Scaffold-based Tissue Engineering Approaches Research Articles

Biomolecule functionalized PVA-chitosan composite nanofibrous scaffold for human endometrium stromal cells attachment and proliferation: An in-vitro study

Human endometrial thickness (>7 mm) has been a prognostic factor for successful embryo implantation in natural pregnancy and assisted reproductive technology (ART). Functionalized scaffold-based tissue engineering approach has shown good potency in the functional repair of endometrium. In this study, we report biomolecule functionalized polyvinyl alcohol-chitosan (PVA-CH) composite nanofibrous scaffold for attachment and proliferation of human endometrial stromal cells (HESC) to improve endometrial thickness. The interconnected nanoporous architecture and sustained release of biomolecules supported HESC attachment, proliferation and extracellular matrix secretion on the developed scaffold. The prepared biomolecule-functionalized PVA-CH composite nanofibrous scaffold was biocompatible and biodegradable making it suitable for endometrial tissue regeneration applications.

Recent scaffold-based tissue engineering approaches in premature ovarian failure treatment.

Recently, tissue engineering and regenerative medicine have received significant attention with outstanding advances. The main scope of this technology is to recover the damaged tissues and organs or to maintain and improve their function. One of the essential fields in tissue engineering is scaffold designing and construction, playing an integral role in damaged tissues reconstruction and repair. However, premature ovarian failure (POF) is a disorder causing many medical and psychological problems in women. POF treatment using tissue engineering and various scaffold has recently made tremendous and promising progress. Due to the importance of the subject, we have summarized the recently examined scaffolds in the treatment of POF in this review.

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches. Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method. The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Genipin crosslinked chitosan/PEO nanofibrous scaffolds exhibiting an improved microenvironment for the regeneration of articular cartilage.

Towards optimizing the growth of extracellular matrix to produce repair cartilage for healing articular cartilage (AC) defects in joints, scaffold-based tissue engineering approaches have recently become a focus of clinical research. Scaffold-based approaches by electrospinning aim to support the differentiation of chondrocytes by providing an ultrastructure similar to the fibrillar meshwork in native cartilage. In a first step, we demonstrate how the blending of chitosan with poly(ethylene oxide) (PEO) allows concentrated chitosan solution to become electrospinnable. The chitosan-based scaffolds share the chemical structure and characteristics of glycosaminoglycans, which are important structural components of the cartilage extracellular matrix. Electrospinning produced nanofibrils of ∼100 nm thickness that are closely mimicking the size of collagen fibrils in human AC. The polymer scaffolds were stabilized in physiological conditions and their stiffness was tuned by introducing the biocompatible natural crosslinker genipin. We produced scaffolds that were crosslinked with 1.0% genipin to obtain values of stiffness that were in between the stiffness of the superficial zone human AC of 600 ± 150 kPa and deep zone AC of 1854 ± 483 kPa, whereas the stiffness of 1.5% genipin crosslinked scaffold was similar to the stiffness of deep zone AC. The scaffolds were degradable, which was indicated by changes in the fibril structure and a decrease in the scaffold stiffness after seven months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes (HACs) showed a cell viability of over 90% on the scaffolds and new extracellular matrix deposited on the scaffolds.

3D printing of tissue engineering scaffolds: a focus on vascular regeneration.

Tissue engineering is an emerging means for resolving the problems of tissue repair and organ replacement in regenerative medicine. Insufficient supply of nutrients and oxygen to cells in large-scale tissues has led to the demand to prepare blood vessels. Scaffold-based tissue engineering approaches are effective methods to form new blood vessel tissues. The demand for blood vessels prompts systematic research on fabrication strategies of vascular scaffolds for tissue engineering. Recent advances in 3D printing have facilitated fabrication of vascular scaffolds, contributing to broad prospects for tissue vascularization. This review presents state of the art on modeling methods, print materials and preparation processes for fabrication of vascular scaffolds, and discusses the advantages and application fields of each method. Specially, significance and importance of scaffold-based tissue engineering for vascular regeneration are emphasized. Print materials and preparation processes are discussed in detail. And a focus is placed on preparation processes based on 3D printing technologies and traditional manufacturing technologies including casting, electrospinning, and Lego-like construction. And related studies are exemplified. Transformation of vascular scaffolds to clinical application is discussed. Also, four trends of 3D printing of tissue engineering vascular scaffolds are presented, including machine learning, near-infrared photopolymerization, 4D printing, and combination of self-assembly and 3D printing-based methods.

