Chapter 19 - Three-dimensional scaffolds

Fabrication of a three-dimensional (3D) scaffold with increased mechanical strength may be an essential requirement for more advanced bone tissue engineering scaffolds. Various material- and chemical-based approaches have been explored to enhance the mechanical properties of engineered bone tissue scaffolds. In this study, the effects of pore architecture and stacking direction on the mechanical and cell proliferation properties of a scaffold were investigated. The 3D scaffold was prepared using solid freeform fabrication technology with a multihead deposition system. Various types of scaffolds with different pore architectures (lattice, stagger, and triangle types) and stacking directions (horizontal and vertical directions) were fabricated with a blend of polycaprolactone and poly lactic-co-glycolic acid. In compression tests, the triangle-type scaffold was the strongest among the experimental groups. Stacking direction affected the mechanical properties of scaffolds. An in vitro cell counting kit-8 assay showed no significant differences in optical density depending on the different pore architectures and stacking directions. In conclusion, mechanical properties of scaffolds can be enhanced by controlling pore architecture and stacking direction.

Tissue engineering applications commonly encompass the use of three-dimensional (3D) scaffolds to provide a suitable microenvironment for the incorporation of cells or growth factors to regenerate damaged tissues or organs. These scaffolds serve to mimic the actual in vivo microenvironment where cells interact and behave according to the mechanical cues obtained from the surrounding 3D environment. Hence, the material properties of the scaffolds are vital in determining cellular response and fate. These 3D scaffolds are generally highly porous with interconnected pore networks to facilitate nutrient and oxygen diffusion and waste removal. This review focuses on the various fabrication techniques (e.g., conventional and rapid prototyping methods) that have been employed to fabricate 3D scaffolds of different pore sizes and porosity. The different pore size and porosity measurement methods will also be discussed. Scaffolds with graded porosity have also been studied for their ability to better represent the actual in vivo situation where cells are exposed to layers of different tissues with varying properties. In addition, the ability of pore size and porosity of scaffolds to direct cellular responses and alter the mechanical properties of scaffolds will be reviewed, followed by a look at nature's own scaffold, the extracellular matrix. Overall, the limitations of current scaffold fabrication approaches for tissue engineering applications and some novel and promising alternatives will be highlighted.

A three-dimensional (3D) scaffold fabricated via electrohydrodynamic jet printing (E-jetting) and thermally uniaxial stretching, has been developed for tendon tissue regeneration in our previous study. In this study, more in-depth biological test showed that the aligned cell morphology guided by the anisotropic geometries of the 3D tendon scaffolds, leading to up-regulated tendious gene expression including collagen type I, decorin, tenascin-C, and biglycan, as compared to the electrospun scaffolds. Given the importance of geometric cues to the biological function of the scaffolds, the degradation behaviors of the 3D scaffolds were investigated. Results from accelerated hydrolysis showed that the E-jetted portion followed bulk-controlled erosion, while the unaixially stretched portion followed surface-controlled erosion. The 3D tendon scaffold exhibited consistency between the weight loss and the decline of mechanical properties, which indicated by a 65% decrease in mass with a corresponding 56% loss in ultimate tensile strength after degradation. This study not only reveals that the anisotropic geometries of 3D tendon scaffold could affect cell morphology and lead to desired gene expression toward tendon tissue but also gives an insight into how the degradation impacts geometric cues and mechanical properties of the as-fabricated scaffold. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1138-1149, 2017.

The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of mesenchymal stem cells expressing BMP7

Tissue engineering (TE) is envisaged to play a vital role in improving quality of life by restoring, maintaining or enhancing tissue and organ functions. TE scaffolds that are two-dimensional in structure suffer from undesirable issues, such as pore blockage, and do not closely mimic the native extra-cellular matrix in tissues. Significant efforts have therefore been channeled to fabricate three-dimensional (3D) scaffolds using various techniques, especially electrospinning. In this study, we propose a modified one-step electrospinning process to arrive at a 3D scaffold with highly interconnected pores. Using a blend of poly (L-lactide)/polycaprolactone/poly (ethylene oxide), this mechanically viable, sponge-like 3D scaffold exhibited sufficiently large pores and enabled cell penetration beyond 500 μm. Dexamethasone (Dex) was loaded into the fibers and a sustained drug release was achieved. Further, the potential of this Dex-loaded 3D scaffold was evaluated for upregulation of osteogenic genes with mesenchymal stem cells. The as-produced Dex-loaded 3D scaffold possesses a unique intertwined sub-micron fibrous morphology that can be tailored for use in bone tissue engineering and beyond.

