Tissue Engineering Design Research Articles

Tissue engineering for compensating short bowel syndrome

Short bowel syndrome is an important clinical problem characterized by a high incidence of serious complications, deaths and socioeconomic consequences. Parenteral nutrition provides only a temporary solution without reducing the risk of complications. This applies equally to surgical treatment, in particular to small intestine transplantation and related concomitant interventions, which only facilitate the adaptation of the intestine to new conditions. Potential approaches have been analyzed in the treatment of the syndrome of the small intestine, which can be offered by dynamically developing tissue engineering. Various types of carriers and cell types that are used in experiments for obtaining tissue engineering designs of the intestine are discussed. A wide range of variants of such constructions is analyzed that can lead to obtaining an organ prosthesis with a cellular organization and mechanical stability similar to those of the native small intestine, which will ensure the necessary biocompatibility. It is established that one of the optimal carriers for today are extracellular matrices obtained by decellularization of the native small intestine. This process allows to preserve the microarchitecture of the small intestine, which greatly facilitates the process of filling the matrix with cells both in vitro and in vivo. It has also been established that mesenchymal stromal multipotent cells and organoid units obtained from the tissue of the native small intestine are particularly prominent among the most promising participants in the cellular ensemble.

A mosaic structure multi-level vascular network design for skull tissue engineering

In human skull tissue engineering scaffolds, cell growth and osteogenesis are limited due to the lack of vascular structure. Therefore, a mosaic structure vascular parameterized design method is proposed according to the scanning characteristics of the diploic vein. Using micro-CT scans of skull samples, the features of the diploic vein were extracted, and a multi-level vascular network model was established based on a power diagram. Considering the characteristics of blood flow in the veins, finite element analysis (FEA) of the fluid-solid coupling was established to analyze the effect of blood on vessels with four-level mosaic structures. The results showed that the deformation and stress distribution of vessels were reasonable, and the blood pressure, velocity and shear stress in the designed vascular structure could meet the cell growth requirements. The mosaic structure was prepared by PDMS and cultured in vitro using HUVECs. It was found that most of the cells survived after 48 h, and some cells were attached to the surface mosaic structure. In this method, different levels of vessels nest together, with a curvature that matches the shape of the skull, forming a similar morphology to the native diploic vein, and the local structures can be adjusted flexibly. This mosaic structure vascular design method can be used for network vascular design and experimental studies in hard tissues.

Matrix stiffness modulates the differentiation of neural crest stem cells in vivo.

Stem cells are often transplanted with scaffolds for tissue regeneration; however, how the mechanical property of a scaffold modulates stem cell fate in vivo is not well understood. Here we investigated how matrix stiffness modulates stem cell differentiation in a model of vascular graft transplantation. Multipotent neural crest stem cells (NCSCs) were differentiated from induced pluripotent stem cells, embedded in the hydrogel on the outer surface of nanofibrous polymer grafts, and implanted into rat carotid arteries by anastomosis. After 3 months, NCSCs differentiated into smooth muscle cells (SMCs) near the outer surface of the polymer grafts; in contrast, NCSCs differentiated into glial cells in the most part of the hydrogel. Atomic force microscopy demonstrated a stiffer matrix near the polymer surface but much lower stiffness away from the polymer graft. Consistently, in vitro studies confirmed that stiff surface induced SMC genes whereas soft surface induced glial genes. These results suggest that the scaffold's mechanical properties play an important role in directing stem cell differentiation in vivo, which has important implications in biomaterials design for stem cell delivery and tissue engineering.

Advances in Porous Scaffold Design for Bone and Cartilage Tissue Engineering and Regeneration.

Challenges in musculoskeletal tissue regeneration affect millions of patients globally. Scaffolds for tissue engineering bone and cartilage provide promising solutions that increase healing and decrease need for complicated surgical procedures. Porous scaffolds have emerged as an attractive alternative to traditional scaffolds. However, the success of advanced materials, use of biological factors, and manufacturing techniques can vary depending on use case. This review provides perspective on porous scaffold manufacturing, characterization and application, and can be used to inform future scaffold design.

Assessment of Migration of Human MSCs through Fibrin Hydrogels as a Tool for Formulation Optimisation.