An Overview of Scaffolds for Retinal Pigment Epithelium Research

The reduced capabilities of retinal pigment epithelium (RPE) marks early retinal degenerative diseases and leads to blindness. Regenerating a functional RPE monolayer in vitro becomes significant and promise for developing new therapies. Many research activities have been done to develop new materials, structures and fabrication technologies to mimic native RPE structure for cell attachment and subsequent tissue development. Scaffold-based tissue engineering approaches attract intensive investigation, due to their capability and potential in building preclinical models and developing possible treatment for retinal degenerative diseases. Scaffolds in facilitating regeneration of RPE tissue in vitro has been proved by recent studies. Various materials have been applied, such as synthetic polymers, plant or animal extracted proteins, and composite materials. This review outlines the most recent fabrication progresses using these materials for RPE scaffolds, including both in vitro and in vivo trials, and the challenges in building a whole tissue model construct and transplantation. Additive manufacturing technologies at micrometer- and nanometer-scale are proposed as promising methods to build biomimetic RPE scaffolds in the current studies.

3D perfusion culture of mouse insulinoma in macro-porous scaffolds enhanced insulin production response.

To address the remaining issue of poor cell immobilization and insufficient mass transfer in scaffold-based tissue engineering approach for future islet transplantation, we employed a macro-porous poly-l-lactide (PLLA) scaffold immobilizing mouse insulinoma cells and studied its function toward an implantable pancreatic tissue in 7-day perfusion culture. The murine pancreatic β cells could be immobilized in the PLLA scaffold at a high density of 107 cells per cm3 close to the estimated range in normal pancreas. The perfusion culture promoted the 3D cellular organization as observed with live/dead staining and histological staining. The insulin production was significantly enhanced in comparison with static 2D culture and 3D rotational suspension culture by two and six folds, respectively (p < 0.001). As enhanced insulin response was only observed where both the perfusion and 3D cellular organization were present, this could represent important elements in engineering a functional bioartificial pancreas.

3D printing of silver-doped polycaprolactone-poly(propylene succinate) composite scaffolds for skin tissue engineering

Scaffold-based tissue engineering approaches have been commonly used for skin regeneration or wound healings caused by diseases or trauma. For an ideal complete healing process, scaffold structures need to meet the criteria of biocompatibility, biodegradability, and antimicrobial properties, as well as to provide geometrical necessities for the regeneration of damaged tissue. In this study, design, synthesis and characterization of a three dimensional (3D) printable copolymer based on polycaprolactone-block-poly(1,3-propylene succinate) (PCL-PPSu) including anti-microbial silver particles is presented. 3D printing of PCL-PPSu copolymers provided a lower processing temperature compared to neat PCL, hence, inclusion of temperature-sensitive bioactive reagents into the developed copolymer could be realized. In addition, 3D printed block copolymer showed an enhanced hydrolytic and enzymatic degradation behavior. Cell viability and cytotoxicity of the developed copolymer were evaluated by using human dermal fibroblast (HDF) cells. The addition of silver nitrate within the polymer matrix resulted in a significant decrease in the adhesion of different types of microorganisms on the scaffold without inducing any cytotoxicity on HDF cells in vitro. The results suggested that 3D printed PCL-PPSu scaffolds containing anti-microbial silver particles could be considered as a promising biomaterial for emerging skin regenerative therapies, in the light of its adaptability to 3D printing technology, low-processing temperature, enhanced degradation behavior and antimicrobial properties.

