-O and collagen staining mimicking native cartilage when compared to other groups. In addition, the immunohistochemical staining results of Collgan X indicated that, after 12 weeks, the ascorbic acid-treated TEC did not exhibit further hypertrophy upon transplantation into the defect site, but maintained an expression profile similar to untreated TECs, while slightly higher than the sham-operated group. Conclusion: These results suggest that CC-derived scaffold-free TEC presents a promising method for articular cartilage regeneration. Ascorbic acid treatment enhances outcomes by promoting cartilage matrix production. This study provides valuable insights and potential advancements in the field of cartilage tissue engineering. The translational potential of this article: Cartilage tissue engineering is an area of research with immense clinical potential. The approach presented in this article offers a cost-effective and straightforward solution, which can minimize the complexity of cell culture and scaffold fabrication. This simplification could offer several translational advantages, such as ease of use, rapid scalability, lower costs, and the potential for patient-specific clinical translation. The use of costal chondrocytes, which are easily obtainable, and the scaffold-free approach, which does not require specialized equipment or membranes, could be particularly advantageous in clinical settings, allowing for in situ regeneration of cartilage.

Auricular Chondrocytes as a Cell Source for Scaffold-Free Elastic Cartilage Tissue Engineering.

Current tissue engineering (TE) methods utilize chondrocytes primarily from costal or articular sources. Despite the robust mechanical properties of neocartilages sourced from these cells, the lack of elasticity and invasiveness of cell collection from these sources negatively impact clinical translation. These limitations invited the exploration of naturally elastic auricular cartilage as an alternative cell source. This study aimed to determine if auricular chondrocytes (AuCs) can be used for TE scaffold-free neocartilage constructs and assess their biomechanical properties. Neocartilages were successfully generated from a small quantity of primary neonatal AuCs of three minipig donors (n = 3). Neocartilage constructs had instantaneous moduli of 200.5 kPa ± 43.34 and 471.9 ± 92.8 kPa at 10% and 20% strain, respectively. TE constructs' relaxation moduli (Er) were 36.99 ± 6.47 kPa Er and 110.3 ± 16.99 kPa at 10% and 20% strain, respectively. The Young's modulus was 2.0 MPa ± 0.63, and the ultimate tensile strength was 0.619 ± 0.177 MPa. AuC-derived neocartilages contained 0.144 ± 0.011 µg collagen, 0.185 µg ± 0.002 glycosaminoglycans per µg dry weight, and 1.7e-3 µg elastin per µg dry weight. In conclusion, this study shows that AuCs can be used as a reliable and easily accessible cell source for TE of biomimetic and mechanically robust elastic neocartilage implants.

Harvest of Cell-Only Muscle Fibers Using Thermally Expandable Hydrogels with Adhesive Patterns.

Muscle tissue engineering has been the focus of extensive research due to its potential for numerous medical applications, including ex vivo actuator development and clinical treatments. In this study, we developed a method for harvesting muscle fiber in a floatable and translocatable manner utilizing thermally expandable hydrogels with a chemically patterned polydopamine (PD) layer generated by microcontact printing (µCP). The µCP of PD on the hydrogel facilitated the formation of stripe patterns with varying widths of printed/nonprinted area (50/50, 100/100, and 200/200 µm). The spatially controlled adhesion of C2C12 myoblasts on the PD patterns produced clearly distinguishable muscle fibers, and translocated muscle fibers exhibited preserved extracellular matrix and junction proteins. Furthermore, the development of anisotropic arrangements and mature myotubes within the fibers suggests the potential for functional control of engineered muscle tissues. Overall, the muscle fiber harvesting method developed herein is suitable for both translocation and floating and is a promising technique for muscle tissue engineering as it mimics the structure-function relationship of natural tissue.

Tissue Engineering of Canine Cartilage from Surgically Debrided Osteochondritis Dissecans Fragments.