Control of cell migration is fundamental to the performance of materials for cell delivery, as for cells to provide any therapeutic effect, they must migrate out from the delivery material. Here the influence of fibrinogen concentration on the migration of encapsulated human mesenchymal stem cells (hMSCs) from a cell spheroid through fibrin hydrogels is tracked over time. Fibrin was chosen as a model material as it is routinely employed as a haemostatic agent and more recently has been applied as a localised delivery vehicle for potential therapeutic cell populations. The hydrogels consisted of 5 U/mL thrombin and between 5 and 50 mg/mL fibrinogen. Microstructural and viscoelastic properties of different compositions were evaluated using SEM and rheometry. Increasing the fibrinogen concentration resulted in a visibly denser matrix with smaller pores and higher stiffness. hMSCs dispersed within the fibrin gels maintained cell viability post-encapsulation, however, the migration of cells from an encapsulated spheroid revealed that denser fibrin matrices inhibit cell migration. This study provides the first quantitative study on the influence of fibrinogen concentration on 3D hMSC migration within fibrin gels, which can be used to guide material selection for scaffold design in tissue engineering and for the clinical application of fibrin sealants.

The fabrication of biomineralized fiber-aligned PLGA scaffolds and their effect on enhancing osteogenic differentiation of UCMSC cells.

The key factor of scaffold design for bone tissue engineering is to mimic the microenvironment of natural bone extracellular matrix (ECM) and guide cell osteogenic differentiation. The biomineralized fiber-aligned PLGA scaffolds (a-PLGA/CaPs) was developed in this study by mimicking the structure and composition of native bone ECM. The aligned PLGA fibers was prepared by wet spinning and then biomineralized via an alternate immersion method. Introduction of a bioceramic component CaP onto the PLGA fibers led to changes in surface roughness and hydrophilicity, which showed to modulate cell adhesion and cell morphology of umbilical cord mesenchymal stem cells (UCMSCs). It was found that organized actin filaments of UCMSCs cultured on both a-PLGA and a-PLGA/CaP scaffolds appeared to follow contact guidance along the aligned fibers, and those cells grown on a-PLGA/CaP scaffolds exhibited a more polarized cellular morphology. The a-PLGA/CaP scaffold with multicycles of mineralization facilitated the cell attachment on the fiber surfaces and then supported better cell adhesion and contact guidance, leading to enhancement in following proliferation and osteogenic differentiation of UCMSCs. Our results give some insights into the regulation of cell behaviors through design of ECM-mimicking structure and composition and provide an alternative wet-spun fiber-aligned scaffold with HA-mineralized layer for bone tissue engineering application.

The multilayered structure of the human corpus spongiosum.

Urethral reconstruction is performed in patients with urethral strictures or for correction of congenital disorders. In the case of shortage of tissue, engineered tissue may enhance urethral reconstruction. As the corpus spongiosum (CS) is important in supporting the function of the urethra, tissue engineering of the urethra should be combined with reconstruction of a CS. For that purpose, detailed knowledge of the composition of the CS, more specifically its extracellular matrix (ECM) and vascularization is needed for scaffold design. The objective of this study is to analyze the microarchitecture of the CS through (immuno) histology and scanning electron microscopy (SEM). The CS including the urethra of patients undergoing male-to-female genital confirming surgery was harvested. This CS was fixed and processed for either (immuno) histology or for SEM. Four layers could be distinguished in the CS; first a transition zone from urethra epithelium to a collagen rich layer, which was highly vascularized, followed by a second, elastin rich layer. The third layer was formed by veins, arteries and vascular spaces and the last layer showed the transition from this vascular rich region to the collagen rich tunica albuginea. In this layer collagen bundles intertwined with elastic fibres. In the CS different components of the ECM were visible and distinguishable. This study provides novel and detailed information on the microarchitecture of the CS and the distribution of vascularization, which is important for scaffold design in tissue engineering.

3D-printing: an emerging and a revolutionary technology in pharmaceuticals.

One of the novel and progressive technology employed in pharmaceutical manufacturing, design of medical device and tissue engineering is three-dimensional (3D) printing. 3D printing technologies provide great advantages in 3D scaffolds fabrication over traditional methods in the control of pore size, porosity, and interconnectivity. Various techniques of 3D-printing include powder bed fusion, fused deposition modeling, binder deposition, inkjet printing, photopolymerization and many others which are still evolving. 3D-printing technique been employed in developing immediate release products, various systems to deliver multiple release modalities etc. 3D printing has opened the door for new generation of customized drug delivery with built-in flexibility for safer and effective therapy. Our mini-review provides a quick snapshot on an overview of 3D printing, various techniques employed, applications and its advancements in pharmaceutical sciences.

