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15 pages, 4641 KiB

Open AccessArticle

Screening of Additive Manufactured Scaffolds Designs for Triple Negative Breast Cancer 3D Cell Culture and Stem-Like Expansion

by Emma Polonio-Alcalá, Marc Rabionet, Antonio J. Guerra, Marc Yeste, Joaquim Ciurana and Teresa Puig

Int. J. Mol. Sci. 2018, 19(10), 3148; https://doi.org/10.3390/ijms19103148 - 12 Oct 2018

Cited by 26 | Viewed by 5609

Abstract

Breast cancer stem cells (BCSCs) are tumor-initiating cells responsible for metastasis and tumor reappearance, but their research is limited by the impossibility to cultivate them in a monolayer culture. Scaffolds are three-dimensional (3D) cell culture systems which avoid problems related with culturing BCSC. [...] Read more.

Breast cancer stem cells (BCSCs) are tumor-initiating cells responsible for metastasis and tumor reappearance, but their research is limited by the impossibility to cultivate them in a monolayer culture. Scaffolds are three-dimensional (3D) cell culture systems which avoid problems related with culturing BCSC. However, a standardized scaffold for enhancing a BCSC population is still an open issue. The main aim of this study is to establish a suitable poly (lactic acid) (PLA) scaffold which will produce BCSC enrichment, thus allowing them to be studied. Different 3D printing parameters were analyzed using Taguchi experimental design methods. Several PLA scaffold architectures were manufactured using a Fused Filament Fabrication (FFF) 3D printer. They were then evaluated by cell proliferation assay and the configurations with the highest growth rates were subjected to BCSC quantification by ALDH activity. The design SS1 (0.2 mm layer height, 70% infill density, Zigzag infill pattern, 45° infill direction, and 100% flow) obtained the highest proliferation rate and was capable of enhancing a ALDH+ cell population compared to 2D cell culture. In conclusion, the data obtained endorse the PLA porous scaffold as useful for culturing breast cancer cells in a microenvironment similar to in vivo and increasing the numbers of BCSCs. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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19 pages, 5928 KiB

Open AccessArticle

Hydrogen Sulfide-Releasing Fibrous Membranes: Potential Patches for Stimulating Human Stem Cells Proliferation and Viability under Oxidative Stress

by Ilaria Cacciotti, Matteo Ciocci, Emilia Di Giovanni, Francesca Nanni and Sonia Melino

Int. J. Mol. Sci. 2018, 19(8), 2368; https://doi.org/10.3390/ijms19082368 - 11 Aug 2018

Cited by 67 | Viewed by 4992

Abstract

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large [...] Read more.

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large surface area-to-volume ratio for an optimal release of gaseous signaling molecules. Recently, the antioxidant and anti-inflammatory properties of the endogenous gasotramsmitter hydrogen sulfide (H₂S), as well as its ability to stimulate relevant biochemical processes on the growth of mesenchymal stem cells (MSC), have been investigated. Therefore, in this work, new poly(lactic) acid fibrous membranes (PFM), doped and functionalized with H₂S slow-releasing donors extracted from garlic, were synthetized. These innovative H₂S-releasing mats were characterized for their morphological, thermal, mechanical, and biological properties. Their antimicrobial activity and effects on the in vitro human cardiac MSC growth, either in the presence or in the absence of oxidative stress, were here assessed. On the basis of the results here presented, these new H₂S-releasing PFM could represent promising and low-cost scaffolds or patches for biomedical applications in tissue repair. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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20 pages, 19826 KiB

Open AccessArticle

Towards a Novel Patch Material for Cardiac Applications: Tissue-Specific Extracellular Matrix Introduces Essential Key Features to Decellularized Amniotic Membrane

by Matthias Becker, Janita A. Maring, Maria Schneider, Aarón X. Herrera Martin, Martina Seifert, Oliver Klein, Thorsten Braun, Volkmar Falk and Christof Stamm

Int. J. Mol. Sci. 2018, 19(4), 1032; https://doi.org/10.3390/ijms19041032 - 29 Mar 2018

Cited by 38 | Viewed by 6991

Abstract

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining [...] Read more.

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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17 pages, 2059 KiB

Open AccessArticle

Comparison of Human Dermal Fibroblasts and HaCat Cells Cultured in Medium with or without Serum via a Generic Tissue Engineering Research Platform

by Christopher Michael Gabbott and Tao Sun

Int. J. Mol. Sci. 2018, 19(2), 388; https://doi.org/10.3390/ijms19020388 - 28 Jan 2018

Cited by 17 | Viewed by 7556

Abstract

A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue [...] Read more.

