Research

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32 pages, 8861 KiB

Open AccessArticle

Fabrication of Adipose-Derived Stem Cell-Based Self-Assembled Scaffold under Hypoxia and Mechanical Stimulation for Urethral Tissue Engineering

by Zahra Rashidbenam, Mohd Hafidzul Jasman, Guan Hee Tan, Eng Hong Goh, Xeng Inn Fam, Christopher Chee Kong Ho, Zulkifli Md Zainuddin, Reynu Rajan, Rizal Abdul Rani, Fatimah Mohd Nor, Mohamad Aznan Shuhaili, Nik Ritza Kosai, Farrah Hani Imran and Min Hwei Ng

Int. J. Mol. Sci. 2021, 22(7), 3350; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073350 - 25 Mar 2021

Cited by 8 | Viewed by 2498

Abstract

Long urethral strictures are often treated with autologous genital skin and buccal mucosa grafts; however, risk of hair ingrowth and donor site morbidity, restrict their application. To overcome this, we introduced a tissue-engineered human urethra comprising adipose-derived stem cell (ASC)-based self-assembled scaffold, human [...] Read more.

Long urethral strictures are often treated with autologous genital skin and buccal mucosa grafts; however, risk of hair ingrowth and donor site morbidity, restrict their application. To overcome this, we introduced a tissue-engineered human urethra comprising adipose-derived stem cell (ASC)-based self-assembled scaffold, human urothelial cells (UCs) and smooth muscle cells (SMCs). ASCs were cultured with ascorbic acid to stimulate extracellular matrix (ECM) production. The scaffold (ECM) was stained with collagen type-I antibody and the thickness was measured under a confocal microscope. Results showed that the thickest scaffold (28.06 ± 0.59 μm) was achieved with 3 × 10⁴ cells/cm² seeding density, 100 μg/mL ascorbic acid concentration under hypoxic and dynamic culture condition. The biocompatibility assessment showed that UCs and SMCs seeded on the scaffold could proliferate and maintain the expression of their markers (CK7, CK20, UPIa, and UPII) and (α-SMA, MHC and Smootheline), respectively, after 14 days of in vitro culture. ECM gene expression analysis showed that the ASC and dermal fibroblast-based scaffolds (control) were comparable. The ASC-based scaffold can be handled and removed from the plate. This suggests that multiple layers of scaffold can be stacked to form the urothelium (seeded with UCs), submucosal layer (ASCs only), and smooth muscle layer (seeded with SMCs) and has the potential to be developed into a fully functional human urethra for urethral reconstructive surgeries. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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20 pages, 5349 KiB

Open AccessArticle

Cell Sheet Comprised of Mesenchymal Stromal Cells Overexpressing Stem Cell Factor Promotes Epicardium Activation and Heart Function Improvement in a Rat Model of Myocardium Infarction

by Konstantin V. Dergilev, Evgeny K. Shevchenko, Zoya I. Tsokolaeva, Irina B. Beloglazova, Ekaterina S. Zubkova, Maria A. Boldyreva, Mikhail Yu. Menshikov, Elizaveta I. Ratner, Dmitry Penkov and Yelena V. Parfyonova

Int. J. Mol. Sci. 2020, 21(24), 9603; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21249603 - 16 Dec 2020

Cited by 11 | Viewed by 2049

Abstract

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived [...] Read more.

Cell therapy of the post-infarcted myocardium is still far from clinical use. Poor survival of transplanted cells, insufficient regeneration, and replacement of the damaged tissue limit the potential of currently available cell-based techniques. In this study, we generated a multilayered construct from adipose-derived mesenchymal stromal cells (MSCs) modified to secrete stem cell factor, SCF. In a rat model of myocardium infarction, we show that transplantation of SCF producing cell sheet induced activation of the epicardium and promoted the accumulation of c-kit positive cells in ischemic muscle. Morphometry showed the reduction of infarct size (16%) and a left ventricle expansion index (0.12) in the treatment group compared to controls (24–28%; 0.17–0.32). The ratio of viable myocardium was more than 1.5-fold higher, reaching 49% compared to the control (28%) or unmodified cell sheet group (30%). Finally, by day 30 after myocardium infarction, SCF-producing cell sheet transplantation increased left ventricle ejection fraction from 37% in the control sham-operated group to 53%. Our results suggest that, combining the genetic modification of MSCs and their assembly into a multilayered construct, we can provide prolonged pleiotropic effects to the damaged heart, induce endogenous regenerative processes, and improve cardiac function. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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13 pages, 5091 KiB

Open AccessArticle

Effects of Helioxanthin Derivative-Treated Human Dental Pulp Stem Cells on Fracture Healing

by Daiki Yamakawa, Yoko Kawase-Koga, Yasuyuki Fujii, Yuki Kanno, Marika Sato, Shinsuke Ohba, Yoshiaki Kitaura, Miki Kashiwagi and Daichi Chikazu

Int. J. Mol. Sci. 2020, 21(23), 9158; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21239158 - 01 Dec 2020

Cited by 10 | Viewed by 2305

Abstract

Bone defects affect patients functionally and psychologically and can decrease quality of life. To resolve these problems, a simple and efficient method of bone regeneration is required. Human dental pulp stem cells (DPSCs) have high proliferative ability and multilineage differentiation potential. In our [...] Read more.