This study in dogs explored the feasibility of using cartilage fragments removed and discarded during routine palliative surgery for osteochondritis dissecans (OCD) as a source of primary chondrocytes for scaffold-free cartilage tissue-engineering. Primary chondrocytes were obtained from three OCD donors and one age-matched healthy articular cartilage (HAC) donor. After monolayer expansion of primary cells, a three-dimensional spherical suspension culture was implemented. Following this stage, cells were seeded at a high density into custom-made agarose molds that allowed for size and shape-specific constructs to be generated via a method of cellular self-assembling in a scaffold-free environment. Fifty-eight neocartilage constructs were tissue-engineered using this methodology. Neocartilage constructs and native cartilage from shoulder joint were subjected to histological, mechanical, and biochemical testing. OCD and HAC chondrocytes-sourced constructs had uniformly flat morphology and histology consistent with cartilage tissue. Constructs sourced from OCD chondrocytes were 1.5-times (32%) stiffer in compression and 1.3 times (23%) stronger in tension than constructs sourced from HAC chondrocytes and only 8.7-times (81%) less stiff in tension than native tissue. Constructs from both cell sources consistently had lower collagen content than native tissue (22.9%/dry weight [DW] for OCD and 4.1%/DW for HAC vs. 51.1%/DW native tissue). To improve the collagen content and mechanical properties of neocartilage, biological and mechanical stimuli, and thyroid hormone (tri-iodothyronine) were applied to the chondrocytes during the self-assembling stage in two separate studies. A 2.6-fold (62%) increase in compressive stiffness was detected with supplementation of biological stimuli alone and 5-fold (81%) increase with combined biological and mechanical stimuli at 20% strain. Application of thyroid hormone improved collagen content (1.7-times, 33%), tensile strength (1.8-times, 43%), and stiffness (1.3-times, 21%) of constructs, relative to untreated controls. Collectively, these data suggest that OCD chondrocytes can serve as a reliable cell source for cartilage tissue-engineering and that canine chondrocytes respond favorably to biological and mechanical stimuli that have been shown effective in chondrocytes from other animal species, including humans.

Effects of Induction Culture on Osteogenesis of Scaffold-Free Engineered Tissue for Bone Regeneration Applications.

BACKGROUND: Restoration of the bone defects caused by infection or disease remains a challenge in orthopedic surgery. In recent studies, scaffold-free engineered tissue with a self-secreted extracellular matrix has been proposed as an alternative strategy for tissue regeneration and reconstruction. Our study aimed to engineer and fabricate self-assembled osteogenic and scaffold-free tissue for bone regeneration. METHODS: Osteogenic scaffold-free tissue was engineered and fabricated using fetal cartilage-derived progenitor cells, which are capable of osteogenic differentiation. They were cultured in osteogenic induction environments or using demineralized bone powder for differentiation. The fabricated tissue was subjected to real-time qPCR, biochemical, and histological analyses to estimate the degree of in vitro osteogenic differentiation. To demonstrate bone formation in an in vivo environment, scaffold-free tissue was transplanted into the dorsal subcutaneous site of nude mice. Bone development was monitored postoperatively over 8 weeks by the observation of calcium deposition in the matrix. RESULTS: In the in vitro experiments, engineered osteogenically induced scaffold-free tissue demonstrated three-dimensional morphological characteristics, and sufficient osteogenic differentiation was confirmed through the quantification of specific osteogenic gene markers expressed and calcium accumulation within the matrix. Following the evaluation of differentiation efficacy, in vivo experiments revealed distinct bone formation, and that blood vessels had penetrated the fabricated tissue. CONCLUSION: The novel engineering of scaffold-free tissue with osteogenic potential can be used as an optimal bone graft substitute for bone regeneration.

Rejuvenation of extensively passaged human chondrocytes to engineer functional articular cartilage.