A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) mono- or co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative in medium without serum than with serum. However, both cell types were successfully co-cultured in 2D using serum-free medium if the initial cell seeding density was higher than 80,000 cells/cm² (with 1:1 ratio). Based on the results from 2D cultures, co-culture of both cell types on modular substrates with small open pores (125 μm) and cellulosic scaffolds with open pores of varying sizes (50–300 µm) were then conducted successfully in serum-free medium. This study demonstrated that the generic research platform had great potential for in-depth understanding of HDFs and HaCat cells cultivated in serum-free medium, which could inform the processes for manufacturing skin cells or tissues for clinical applications. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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18 pages, 4276 KiB

Open AccessArticle

Biotherapeutic Effect of Gingival Stem Cells Conditioned Medium in Bone Tissue Restoration

by Francesca Diomede, Agnese Gugliandolo, Domenico Scionti, Ilaria Merciaro, Marcos FXB Cavalcanti, Emanuela Mazzon and Oriana Trubiani

Int. J. Mol. Sci. 2018, 19(2), 329; https://doi.org/10.3390/ijms19020329 - 23 Jan 2018

Cited by 74 | Viewed by 6580

Abstract

Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal [...] Read more.

Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal stem cells (hGMSCs) may present advantages compared to other MSCs, including the easier isolation. However, MSCs’ secretome has attracted much attention for its potential use in tissue regeneration, such as conditioned medium (CM) that contains different soluble factors proved to be useful for the regenerative purposes. In this study, we evaluated the osteogenic capacity of a poly-(lactide) (3D-PLA) scaffold enriched with hGMSCs and hGMSCs derived CM and its ability to regenerate bone defects in rat calvarias. 3D-PLA alone, 3D-PLA + CM or 3D-PLA + hGMSCs with/without CM were implanted in Wistar male rats subjected to calvarial defects. We observed that 3D-PLA scaffold enriched with hGMSCs and CM showed a better osteogenic capacity, being able to repair the calvarial defect as revealed in vivo by morphological evaluation. Moreover, transcriptomic analysis in vitro revealed the upregulation of genes involved in ossification and regulation of ossification in the 3D-PLA + CM + hGMSCs group. All of these results indicate the great osteogenic ability of 3D-PLA + CM + hGMSCs supporting its use in bone regenerative medicine, in particular in the repair of cranial bone defects. Especially, hGMSCs derived CM played a key role in the induction of the osteogenic process and in bone regeneration. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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15 pages, 6294 KiB

Open AccessArticle

Development of a Cytocompatible Scaffold from Pig Immature Testicular Tissue Allowing Human Sertoli Cell Attachment, Proliferation and Functionality

by Maxime Vermeulen, Federico Del Vento, Francesca De Michele, Jonathan Poels and Christine Wyns

Int. J. Mol. Sci. 2018, 19(1), 227; https://doi.org/10.3390/ijms19010227 - 12 Jan 2018

Cited by 55 | Viewed by 6406

Abstract

Cryopreservation of immature testicular tissue before chemo/radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cells, development of a transplantable scaffold by decellularization of pig immature testicular tissue (ITT) able to support decontaminated testicular cells [...] Read more.

Cryopreservation of immature testicular tissue before chemo/radiotherapy is the only option to preserve fertility of cancer-affected prepubertal boys. To avoid reintroduction of malignant cells, development of a transplantable scaffold by decellularization of pig immature testicular tissue (ITT) able to support decontaminated testicular cells could be an option for fertility restoration in these patients. We, therefore, compared decellularization protocols to produce a cytocompatible scaffold. Fragments of ITT from 15 piglets were decellularized using three protocols: sodium dodecyl sulfate (SDS)-Triton (ST), Triton-SDS-Triton (TST) and trypsin 0.05%/ethylenediaminetetraacetic acid (EDTA) 0.02%-Triton (TET) with varying detergent concentrations. All protocols were able to lower DNA levels. Collagen retention was demonstrated in all groups except ST 1%, and a significant decrease in glycosaminoglycans was observed in the TST 1% and TET 1% groups. When Sertoli cells (SCs) were cultured with decellularized tissue, no signs of cytotoxicity were detected. A higher SC proliferation rate and greater stem cell factor secretion were observed than with SCs cultured without scaffold. ST 0.01% and TET 3% conditions offered the best compromise in terms of DNA elimination and extracellular matrix (ECM) preservation, while ensuring good attachment, proliferation and functionality of human SCs. This study demonstrates the potential of using decellularized pig ITT for human testicular tissue engineering purposes. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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17 pages, 5252 KiB

Open AccessArticle

Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

by Nor Kamalia Zahari, Ruszymah Binti Haji Idrus and Shiplu Roy Chowdhury

Int. J. Mol. Sci. 2017, 18(11), 2242; https://doi.org/10.3390/ijms18112242 - 30 Oct 2017

Cited by 30 | Viewed by 6410

Abstract

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these [...] Read more.