Bone defects affect patients functionally and psychologically and can decrease quality of life. To resolve these problems, a simple and efficient method of bone regeneration is required. Human dental pulp stem cells (DPSCs) have high proliferative ability and multilineage differentiation potential. In our previous study, we reported a highly efficient method to induce osteogenic differentiation using DPSC sheets treated with a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH)) in a mouse calvarial defect model. However, the localization of the DPSCs after transplantation remains unknown. Therefore, in this study, we investigated the localization of transplanted DPSCs in a mouse fracture model. DPSCs were collected from six healthy patients aged 18–29 years, cultured in normal medium (NM), osteogenic medium (OM), or OM with TH, and fabricated them into cell sheets. To evaluate the efficacy of fracture healing using DPSCs treated with OM+TH, and to clarify the localization of the transplanted DPSC sheets in vivo, we transplanted OM+TH-treated DPSC sheets labeled with PKH26 into mouse tibiae fractures. We demonstrated that transplanted OM+TH-treated DPSCs sheets were localized to the fracture site and facilitated bone formation. These results indicated that transplanted OM+TH-treated DPSCs were localized at fracture sites and directly promoted fracture healing. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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14 pages, 4411 KiB

Open AccessArticle

Bone Regeneration Potential of Human Dental Pulp Stem Cells Derived from Elderly Patients and Osteo-Induced by a Helioxanthin Derivative

by Marika Sato, Yoko Kawase-Koga, Daiki Yamakawa, Yasuyuki Fujii and Daichi Chikazu

Int. J. Mol. Sci. 2020, 21(20), 7731; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21207731 - 19 Oct 2020

Cited by 9 | Viewed by 2044

Abstract

Human dental pulp stem cells (DPSCs) have high clonogenic and proliferative potential. We previously reported that a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2–b]pyridine-2-carboxamide (TH)) enhances osteogenic differentiation of DPSCs derived from young patients. However, in the clinical field, elderly patients more frequently require bone regenerative therapy [...] Read more.

Human dental pulp stem cells (DPSCs) have high clonogenic and proliferative potential. We previously reported that a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2–b]pyridine-2-carboxamide (TH)) enhances osteogenic differentiation of DPSCs derived from young patients. However, in the clinical field, elderly patients more frequently require bone regenerative therapy than young patients. In this study, we examined and compared the osteogenic differentiation potential of TH-induced DPSCs from elderly patients and young patients to explore the potential clinical use of DPSCs for elderly patients. DPSCs were obtained from young and elderly patients and cultured in osteogenic medium with or without TH. We assessed the characteristics and osteogenic differentiation by means of specific staining and gene expression analyses. Moreover, DPSC sheets were transplanted into mouse calvarial defects to investigate osteogenesis of TH-induced DPSCs by performing micro-computed tomography (micro-CT). We demonstrated that osteogenic conditions with TH enhance the osteogenic differentiation marker of DPSCs from elderly patients as well as young patients in vitro. In vivo examination showed increased osteogenesis of DPSCs treated with TH from both elderly patients and young patients. Our results suggest that the osteogenic differentiation potential of DPSCs from elderly patients is as high as that of DPSCs from young patients. Moreover, TH-induced DPSCs showed increased osteogenic differentiation potential, and are thus a potentially useful cell source for bone regenerative therapy for elderly patients. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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21 pages, 5679 KiB

Open AccessArticle

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization

by Natalya Alexandrushkina, Peter Nimiritsky, Roman Eremichev, Vladimir Popov, Mikhail Arbatskiy, Natalia Danilova, Pavel Malkov, Zhanna Akopyan, Vsevolod Tkachuk and Pavel Makarevich

Int. J. Mol. Sci. 2020, 21(15), 5567; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155567 - 04 Aug 2020

Cited by 18 | Viewed by 3972

Abstract

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction [...] Read more.

We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3–7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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Review

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18 pages, 3356 KiB

Open AccessReview

Fundamental Technologies and Recent Advances of Cell-Sheet-Based Tissue Engineering

by Chikahiro Imashiro and Tatsuya Shimizu

Int. J. Mol. Sci. 2021, 22(1), 425; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010425 - 03 Jan 2021

Cited by 43 | Viewed by 6469

Abstract

Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this [...] Read more.