Scaffold-based tissue engineering approaches in treating infertility

Tissue engineering and the use of scaffolds have shown high therapeutic potentialities about male and female infertility. Nowadays, many couples are suffering from infertility problems. There are different causes for infertility including chemotherapy (for male and female), uterine injuries, and intrauterine adhesions. Extra-cellular matrix in tissue engineering provides a supportive medium for blood or lymphatic vessels making it a suitable substrate for cell implantation and growth. Dominant successes in this branch have been in use of patient-derived primary cells, these cells loaded in scaffolds and used to generate tissue for re-implantation. However, this method has limitations, because of the invasive nature of cell collection, also the cells patient-derived may be not healthy and become the source of disease. Therefore, use of stem cells, including embryonic stem (ES) cells, bone marrow mesenchymal stem cells (BM-MSCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been considered. Cell/scaffold systems have a substantial role in fertility organs or agents repair or regeneration. This review summarizes the novel scaffold-based tissue engineering approaches to treat infertility.

Replacement of Rat Tracheas by Layered, Trachea-Like, Scaffold-Free Structures of Human Cells Using a Bio-3D Printing System.

Current scaffold-based tissue engineering approaches are subject to several limitations, such as design inflexibility, poor cytocompatibility, toxicity, and post-transplant degradation. Thus, scaffold-free tissue-engineered structures can be a promising solution to overcome the issues associated with classical scaffold-based materials in clinical transplantation. The present study seeks to optimize the culture conditions and cell combinations used to generate scaffold-free structures using a Bio-3D printing system. Human cartilage cells, human fibroblasts, human umbilical vein endothelial cells, and human mesenchymal stem cells from bone marrow are aggregated into spheroids and placed into a Bio-3D printing system with dedicated needles positioned according to 3D configuration data, to develop scaffold-free trachea-like tubes. Culturing the Bio-3D-printed structures with proper flow of specific medium in a bioreactor facilitates the rearrangement and self-organization of cells, improving physical strength and tissue function. The Bio-3D-printed tissue forms small-diameter trachea-like tubes that are implanted into rats with the support of catheters. It is confirmed that the tubes are viable in vivo and that the tracheal epithelium and capillaries proliferate. This tissue-engineered, scaffold-free, tubular structure can represent a significant step toward clinical application of bioengineered organs.

Fibre-based scaffolding techniques for tendon tissue engineering.

Tendon refers to a band of tough, regularly arranged, and connective tissue connecting muscle and bone, transferring strength from muscle to bone, and enabling articular stability and movement. The limitations of natural tendon grafts motivate the scaffold-based tissue engineering (TE) approaches, which aim to build patient-specific biological substitutes that can repair the damaged or diseased tissues. Advances in engineering and knowledge of chemistry and biology have brought forth numerous fibre-based technologies, including electrospinning, electrohydrodynamic jet printing, electrochemical alignment technique, and other fibre-assembly technologies, which enable the fabrication of tendon tissue structure in 3-dimension. Textile techniques such as knitting and braiding have also been performed based on the fibrous materials to produce more complex structure. These scaffolds showed great similarity with native tendons in architectural features, mechanical properties, and facilitate biological functionality such as cellular adhesion, ingrowth, proliferation, and differentiation towards tendon tissue. Herein, we review the techniques that have been used to assemble fibres into scaffolds for tendon TE application. The morphological structures, mechanical properties, materials, degradation characteristics, and biological activities of the induced scaffolds were compared. The existing challenges and future prospects of fibre-based tendon TE have also been discussed.