Carboxymethyl guar gum synthesis in homogeneous phase and macroporous 3D scaffolds design for tissue engineering

Guar gum (GG) is a galactomannan obtained directly from the Cyamopsis tetragonoloba seeds pericarb. The biopolymer hydrates hugely in three chain associated coil formations. Chaotropic Hofmeister ion like lithium interacts at the hydrogen bonding sites and render GG homogenization in polar solvents like dimethyl sulfoxide. This phenomenon was used for the first time for galactomannan derivatisations in homogeneous phase. Higher degree of substitution (DS) that was hereto unattainable in GG was achieved due to Hofmeister ion assisted assembly deformations. Furthermore, carboxymethyl guar gum (CMGG, DS = 1.10) blends well in poly-vinyl alcohol (PVA) at 2:1 mass ratio and enabled hydrophilic porous scaffold design for cell propagation. CMGG-PVA scaffolds porosity was 70–90% and the tensile strength was 6.32 MPa. CMGG-PVA scaffolds were useful as cell factories and in tissue engineering. New generation guar gum derivative scaffolds were non cytotoxic and permitted cell propagation in growth medium.

Trachea Mechanics for Tissue Engineering Design.

Trachea replacement for nonoperable defects remains an unsolved problem due to complications with stenosis and mechanical insufficiency. While native trachea has anisotropic mechanical properties, the vast majority of engineered constructs focus on uniform cartilaginous-like conduits. These conduits often lack quantitative mechanical analysis at the construct level, which limits analysis of functional outcomes in vivo, as well as comparisons across studies. This review aims to present a clear picture of native tracheal mechanics at the tissue and organ level, as well as loading conditions to establish design criteria for trachea replacements. We further explore the implications of failing to match native properties with regards to implant collapse, stenosis, and infection.

Endothelial Progenitor Cells for the Vascularization of Engineered Tissues.

Self-assembled microvasculature from cocultures of endothelial cells (ECs) and stromal cells has significantly advanced efforts to vascularize engineered tissues by enhancing perfusion rates in vivo and producing investigative platforms for microvascular morphogenesis in vitro. However, to clinically translate prevascularized constructs, the issue of EC source must be resolved. Endothelial progenitor cells (EPCs) can be noninvasively supplied from the recipient through adult peripheral and umbilical cord blood, as well as derived from induced pluripotent stem cells, alleviating antigenicity issues. EPCs can also differentiate into all tissue endothelium, and have demonstrated potential for therapeutic vascularization. Yet, EPCs are not the standard EC choice to vascularize tissue constructs in vitro. Possible reasons include unresolved issues with EPC identity and characterization, as well as uncertainty in the selection of coculture, scaffold, and culture media combinations that promote EPC microvessel formation. This review addresses these issues through a summary of EPC vascular biology and the effects of tissue engineering design parameters upon EPC microvessel formation. Also included are perspectives to integrate EPCs with emerging technologies to produce functional, organotypic vascularized tissues.

Proteomic Analysis of Nucleus Pulposus Cell-derived Extracellular Matrix Niche and Its Effect on Phenotypic Alteration of Dermal Fibroblasts

Reconstituting biomimetic matrix niche in vitro and culturing cells at the cell niche interface is necessary to understand the effect and function of the specific matrix niche. Here we attempted to reconstitute a biomimetic extracellular matrix (ECM) niche by culturing nucleus pulposus cells (NPCs) in a collagen microsphere system previously established and allowing them to remodel the template matrix. The reconstituted NPC-derived complex ECM was obtained after decellularization and the composition of such niche was evaluated by proteomic analysis. Results showed that a complex acellular matrix niche consisting of ECM proteins and cytoskeletal proteins by comparing with the template collagen matrix starting material. In order to study the significance of the NPC-derived matrix niche, dermal fibroblasts were repopulated in such niche and the phenotypes of these cells were changed, gene expression of collagen type II and CA12 increased significantly. A biomimetic NPC-derived cell niche consisting of complex ECM can be reconstituted in vitro, and repopulating such matrix niche with fibroblasts resulted in changes in phenotypic markers. This work reports a 3D in vitro model to study cell niche factors, contributing to future understanding of cellular interactions at the cell-niche interface and rationalized scaffold design for tissue engineering.