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h⁻¹) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h⁻¹) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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Review

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79 pages, 3140 KiB

Open AccessReview

Tissue-Engineered Grafts from Human Decellularized Extracellular Matrices: A Systematic Review and Future Perspectives

by Andrea Porzionato, Elena Stocco, Silvia Barbon, Francesca Grandi, Veronica Macchi and Raffaele De Caro

Int. J. Mol. Sci. 2018, 19(12), 4117; https://doi.org/10.3390/ijms19124117 - 18 Dec 2018

Cited by 265 | Viewed by 16331

Abstract

Tissue engineering and regenerative medicine involve many different artificial and biologic materials, frequently integrated in composite scaffolds, which can be repopulated with various cell types. One of the most promising scaffolds is decellularized allogeneic extracellular matrix (ECM) then recellularized by autologous or stem [...] Read more.

Tissue engineering and regenerative medicine involve many different artificial and biologic materials, frequently integrated in composite scaffolds, which can be repopulated with various cell types. One of the most promising scaffolds is decellularized allogeneic extracellular matrix (ECM) then recellularized by autologous or stem cells, in order to develop fully personalized clinical approaches. Decellularization protocols have to efficiently remove immunogenic cellular materials, maintaining the nonimmunogenic ECM, which is endowed with specific inductive/differentiating actions due to its architecture and bioactive factors. In the present paper, we review the available literature about the development of grafts from decellularized human tissues/organs. Human tissues may be obtained not only from surgery but also from cadavers, suggesting possible development of Human Tissue BioBanks from body donation programs. Many human tissues/organs have been decellularized for tissue engineering purposes, such as cartilage, bone, skeletal muscle, tendons, adipose tissue, heart, vessels, lung, dental pulp, intestine, liver, pancreas, kidney, gonads, uterus, childbirth products, cornea, and peripheral nerves. In vitro recellularizations have been reported with various cell types and procedures (seeding, injection, and perfusion). Conversely, studies about in vivo behaviour are poorly represented. Actually, the future challenge will be the development of human grafts to be implanted fully restored in all their structural/functional aspects. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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20 pages, 1171 KiB

Open AccessReview

Challenges in Fabrication of Tissue-Engineered Cartilage with Correct Cellular Colonization and Extracellular Matrix Assembly

by Mikko J. Lammi, Juha Piltti, Juha Prittinen and Chengjuan Qu

Int. J. Mol. Sci. 2018, 19(9), 2700; https://doi.org/10.3390/ijms19092700 - 11 Sep 2018

Cited by 35 | Viewed by 5748

Abstract

A correct articular cartilage ultrastructure regarding its structural components and cellularity is important for appropriate performance of tissue-engineered articular cartilage. Various scaffold-based, as well as scaffold-free, culture models have been under development to manufacture functional cartilage tissue. Even decellularized tissues have been considered [...] Read more.

A correct articular cartilage ultrastructure regarding its structural components and cellularity is important for appropriate performance of tissue-engineered articular cartilage. Various scaffold-based, as well as scaffold-free, culture models have been under development to manufacture functional cartilage tissue. Even decellularized tissues have been considered as a potential choice for cellular seeding and tissue fabrication. Pore size, interconnectivity, and functionalization of the scaffold architecture can be varied. Increased mechanical function requires a dense scaffold, which also easily restricts cellular access within the scaffold at seeding. High pore size enhances nutrient transport, while small pore size improves cellular interactions and scaffold resorption. In scaffold-free cultures, the cells assemble the tissue completely by themselves; in optimized cultures, they should be able to fabricate native-like tissue. Decellularized cartilage has a native ultrastructure, although it is a challenge to obtain proper cellular colonization during cell seeding. Bioprinting can, in principle, provide the tissue with correct cellularity and extracellular matrix content, although it is still an open question as to how the correct molecular interaction and structure of extracellular matrix could be achieved. These are challenges facing the ongoing efforts to manufacture optimal articular cartilage. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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27 pages, 295 KiB

Open AccessReview

In Vivo Performance of Decellularized Vascular Grafts: A Review Article

by Chih-Hsun Lin, Kai Hsia, Hsu Ma, Hsinyu Lee and Jen-Her Lu

Int. J. Mol. Sci. 2018, 19(7), 2101; https://doi.org/10.3390/ijms19072101 - 19 Jul 2018

Cited by 120 | Viewed by 7724

Abstract

Due to poor vessel quality in patients with cardiovascular diseases, there has been an increased demand for small-diameter tissue-engineered blood vessels that can be used as replacement grafts in bypass surgery. Decellularization techniques to minimize cellular inflammation have been applied in tissue engineering [...] Read more.