Adipose-derived stromal cells (ADSCs) have been shown to increase angiogenesis in ischemic tissue. Maintaining cell survival and facilitating angiogenesis in ischemic tissue, however, continues to be the major challenge of ADSCs implantation. Recently, bioengineered scaffolds were introduced to support and facilitate cell culture and differentiation. The effects of a surface modified three-dimensional (3D) scaffold on ADSC function have not been investigated. Accordingly, the objective of this study was to determine the influence of a gas-plasma treated scaffold on ADSC growth, differentiation into endothelial cell, and angiogenic gene expression. Freshly isolated mouse ADSCs were characterized by flow cytometry and cultured into wells containing gas-plasma treated scaffolds, non-treated scaffolds, or control wells. Either endothelial growth media or differentiation media was used to alter cell environment. After 3 and 6 days, cell proliferation was analyzed. VEGF concentration in the medium was measured by ELISA. Gene expression was quantified by real-time PCR for VEGF receptor-2 (KDR), cyclooxygenase-2 (COX-2) and matrix metalloproteinases-2 (MMP-2). ADSCs expressed stem/endothelial progenitor markers CD34 and CD133 and endothelial cell marker CD31. ADSCs grew in the 3D scaffold. Cells grown on gas-plasma treated scaffolds displayed significantly increased expression of VEGF, COX-2, and MMP-2 when grown in differentiation but not growth media. When cultured in endothelial growth media, VEGF secretion and the expression of KDR, COX-2 and MMP-2 were lower in 3D scaffolds than controls. This study suggests that 3D scaffolds, especially gas-plasma treated scaffolds, support ADSC growth and support differentiation into endothelial cells.

Stem cell-based tissue engineering holds much hope for the development of multifunctional tissues to replace diseased organs. The attachment and survival of stem cells on a three-dimensional (3D) scaffold must be enhanced for faster progression of stem cell based tissue engineering. This study evaluate the stability of mesenchymal stem cells (MSCs) in 3D porous scaffolds composed of a collagen and chitosan blend impregnated with epidermal growth factor incorporated chitosan nanoparticles (EGF-CNP). The EGF-CNP scaffolds were characterized by transmission electron microscopy, which revealed that the nanoparticles were round in shape and 20 ∼ 50 nm in size. The scaffolds were prepared by freeze drying. A Fourier-transform infrared spectrum study revealed that the linkage between collagen and chitosan was through an ionic interaction. Thermal analysis and degradation studies showed that the scaffold could be used in tissue engineering application. MSCs proliferated well in the EGF-CNP impregnated scaffold. A scanning electron microscope study showed anchored and elongated MSCs on the EGF-CNP impregnated scaffold. A 3D biodegradable collagen chitosan scaffold impregnated with EGF-CNP is a promising transportable candidate for MSC-based tissue engineering, and this scaffold could be used as an in vitro model for subsequent clinical applications.

Design and characterization of microcapsules-integrated collagen matrixes as multifunctional three-dimensional scaffolds for soft tissue engineering

Simple SummaryThis study aims to overcome the current methodological limitations in discovering new therapeutic targets. Therefore, we optimized and validated a co-culture system using decellularized human liver three-dimensional (3D) scaffolds obtained from healthy and cirrhotic human livers for anti-fibrotic and anti-cancer dual drug screening. Both platforms mimic the naturally healthy and physio-pathological microenvironment and are able to recapitulate the key cellular and molecular events leading to liver fibrogenesis and cancer. This study demonstrates the differences between single versus co-cultures and the usage of human-derived liver 3D ECM scaffolds from healthy and cirrhotic livers. As lead compounds, we used Sorafenib and Regorafenib, first- and second-line drugs, and identified two different drug-induced mechanisms depending on the 3D ECM microenvironment. The 3D ECM scaffolds may represent innovative platforms for disease modeling, biomarker discovery, and drug testing in fibrosis and primary cancer.The introduction of new preclinical models for in vitro drug discovery and testing based on 3D tissue-specific extracellular matrix (ECM) is very much awaited. This study was aimed at developing and validating a co-culture model using decellularized human liver 3D ECM scaffolds as a platform for anti-fibrotic and anti-cancer drug testing. Decellularized 3D scaffolds obtained from healthy and cirrhotic human livers were bioengineered with LX2 and HEPG2 as single and co-cultures for up to 13 days and validated as a new drug-testing platform. Pro-fibrogenic markers and cancer phenotypic gene/protein expression and secretion were differently affected when single and co-cultures were exposed to TGF-β1 with specific ECM-dependent effects. The anti-fibrotic efficacy of Sorafenib significantly reduced TGF-β1-induced pro-fibrogenic effects, which coincided with a downregulation of STAT3 phosphorylation. The anti-cancer efficacy of Regorafenib was significantly reduced in 3D bioengineered cells when compared to 2D cultures and dose-dependently associated with cell apoptosis by cleaved PARP-1 activation and P-STAT3 inhibition. Regorafenib reversed TGF-β1-induced P-STAT3 and SHP-1 through induction of epithelial mesenchymal marker E-cadherin and downregulation of vimentin protein expression in both co-cultures engrafting healthy and cirrhotic 3D scaffolds. In their complex, the results of the study suggest that this newly proposed 3D co-culture platform is able to reproduce the natural physio-pathological microenvironment and could be employed for anti-fibrotic and anti-HCC drug screening.