Human articular chondrocytes (hACs) are scarce and lose their chondrogenic potential during monolayer passaging, impeding their therapeutic use. This study investigated (a) the translatability of conservative chondrogenic passaging and aggregate rejuvenation on restoring chondrogenic properties of hACs passaged up to P9; and (b) the efficacy of a combined treatment of transforming growth factor-beta 1 (TGF-ß1) (T), chondroitinase-ABC (C), and lysyl oxidase-like 2 (L), collectively termed TCL, on engineering functional human neocartilage via the self-assembling process, as a function of passage number up to P11. Here, we show that aggregate rejuvenation enhanced glycosaminoglycan (GAG) content and type II collagen staining at all passages and yielded human neocartilage with chondrogenic phenotype present up to P7. Addition of TCL extended the chondrogenic phenotype to P11 and significantly enhanced GAG content and type II collagen staining at all passages. Human neocartilage derived from high passages, treated with TCL, displayed mechanical properties that were on par with or greater than those derived from low passages. Conservative chondrogenic passaging and aggregate rejuvenation may be a viable new strategy (a) to address the perennial problem of chondrocyte scarcity and (b) to successfully rejuvenate the chondrogenic phenotype of extensively passaged cells (up to P11). Furthermore, tissue engineering human neocartilage via self-assembly in conjunction with TCL treatment advances the clinical use of extensively passaged human chondrocytes for cartilage repair.

Fabrication of Tunable 3D Cellular Structures in High Volume Using Magnetic Levitation Guided Assembly.

Tunable and reproducible size with high circularity is an important limitation to obtain three-dimensional (3D) cellular structures and spheroids in scaffold free tissue engineering approaches. Here, we present a facile methodology based on magnetic levitation (MagLev) to fabricate 3D cellular structures rapidly and easily in high-volume and low magnetic field. In this study, 3D cellular structures were fabricated using magnetic levitation directed assembly where cells are suspended and self-assembled by contactless magnetic manipulation in the presence of a paramagnetic agent. The effect of cell seeding density, culture time, and paramagnetic agent concentration on the formation of 3D cellular structures was evaluated for NIH/3T3 mouse fibroblast cells. In addition, magnetic levitation guided cellular assembly and 3D tumor spheroid formation was examined for five different cancer cell lines: MCF7 (human epithelial breast adenocarcinoma), MDA-MB-231 (human epithelial breast adenocarcinoma), SH-SY5Y (human bone-marrow neuroblastoma), PC-12 (rat adrenal gland pheochromocytoma), and HeLa (human epithelial cervix adenocarcinoma). Moreover, formation of a 3D coculture model was successfully observed by using MDA-MB-231 dsRED and MDA-MB-231 GFP cells. Taken together, these results indicate that the developed MagLev setup provides an easy and efficient way to fabricate 3D cellular structures and may be a feasible alternative to conventional methodologies for cellular/multicellular studies.

Soft Ring-Shaped Cellu-Robots with Simultaneous Locomotion in Batches.

Untethered mini-robots can move single cells or aggregates to build complex constructs in confined spaces and may enable various biomedical applications such as regenerative repair in medicine and biosensing in bioengineering. However, a significant challenge is the ability to control multiple microrobots simultaneously in the same space to operate toward a common goal in a distributed operation. A locomotion strategy that can simultaneously guide the formation and operation of multiple robots in response to a common acoustic stimulus is developed. The scaffold-free cellu-robots comprise only highly packed cells and eliminate the influence of supportive materials, making them less cumbersome during locomotion. The ring shape of the cellu-robot contributes to anisotropic cellular interactions which induce radial cellular orientation. Under a single stimulus, several cellu-robots form predetermined complex structures such as bracelet-like ring-chains which transform into a single new living entity through cell-cell interactions, migration or cellular extensions between cellu-robots.

Multi-layer pre-vascularized magnetic cell sheets for bone regeneration.