Due to poor vessel quality in patients with cardiovascular diseases, there has been an increased demand for small-diameter tissue-engineered blood vessels that can be used as replacement grafts in bypass surgery. Decellularization techniques to minimize cellular inflammation have been applied in tissue engineering research for the development of small-diameter vascular grafts. The biocompatibility of allogenic or xenogenic decellularized matrices has been evaluated in vitro and in vivo. Both short-term and long-term preclinical studies are crucial for evaluation of the in vivo performance of decellularized vascular grafts. This review offers insight into the various preclinical studies that have been performed using decellularized vascular grafts. Different strategies, such as surface-modified, recellularized, or hybrid vascular grafts, used to improve neoendothelialization and vascular wall remodeling, are also highlighted. This review provides information on the current status and the future development of decellularized vascular grafts. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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26 pages, 37748 KiB

Open AccessReview

Bioengineering Approaches for Bladder Regeneration

by Ángel Serrano-Aroca, César David Vera-Donoso and Victoria Moreno-Manzano

Int. J. Mol. Sci. 2018, 19(6), 1796; https://doi.org/10.3390/ijms19061796 - 17 Jun 2018

Cited by 72 | Viewed by 11553

Abstract

Current clinical strategies for bladder reconstruction or substitution are associated to serious problems. Therefore, new alternative approaches are becoming more and more necessary. The purpose of this work is to review the state of the art of the current bioengineering advances and obstacles [...] Read more.

Current clinical strategies for bladder reconstruction or substitution are associated to serious problems. Therefore, new alternative approaches are becoming more and more necessary. The purpose of this work is to review the state of the art of the current bioengineering advances and obstacles reported in bladder regeneration. Tissue bladder engineering requires an ideal engineered bladder scaffold composed of a biocompatible material suitable to sustain the mechanical forces necessary for bladder filling and emptying. In addition, an engineered bladder needs to reconstruct a compliant muscular wall and a highly specialized urothelium, well-orchestrated under control of autonomic and sensory innervations. Bioreactors play a very important role allowing cell growth and specialization into a tissue-engineered vascular construct within a physiological environment. Bioprinting technology is rapidly progressing, achieving the generation of custom-made structural supports using an increasing number of different polymers as ink with a high capacity of reproducibility. Although many promising results have been achieved, few of them have been tested with clinical success. This lack of satisfactory applications is a good reason to discourage researchers in this field and explains, somehow, the limited high-impact scientific production in this area during the last decade, emphasizing that still much more progress is required before bioengineered bladders become a commonplace in the clinical setting. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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14 pages, 1828 KiB

Open AccessReview

Adult Stem Cells Spheroids to Optimize Cell Colonization in Scaffolds for Cartilage and Bone Tissue Engineering

by Leandra Santos Baptista, Gabriela Soares Kronemberger, Isis Côrtes, Letícia Emiliano Charelli, Renata Akemi Morais Matsui, Thiago Nunes Palhares, Jerome Sohier, Alexandre Malta Rossi and José Mauro Granjeiro

Int. J. Mol. Sci. 2018, 19(5), 1285; https://doi.org/10.3390/ijms19051285 - 25 Apr 2018

Cited by 61 | Viewed by 8860

Abstract

Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells [...] Read more.

Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells are responsible for their extracellular matrix synthesis, re-creating structures at the tissue level. Spheroids from adult stem cells can be considered as organoids, since stem cells recapitulate differentiation pathways and also represent a promising approach for identifying new molecular targets (biomarkers) for diagnosis and therapy. Currently, spheroids can be used for scaffold-free (developmental engineering) or scaffold-based approaches. The scaffold promotes better spatial organization of individual spheroids and provides a defined geometry for their 3D assembly in larger and complex tissues. Furthermore, spheroids exhibit potent angiogenic and vasculogenic capacity and serve as efficient vascularization units in porous scaffolds for bone tissue engineering. An automated combinatorial approach that integrates spheroids into scaffolds is starting to be investigated for macro-scale tissue biofabrication. Full article

(This article belongs to the Special Issue Cell Colonization in Scaffolds)

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