Fabrication of a thick three-dimensional scaffold with an open cellular-like structure using airbrushing and thermal cross-linking of molded short nanofibers

BackgroundHydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM.ResultsThe physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope.ConclusionsOur findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.

Three-dimensional CaP/gelatin lattice scaffolds with integrated osteoinductive surface topographies for bone tissue engineering

Three-dimensional (3D) scaffolds with chemical diversity are significant to direct cell adhesion onto targeted surfaces, which provides solutions to further control over cell fates and even tissue formation. However, the site-specific modification of specific biomolecules to realize selective cell adhesion has been a challenge with the current methods when building 3D scaffolds. Conventional methods of immersing as-prepared structures in solutions of biomolecules lead to nonselective adsorption; recent printing methods have to address the problem of switching multiple nozzles containing different biomolecules. The recently developed concept of macroscopic supramolecular assembly (MSA) based on the idea of "modular assembly" is promising to fabricate such 3D scaffolds with advantages of flexible design and combination of diverse modules with different surface chemistry. Herein we report an MSA method to fabricate 3D ordered structures with internal chemical diversity for site-selective cell adhesion. The 3D structure is prepared via 3D alignment of polydimethylsiloxane (PDMS) building blocks with magnetic pick-and-place operation and subsequent interfacial bindings between PDMS based on host/guest molecular recognition. The site-specific cell affinity is realized by distributing targeted building blocks that are modified with polylysine molecules of opposite chiralities: PDMS modified with films containing poly-l-lysine (PLL) show higher cell density than those with poly-d-lysine (PDL). This principle of selective cell adhesion directed simply by spatial distribution of chiral molecules has been proven effective for five different cell lines. This facile MSA strategy holds promise to build complex 3D microenvironment with on-demand chemical/biological diversities, which is meaningful to study cell/material interactions and even tissue formation.

Studies have been conducted on the pathogenicity of periodontopathogens in cultures of endothelial cells on two-dimensional (2D) polystyrene surfaces, where the monolayer formed is not exposed to proteins of the subendothelial matrix. In this work, we developed a culture system by seeding human coronary artery endothelial cells (HCAECs) onto three-dimensional (3D) scaffolds of collagen type I, a subendothelial protein. The inflammatory responses of the HCAEC monolayers, formed either on 3D scaffolds or directly on a 2D polystyrene plate, to lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) were evaluated. The transcription of 3 genes, the secretion of 40 cytokines and 2 prostanoids, and the adhesion of monocytes to 2D and 3D cultures with or without exposure to lipopolysaccharides (control) were assessed. HCAECs exhibited differences in transcriptional and secretory profiles between the 3D and 2D models. In addition, the inflammatory responses of HCAEC to Aa-LPS and Pg-LPS differed between the two models. In 3D cultures treated with Aa-LPS, the levels of IL-8, RANTES, G-CSF, ICAM-1, IL-6, and TXA2 were significantly higher than those in the controls. In 2D cultures treated with Aa-LPS, IL-8, RANTES, G-CSF, ICAM-1, TNF-RI, PGI2, and TXA2 levels were significantly higher than those in their controls. In the presence of Aa-LPS, monocyte adhesion did not differ between treated and control 3D cultures but was significantly higher in treated 2D cultures than in the controls. In response to Pg-LPS, cytokine-prostaglandin secretion and monocyte adhesion did not differ between 3D and 2D cultures. These data indicate that HCAECs respond differently to these two types of LPS.

The objective of this study was to evaluate the effect of beta-tricalcium phosphate (beta-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the beta-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from beta-TCP beads with diameters ranging from 300 to 500 microm (small beads), 500-700 microm (medium beads) and 700-850 microm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context.