Complete tissue coverage achieved by scaffold-based tissue engineering in the fetal sheep model of Myelomeningocele

Myelomeningocele (MMC) is the most severe form of spina bifida, one of the most common congenital anomalies. Although open fetal surgical repair of the MMC defect has been shown to result in improved outcomes, a less invasive approach applicable earlier in gestation than the current open surgical approach between 19 and 26 weeks of gestation is desirable for further improvement of neurological symptoms, as well as reduction of maternal and fetal risks. We previously reported the therapeutic potential of a scaffold-based tissue engineering approach in a fetal rat MMC model. The objective of this study was to confirm the long-term efficacy of this approach in the surgically created fetal sheep MMC model. Gelatin-based or gelatin/collagen hybrid sponges were prepared with and without basic fibroblast growth factor (bFGF) incorporation. The defect was covered by a sponge and secured by a supporting sheet with adhesive at 100 days of gestation or the gelatin/collagen hybrid with bFGF was secured with adhesive without the sheet. Although sheets were found detached at term (140 days' gestation), both gelatin-based and gelatin/collagen hybrid sponges had integrated within the newly formed granulation tissue, resulting in complete coverage of the MMC defect. The release of bFGF from sponges resulted in enhanced formation of granulation tissue and epithelialization. There was also evidence of improved preservation of the spinal cord with less associated damage on histological analysis and reversal of hindbrain herniation. These experiments provide important proof-of-principle evidence of the efficacy of scaffold-based tissue engineered coverage for the prenatal treatment of MMC.

Silicon nanowire-induced maturation of cardiomyocytes derived from human induced pluripotent stem cells.

The current inability to derive mature cardiomyocytes from human pluripotent stem cells has been the limiting step for transitioning this powerful technology into clinical therapies. To address this, scaffold-based tissue engineering approaches have been utilized to mimic heart development in vitro and promote maturation of cardiomyocytes derived from human pluripotent stem cells. While scaffolds can provide 3D microenvironments, current scaffolds lack the matched physical/chemical/biological properties of native extracellular environments. On the other hand, scaffold-free, 3D cardiac spheroids (i.e., spherical-shaped microtissues) prepared by seeding cardiomyocytes into agarose microwells were shown to improve cardiac functions. However, cardiomyocytes within the spheroids could not assemble in a controlled manner and led to compromised, unsynchronized contractions. Here, we show, for the first time, that incorporation of a trace amount (i.e., ∼0.004% w/v) of electrically conductive silicon nanowires (e-SiNWs) in otherwise scaffold-free cardiac spheroids can form an electrically conductive network, leading to synchronized and significantly enhanced contraction (i.e., >55% increase in average contraction amplitude), resulting in significantly more advanced cellular structural and contractile maturation.

Emergence of scaffold-free approaches for tissue engineering musculoskeletal cartilages.

This review explores scaffold-free methods as an additional paradigm for tissue engineering. Musculoskeletal cartilages-for example articular cartilage, meniscus, temporomandibular joint disc, and intervertebral disc-are characterized by low vascularity and cellularity, and are amenable to scaffold-free tissue engineering approaches. Scaffold-free approaches, particularly the self-assembling process, mimic elements of developmental processes underlying these tissues. Discussed are various scaffold-free approaches for musculoskeletal cartilage tissue engineering, such as cell sheet engineering, aggregation, and the self-assembling process, as well as the availability and variety of cells used. Immunological considerations are of particular importance as engineered tissues are frequently of allogeneic, if not xenogeneic, origin. Factors that enhance the matrix production and mechanical properties of these engineered cartilages are also reviewed, as the fabrication of biomimetically suitable tissues is necessary to replicate function and ensure graft survival in vivo. The concept of combining scaffold-free and scaffold-based tissue engineering methods to address clinical needs is also discussed. Inasmuch as scaffold-based musculoskeletal tissue engineering approaches have been employed as a paradigm to generate engineered cartilages with appropriate functional properties, scaffold-free approaches are emerging as promising elements of a translational pathway not only for musculoskeletal cartilages but for other tissues as well.