Designing 3D Biological Surfaces via the Breath‐Figure Method

The fabrication of biointerfaces that mimic cellular physiological environments is critical to understanding cell behaviors in vitro and for the design of tissue engineering. Breath figure is a self-assemble method that uses water droplets condensed from moisture as template and ends up with a highly ordered hexagonal pore array; this approach is used to fabricate various biological substrates. This progress report provides an overview of strategies to achieve topographical modifications and chemical-patterned arrays, such as modulation of the pore size, shape and selective decoration of the honeycomb holes. Using recent results in the biological fields, potential future applications and developments of honeycomb structures are commented upon.

The importance of factorial design in tissue engineering and biomaterials science: Optimisation of cell seeding efficiency on dermal scaffolds as a case study

This article presents a case study to show the usefulness and importance of using factorial design in tissue engineering and biomaterials science. We used a full factorial experimental design (2 × 2 × 2 × 3) to solve a routine query in every biomaterial research project: the optimisation of cell seeding efficiency for pre-clinical in vitro cell studies, the importance of which is often overlooked. In addition, tissue-engineered scaffolds can be cellularised with relevant cell type(s) to form implantable tissue constructs, where the cell seeding method must be reliable and robust. Our results show the complex relationship between cells and scaffolds and suggest that the optimum seeding conditions for each material may be different due to different material properties, and therefore, should be investigated for individual scaffolds. Our factorial experimental design can be easily translated to other cell types and three-dimensional biomaterials, where multiple interacting variables can be thoroughly investigated for better understanding of cell–biomaterial interactions.

Cells may feel a hard substrate even on a grafted layer of soft hydrogel.

Introducing or grafting molecules onto biomaterial surfaces to regulate cell destination via biophysical cues is one of the important steps for biomaterial design in tissue engineering. Understanding how cells feel the substrate makes it easier to learn the mechanism behind cell-material interaction. In this study, on a glass substrate, we constructed poly-phenoxyethyl methacrylate (PHEMA) brushes having different lengths via a surface-induced atom transfer radical polymerization (SI-ATRP) method. FTIR-ATR and XPS tests of the formed polymer brushes indicate that these brushes have characteristic chemical structures of PHEMA; the polymer brush length revealed by the AFM tests increases linearly with reaction time. Cell lines of BMSCs, ATDC5, and human chondrocytes (HC) were cultured on these substrates to evaluate proliferation, adhesion, and differentiation. Our results demonstrated that the cells cultured on the substrates with short PHEMA brushes developed a spread morphology and organized actin fibers as compared to the cells cultured on those with long brushes. Different cell lines showed different responses depending on the PHEMA brush length. Cells cultured on long PHEMA brushes displayed a more rounded shape, higher gene expression of FAK and integrin, and lower gene expression of NCAM and N-cadherin as compared to those, especially ATDC5 cells, cultured on short PHEMA brushes. On PHEMA brushes with a long length, the cell lines express higher cartilage-specific genes including Sox9 and Col2 and GAG in ECM. The results suggest that polymer brushes having different lengths may interfere with the behavior of the cells cultured on them.

Osteoinductive Activity of Bone Scaffold Bioceramic Companied with Control Release of VEGF Protein Treated Dental stem cells as A New Concept for Bone Regeneration: Part II

In bone tissue engineering, angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration. Over-expression of the angiogenesis-related gene due to a higher dose of vascular endothelial growth factor (VEGF) protein can inhibit osteogenesis process at mRNA level. To study the effect of controlled released of the VEGF protein incorporating fibrin glue (FG) treated with fabricated porous biphasic calcium phosphate (BCP) on osteogenesis gene (BMP-2) and angiogenesis gene (VEGF) on dental stem cells (DSCs) at mRNA level. DSCs were treated with two different modalities; VEGF protein incorporated FG, and VEGF protein incorporated FG added-BCP treated media. The cells were harvested at four different time intervals (day 3, day 7, day 10 and day 14) and were subjected to RNA isolation using the RNA extraction kit. This was followed by performing one step-reverse transcriptase-PCR (RT-PCR) to amplify the osteogenesis BMP-2 gene, angiogenesis VEGF gene and the osteoblast- specific transcription factor expression Osterix (Osx) with and without the controlled release of VEGF protein. The RT-PCR products were then electrophoresed. The gel image was captured using Image Analyser. Controlled release of VEGF protein using FG as a natural delivery system, using a single growth factor, show a significantly enhanced osteogenesis BMP-2 gene and angiogenesis VEGF gene with a high expression of Osx compare with non-delivered free VEGF protein treated groups. FG is a biocompatible material that could be employed as a delivery vehicle for controlled release of VEGF protein single or dual release in bone tissue engineering strategy and design of the study. Application of this method for using FG is mixing with a porous ceramic scaffold loading with the growth factors is a convenient and promising strategy for improving osteogenesis and angiogenesis processes of reconstruction critical-sized bone defects and might change the scope of modern surgery.