The lack of effective strategies to produce vascularized 3D bone transplants in vitro, hampers the development of thick-constructed bone, limiting the translational of lab-based engineered system to clinical practices. Cell sheet (CS) engineering techniques provide an excellent microenvironment for vascularization since the technique can maintain the intact cell matrix, crucial for angiogenesis. In an attempt to develop hierarchical vascularized 3D cellular constructs, we herein propose the construction of stratified magnetic responsive heterotypic CSs by making use of iron oxide nanoparticles previously internalized within cells. Magnetic force-based CS engineering allows for the construction of thick cellular multilayers. Results show that osteogenesis is achieved due to a synergic effect of human umbilical vein endothelial cells (HUVECs) and adipose-derived stromal cells (ASCs), even in the absence of osteogenic differentiating factors. Increased ALP activity, matrix mineralization, osteopontin and osteocalcin detection were achieved over a period of 21 days for the heterotypic CS conformation (ASCs/HUVECs/ASCs), over the homotypic one (ASCs/ASCs), corroborating our findings. Moreover, the validated crosstalk between BMP-2 and VEGF releases triggers not only the recruitment of blood vessels, as demonstrated in an in vivo CAM assay, as well as the osteogenesis of the 3D cell construct. The in vivo angiogenic profile also demonstrated preserved human vascular structures and human cells showed the ability to migrate and integrate within the chick vasculature.

Magnetic Tissue Engineering of the Vocal Fold Using Superparamagnetic Iron Oxide Nanoparticles.

Losing one's ability to speak, because of tissue deficiency at the vocal fold (VF), leads to serious impairment in the quality of life. Until now, there is no successful approach for regenerating the VF. The aim of this study was to show the advantage of magnetic nanoparticles in the generation of scaffold-free three-dimensional (3D) VF cell constructs by magnetic tissue engineering (MTE). Rabbit VF fibroblasts were used to establish MTE: after cellular uptake of superparamagnetic iron oxide nanoparticles (SPIONs), cells can be controlled with a magnetic field thereby forming solid 3D cell structures. To transfer this method into human cells, SPIONs were adapted accordingly and tested for their influence on human VF (hVF) cells and for their ability to perform MTE with hVF cells. Of interest, the cell number and the magnet's shape influence the form of the rabbit VF cell construct. After successful characterization of hVF cells, biocompatibility analyses revealed no significant influence of SPIONs on them, thus 3D hVF cell constructs could be successfully generated by MTE. These basic results are important to develop MTE as an innovative method to regenerate functional VFs. We expect that in vivo studies, including MTE as an elegant, far-field controlled and touchless technology, will translate MTE VF bioconstructs into reconstructive laryngeal medicine. Impact Statement This study aims at nanotechnology for regenerative medicine by magnetic tissue engineering (MTE). New approaches for vocal fold (VF) reconstruction are desperately needed. Superparamagnetic iron oxide nanoparticles offer innovative, scaffold-free potentials for tissue engineering: MTE. By using MTE we could generate functional multilayered human VF cell constructs, which can consequently be used to regenerate the voice in patients with VF injuries.

Vascular Tissue Engineering Using Scaffold-Free Prevascular Endothelial-Fibroblast Constructs.

Vascularization remains a substantial limitation to the viability of engineered tissue. By comparing in vivo vascularization dynamics of a self-assembled prevascular endothelial-fibroblast model to avascular grafts, we explore the vascularization rate limitations in implants at early time intervals, during which tissue hypoxia begins to affect cell viability. Scaffold-free prevascular endothelial-fibroblast constructs (SPECs) may serve as a modular and reshapable vascular bed in replacement tissues. SPECs, fibroblast-only spheroids (FOS), and silicone implants were implanted in 54 Sprague Dawley rats and harvested at 6, 12, and 24 h (n = 5 per time point and implant type). We hypothesized that the primary endothelial networks of the SPECs allow earlier anastomosis and increased vessel formation in the interior of the implant compared to FOS and silicone implants within a 24 h window. All constructs were encapsulated by an endothelial lining at 6 h postimplantation and SPEC internal cords inosculated with the host vascular network by this time point. SPECs had a significantly higher microvascular area fraction and branch/junction density of penetrating cords at 6-12 h compared with other constructs. In addition, SPECs demonstrated perivascular cell recruitment, lumen formation, and network remodeling consistent with vessel maturation at 12-24 h; however, these implants were poorly perfused within our observation window, suggesting poor lumen patency. FOS vascular characteristics (microvessel area and penetrating cord density) increased within the 12-24 h period to represent those of the SPEC implants, suggesting a 12 h latency in host response to avascular grafts compared to prevascular grafts. Knowledge of this temporal advantage in in vitro prevascular network self-assembly as well as an understanding of the current limitations of SPEC engraftment builds on our theoretical temporal model of tissue graft vascularization and suggests a crucial time window, during which technological improvements and vascular therapy can improve engineered tissue survival.