Tissue engineering has attracted significant attention since the 1980s, and the applications of tissue engineering have been expanding. To produce a cell-dense tissue, cell sheet technology has been studied as a promising strategy. Fundamental techniques involving tissue engineering are mainly introduced in this review. First, the technologies to fabricate a cell sheet were reviewed. Although temperature-responsive polymer-based technique was a trigger to establish and spread cell sheet technology, other methodologies for cell sheet fabrication have also been reported. Second, the methods to improve the function of the cell sheet were investigated. Adding electrical and mechanical stimulation on muscle-type cells, building 3D structures, and co-culturing with other cell species can be possible strategies for imitating the physiological situation under in vitro conditions, resulting in improved functions. Finally, culture methods to promote vasculogenesis in the layered cell sheets were introduced with in vivo, ex vivo, and in vitro bioreactors. We believe the present review that shows and compares the fundamental technologies and recent advances for cell-sheet-based tissue engineering should promote further development of tissue engineering. The development of cell sheet technology should promote many bioengineering applications. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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17 pages, 1105 KiB

Open AccessReview

Clinical Trials of Limbal Stem Cell Deficiency Treated with Oral Mucosal Epithelial Cells

by Joan Oliva, Fawzia Bardag-Gorce and Yutaka Niihara

Int. J. Mol. Sci. 2020, 21(2), 411; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21020411 - 09 Jan 2020

Cited by 17 | Viewed by 5162

Abstract

The corneal surface is an essential organ necessary for vision, and its clarity must be maintained. The corneal epithelium is renewed by limbal stem cells, located in the limbus and in palisades of Vogt. Palisades of Vogt maintain the clearness of the corneal [...] Read more.

The corneal surface is an essential organ necessary for vision, and its clarity must be maintained. The corneal epithelium is renewed by limbal stem cells, located in the limbus and in palisades of Vogt. Palisades of Vogt maintain the clearness of the corneal epithelium by blocking the growth of conjunctival epithelium and the invasion of blood vessels over the cornea. The limbal region can be damaged by chemical burns, physical damage (e.g., by contact lenses), congenital disease, chronic inflammation, or limbal surgeries. The degree of limbus damage is associated with the degree of limbal stem cells deficiency (partial or total). For a long time, the only treatment to restore vision was grafting part of the healthy cornea from the other eye of the patient or by transplanting a cornea from cadavers. The regenerative medicine and stem cell therapies have been applied to restore normal vision using different methodologies. The source of stem cells varies from embryonic stem cells, mesenchymal stem cells, to induced pluripotent stem cells. This review focuses on the use of oral mucosa epithelial stem cells and their use in engineering cell sheets to treat limbal stem cell deficient patients. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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12 pages, 1177 KiB

Open AccessReview

Recent Advances in ROS-Responsive Cell Sheet Techniques for Tissue Engineering

by Min-Ah Koo, Mi Hee Lee and Jong-Chul Park

Int. J. Mol. Sci. 2019, 20(22), 5656; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20225656 - 12 Nov 2019

Cited by 13 | Viewed by 4558

Abstract

Cell sheet engineering has evolved rapidly in recent years as a new approach for cell-based therapy. Cell sheet harvest technology is important for producing viable, transplantable cell sheets and applying them to tissue engineering. To date, most cell sheet studies use thermo-responsive systems [...] Read more.

Cell sheet engineering has evolved rapidly in recent years as a new approach for cell-based therapy. Cell sheet harvest technology is important for producing viable, transplantable cell sheets and applying them to tissue engineering. To date, most cell sheet studies use thermo-responsive systems to detach cell sheets. However, other approaches have been reported. This review provides the progress in cell sheet detachment techniques, particularly reactive oxygen species (ROS)-responsive strategies. Therefore, we present a comprehensive introduction to ROS, their application in regenerative medicine, and considerations on how to use ROS in cell detachment. The review also discusses current limitations and challenges for clarifying the mechanism of the ROS-responsive cell sheet detachment. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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13 pages, 461 KiB

Open AccessReview

Scaffold-Free 3-D Cell Sheet Technique Bridges the Gap between 2-D Cell Culture and Animal Models

by Ayidah Alghuwainem, Alaa T. Alshareeda and Batla Alsowayan

Int. J. Mol. Sci. 2019, 20(19), 4926; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms20194926 - 04 Oct 2019

Cited by 44 | Viewed by 5383

Abstract

Various tissue engineering techniques have been created in research spanning two centuries, resulting in new opportunities for growing cells in culture and the creation of 3-D tissue-like constructs. These techniques are classified as scaffold-based and scaffold-free techniques. Cell sheet, as a scaffold-free technique, [...] Read more.

Various tissue engineering techniques have been created in research spanning two centuries, resulting in new opportunities for growing cells in culture and the creation of 3-D tissue-like constructs. These techniques are classified as scaffold-based and scaffold-free techniques. Cell sheet, as a scaffold-free technique, has attracted research interest in the context of drug discovery and tissue repair, because it provides more predictive data for in vivo testing. It is one of the most promising techniques and has the potential to treat degenerative tissues such as heart, kidneys, and liver. In this paper, we argue the advantages of cell sheets as a scaffold-free approach, compared to other techniques, including scaffold-based and scaffold-free techniques such as the classic systemic injection of cell suspension. Full article

(This article belongs to the Special Issue Potential Application of Cell Sheets, for Tissue Engineering and Repairing Tissue)

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