Micropatterned Geometry Shape Oligodendrocyte and Microglia Plasticity.

Cellular adhesion is essential in maintaining multicellular structure by connecting cells to each other in vivo and to a biomimetic substrate in vitro. In this context, one of the first steps toward the comprehension, for instance, of oligodendrocyte and microglia adhesion and migrating behavior consists in discriminating the different morphological features that can be acquired when cells are cultured on diverse surface topographies that mimic an in vivo three-dimensional environment. With this in mind, in this chapter, we describe how to exploit the silicon isotropic topography consisting of line-grating geometries and micropillar structures fabricated on polydimethylsiloxane by soft lithography. By reproducing a specialized niche for the cells, micropatterned biomimetic substrates can help to understand the role of structural determinants in priming morphogenesis of oligodendrocytes and microglia and can be exploited for translational research on functional tissue engineering and implantable device design.

Multiphoton Fabrication of Fibronectin-Functionalized Protein Micropatterns: Stiffness-Induced Maturation of Cell-Matrix Adhesions in Human Mesenchymal Stem Cells.

Cell-matrix adhesions are important structures governing the interactions between cells and their microenvironment at the cell-matrix interface. The focal complex (FC) and focal adhesion (FA) have been substantially investigated in conventional planar culture systems using fibroblasts as an in vitro model. However, the formation of more mature types of cell-matrix adhesion in human mesenchymal stem cells (hMSCs), including fibrillar adhesion (FBA) and 3D matrix adhesion (3DMA), have not been fully elucidated. Here we investigate the niche factor(s) that influence(s) the maturation of FBA and 3DMA by using multiphoton fabrication-based micropatterning. First, the bovine serum albumin (BSA)-made protein micropatterns were functionalized by incorporating various concentrations of fibronectin (FN) in fabrication solution. The amount of cross-linked FN is positively correlated with the initial concentration of FN in the reaction liquid, as verified by immunofluorescence staining. On the other hand, the anisotropic FN-functionalized micropatterns were fabricated by varying the length (i.e., in-plane stiffness) and height (i.e., bending stiffness) of micropatterns, respectively. Finally, hMSCs were cultured on these micropatterns for 2 h and 1 day to determine the formation of FBA and 3DMA, respectively, using immunofluorescence staining. Results demonstrated that FN-functionalized micropatterns with high anisotropy in x-y dimension benefit FBA maturation. Furthermore, niche factors such as higher bending and in-plane stiffness and the presence of abundant fibronectin have a positive effect on the maturation of FN-based cell-matrix adhesion. These findings could provide some new perspectives on designing platforms for further cell niche study and rationalizing scaffold design for tissue engineering.

Synthetic sutures: Clinical evaluation and future developments.

Today's sutures are the result of a 4000-year innovation process with regard to their materials and manufacturing techniques, yet little has been done to enhance the therapeutic value of the suture itself. In this review, we explore the historical development, regulatory database and clinical literature of sutures to gain a fuller picture of suture advances to date. First, we examine historical shifts in suture manufacturing companies and review suture regulatory databases to understand the forces driving suture development. Second, we gather the existing clinical evidence of suture efficacy from reviewing the clinical literature and the Food and Drug Administration database in order to identify to what extent sutures have been clinically evaluated and the key clinical areas that would benefit from improved suture materials. Finally, we apply tissue engineering and regenerative medicine design hypotheses to suture materials to identify routes by which bioactive sutures can be designed and passed through regulatory hurdles, to improve surgical outcomes. Our review of the clinical literature revealed that many of the sutures currently in use have been available for decades, yet have never been clinically evaluated. Since suture design and development is industry driven, incremental modifications have allowed for a steady outflow of products while maintaining a safe regulatory position and limiting costs. Until recently, there has been little academic interest in suture development, however the rise of regenerative medicine strategies is shifting the suture paradigm from an inert material, which mechanically approximates tissue, to a bioactive material, which also actively promotes cell-directed repair and a positive healing response. These materials hold significant therapeutic potential, but could be associated with an increased regulatory burden, cost, and clinical evaluation compared with current devices.

A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice

Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle–scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.