Design and fabrication of scaffold-based tissue engineering

Scaffold-based tissue engineering approaches have been under investigation for more than 30 years now and many different techniques have been developed in order to engineer various tissues of the body. Some of them have been translated from bench to bedside, yet many are still under intensive examination. Biodegradable scaffolds applied in tissue engineering aim to temporarily substitute for the extracellular matrix and its complex biological functions during the regeneration and/or remodelling period, and are subsequently degraded and replaced by new tissue. We herein discuss how knowledge from developmental biology and collective tissue healing biology can be applied to optimise the design of scaffolds that promote fundamental steps in tissue regeneration by creating a suitable microenvironment. The review focuses on the design and fabrication of scaffolds for bone tissue engineering, yet also highlights general considerations which apply to scaffolds used in any tissue engineering strategy. We then turn to scaffold manufacturing where a plethora of design and fabrication technologies have been applied to process biomaterials into scaffolds for bone tissue engineering and other applications. Each manufacturing technology has its advantages and disadvantages from a processing, material science and biological point of view. We herein review several additive manufacturing technologies, which in the authors' opinion currently are most relevant for scaffold-based bone tissue engineering. We also provide a future outlook on Image-guided Tissue Engineering, Additive Tissue Manufacturing and Guided Tissue Regeneration with respect to tissue engineering in general and bone tissue engineering in particular.

Repair of meniscal defect using an induced myoblast-loaded polyglycolic acid mesh in a canine model

Defects of the meniscus greatly alter knee function and predispose the joint to degenerative changes. The purpose of this study was to test a recently developed cell-scaffold combination for the repair of a critical-size defect in the canine medial meniscus. A bilateral, complete resection of the anterior horn of the medial meniscus was performed in 18 Beagle canines. A PLGA scaffold was implanted into the defect of one knee of 6 canines and the contralateral defect was left untreated. Scaffolds loaded with autologous myoblasts and cultured in a chondrogenic medium for 14 days were implanted in a second series of 12 canines. Empty scaffolds were implanted in the contralateral knees. Menisci were harvested at 12 weeks. Untreated defects had a muted fibrous healing response. Defects treated with cell-free implants also showed predominantly fibrous tissue, whereas fibrocartilage was present in several scaffolds. The thickness of the repair tissue after treatment with cell-free scaffolds was significantly greater compared to the controls (p<0.05). Pre-cultured implants integrated with the host tissue, and 9 of 12 contained meniscus-like fibrocartilage when compared to 2 of the 12 controls (p<0.05). The thickness of the pre-cultured implant repair tissue was greater compared to the controls (p<0.05). This study demonstrates the repair of a critical size meniscal defect using a stem cell and scaffold-based tissue engineering approach.

Thermoreversible Hydrogel for In Situ Generation and Release of HepG2 Spheroids

Organ printing is an alternative to the classic scaffold-based tissue engineering approach in which functional living macrotissues and organ constructs are fabricated by assembly of the building blocks: microtissue spheroids. However, the method for scalable fabrication of cell spheroids does not exist yet. We propose here that it may be a suitable one to generate cell spheroids in thermoreversible hydrogel scaffold, followed by liquefying the scaffold and releasing the generated spheroids. We show that concentrated poly(N-isopropylacrylamide-co-acrylic acid) microgel dispersions solidify upon heating and liquefy upon cooling. A hysteresis in the cooling process was observed and explained by the slow kinetics of the dissolution of the aggregated polymer chains in the cooling process due to additional intra- and interchain interactions. Hep G2 cells are seeded by simple mixing the cells with the microgel dispersions at room temperature. Cell/scaffold constructs form in situ when heated to 37 °C. The cells proliferate and form multicellular spheroids. When brought back to room temperature, the hydrogel scaffolds liquefy, thus, releasing the generated cell spheroids. The released spheroids can attach on the cell culture plate, disassemble, and spread on the substrate, confirming the cell viability. The whole process is carried out under mild conditions and does not involve any toxic additives, which may introduce injury to the cells or DNA. It is scalable and may meet the need for large scale fabrication of cell spheroids for organ printing.