Magnetically Guided Self-Assembly and Coding of 3D Living Architectures.

In nature, cells self-assemble at the microscale into complex functional configurations. This mechanism is increasingly exploited to assemble biofidelic biological systems in vitro. However, precise coding of 3D multicellular living materials is challenging due to their architectural complexity and spatiotemporal heterogeneity. Therefore, there is an unmet need for an effective assembly method with deterministic control on the biomanufacturing of functional living systems, which can be used to model physiological and pathological behavior. Here, a universal system is presented for 3D assembly and coding of cells into complex living architectures. In this system, a gadolinium-based nonionic paramagnetic agent is used in conjunction with magnetic fields to levitate and assemble cells. Thus, living materials are fabricated with controlled geometry and organization and imaged in situ in real time, preserving viability and functional properties. The developed method provides an innovative direction to monitor and guide the reconfigurability of living materials temporally and spatially in 3D, which can enable the study of transient biological mechanisms. This platform offers broad applications in numerous fields, such as 3D bioprinting and bottom-up tissue engineering, as well as drug discovery, developmental biology, neuroscience, and cancer research.

Three-dimensional direct cell bioprinting for tissue engineering.

Bioprinting is a relatively new technology where living cells with or without biomaterials are printed layer-by-layer in order to create three-dimensional (3D) living structures. In this article, novel bioprinting methodologies are developed to fabricate 3D biological structures directly from computer models using live multicellular aggregates. Multicellular aggregates made out of at least two cell types from fibroblast, endothelial and smooth muscle cells are prepared and optimized. A novel bioprinting approach is proposed in order to continuously extrude cylindrical multicellular aggregates through the bioprinter's glass microcapillaries. The multicellular aggregates are first aspirated into a capillary and then compressed to form a continuous cylindrical multicellular bioink. To overcome surface tension-driven droplet formation, the required compression ratio is calculated. Based on the developed bioprinting strategies, multicellular aggregates and their support structures are bioprinted to form 3D tissue constructs with predefined shapes. The effect of the bioprinting process was examined for fusion, cell viability at different compression ratios, and f-actin cytoskeletal organization. The results show that the bioprinted 3D constructs fuse rapidly and have high cell viability after printing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2530-2544, 2017.

Peritoneal cell sheets composed of mesothelial cells and fibroblasts prevent intra-abdominal adhesion formation in a rat model.

Postoperative intra-abdominal adhesions remain an unsolved problem despite significant progress in the surgical procedures themselves. They often lead to small-bowel obstruction, chronic abdominal and pelvic pain, as well as female infertility. The loss of mesothelial cells and several components of the inflammatory system following injury to the peritoneum results in fibrin formation and angiogenesis. The remaining fibrin matrix and angiogenesis lead to replacement by fibroblasts and fibrous band formation. The aim of this study was to develop a new therapeutic method of preventing intra-abdominal adhesions. We fabricated transplantable peritoneal cell sheets from the rat peritoneum by cell sheet engineering using a temperature-responsive culture system. The peritoneal cell sheets developed were composed of an upper monolayer of mesothelial cells and underlying multilayered fibroblasts, similar to the peritoneum in vivo. Transplantation of peritoneal cell sheets prevented tissue adhesion, fibrin deposition and angiogenesis, and, moreover, lymphangiogenesis and macrophage infiltration in a rat caecum cauterization adhesion model. Copyright © 2013 John Wiley & Sons, Ltd.