Additive and synergistic effects of bFGF and hypoxia on leporine meniscus cell-seeded PLLA scaffolds.

Injuries to avascular regions of menisci do not heal and result in significant discomfort to patients. Current treatments, such as partial meniscectomy, alleviate these symptoms in the short term but lead to premature osteoarthritis as a result of compromised stability and changes in knee biomechanics. Thus, tissue engineering of the meniscus may provide an alternative treatment modality to overcome this problem. In this experiment, a scaffold-based tissue-engineering approach was utilized to regenerate the meniscus. Meniscus cells were cultured on poly-L-lactic acid scaffolds in normoxic (approximately 21% oxygen) or hypoxic (approximately 2% oxygen) conditions in the presence or absence of the growth factor, basic fibroblast growth factor (bFGF). At t = 4 weeks, histological sections of constructs showed presence of collagen and glycosaminoglycan (GAG) in all groups. Immunohistochemical staining showed the presence of collagen I in all groups and collagen II in groups cultured under hypoxic conditions. bFGF in the culture medium significantly increased cell number/construct by 25%, regardless of culture conditions. For GAG/construct, synergistic increases were observed in constructs cultured in hypoxic conditions and bFGF (two-fold) when compared to constructs cultured in normoxic conditions. Compressive tests showed synergistic increases in the relaxation modulus and coefficient of viscosity and additive increases in the instantaneous modulus for constructs cultured under hypoxic conditions and bFGF, when compared to constructs cultured under normoxic conditions. Overall, these results demonstrate that bFGF and hypoxia can significantly enhance the ability of meniscus cells to produce GAGs and improve the compressive properties of tissue-engineered meniscus constructs in vitro.

Stem cell based tissue engineering for meniscus repair

Defects of the meniscus greatly alter knee function and predispose the joint to degenerative changes. The purpose of this study was to test a recently developed cell-scaffold combination for the repair of a critical-size defect of the rabbit medial meniscus. A bilateral, complete resection of the pars intermedia of the medial meniscus was performed in 18 New Zealand White rabbits. A hyaluronan/gelatin composite scaffold was implanted into the defect of one knee of 6 rabbits and the contralateral defect was left untreated. Scaffolds loaded with autologous marrow-derived mesenchymal stem cells and cultured in a chondrogenic medium for 14 days were implanted in a second series of 12 rabbits. Empty scaffolds were implanted in the contralateral knees. Meniscii were harvested at 12 weeks. Untreated defects had a muted fibrous healing response. Defects treated with cell-free implants showed also predominantly fibrous tissue whereas fibrocartilage was present in some scaffolds. The cross-sectional width of the repair tissue after treatment with cell-free scaffolds was significantly greater than controls (p < 0.05). Pre-cultured implants integrated with the host tissue and 8 of 11 contained meniscus-like fibrocartilage, compared with 2 of 11 controls (p < 0.03). The mean cross-sectional width of the pre-cultured implant repair tissue was greater than controls (p < 0.004). This study demonstrates the repair of a critical size meniscal defect with a stem cell and scaffold based tissue engineering approach.

Microrobotics and MEMS‐Based Fabrication Techniques for Scaffold‐Based Tissue Engineering

Scaffold based tissue engineering strategies use cells, biomolecules and a scaffold to promote the repair and regeneration of tissues. Although scaffold-based tissue engineering approaches are being actively developed, most are still experimental, and it is not yet clear what defines an ideal scaffold/cell construct. Solid free form fabrication (SFF) techniques can precisely control matrix architecture (size, shape, interconnectivity, branching, geometry and orientation). The SFF methods enable the fabrication of scaffolds with various designs and material compositions, thus providing a control of mechanical properties, biological effects and degradation kinetics. This paper reviews the application of micro-robotics and MEMS-based fabrication techniques for scaffold design and fabrication. It also presents a novel robotic technique to fabricate scaffold/cell constructs for tissue engineering by the assembly of microscopic building blocks.