Biomimetic Bilayered Scaffolds for Tissue Engineering: From Current Design Strategies to Medical Applications

Abstract

Tissue damage due to cancer, congenital anomalies, and injuries needs new efficient treatments that allow tissue regeneration. In this context, tissue engineering shows a great potential to restore the native architecture and function of damaged tissues, by combining cells with specific scaffolds. Scaffolds made of natural and/or synthetic polymers and sometimes ceramics play a key role in guiding cell growth and formation of the new tissues. Monolayered scaffolds, which consist of uniform material structure, are reported as not being sufficient to mimic complex biological environment of the tissues. Osteochondral, cutaneous, vascular, and many other tissues all have multilayered structures, therefore multilayered scaffolds seem more advantageous to regenerate these tissues. In this review, recent advances in bilayered scaffolds design applied to regeneration of vascular, bone, cartilage, skin, periodontal, urinary bladder, and tracheal tissues are focused on. After a short introduction on tissue anatomy, composition and fabrication techniques of bilayered scaffolds are explained. Then, experimental results obtained in vitro and in vivo are described, and their limitations are given. Finally, difficulties in scaling up production of bilayer scaffolds and reaching the stage of clinical studies are discussed when multiple scaffold components are used.

Keywords: bilayered scaffolds; biomaterials; biomimetism; material design; medical applications; tissue engineering.

Figure 1

Overview of the strategies to design bilayered scaffolds for TE. TE refers to the combination of cells and scaffolds to build a structure, which would be able to restore and maintain the native architecture and function of damaged tissues and/or organs. Scaffolds are supporting structures for the cells and must be appropriately designed, in terms of properties and functionality, to mimic the architecture of the native tissue of interest. It exists various scaffold design strategies, i.e., selection of raw materials and fabrication techniques. Scaffolds can be made of natural and/or synthetic polymers, as well as bioceramics and can be built with several fabrication techniques. Selecting the appropriate scaffold design strategies is crucial.

Figure 2

General structure of blood vessels composed of three main layers: tunica intima, tunica media, and tunica adventitia. Reproduced under the terms of the Creative Commons License.^{[ 27 ]} Copyright 2021, the Author(s). Published by Frontiers Media S.A.

Figure 3

Schematic illustration of the fabrication process set up by Zhu et al. a) Hypothesis orientation of circumferentially aligned PCL microfibers which could guide VSMCs regeneration. b) The two‐step fabrication process that has been used to manufacture the inner layer with circumferentially oriented PCL fibers by wet‐spinning and the outer layer randomly with oriented nanofibers by electrospinning. Reproduced with permission.^{[ 35 ]} Copyright 2015, Elsevier. Schematic illustration of the fabrication process set up by Li et al. c) The custom‐made electrospinning technique to develop a PCL‐based two‐layer‐tubular scaffold in which the directions of the fibers of these two layers were orthogonal. d) Heparinization of PCL to improve its hydrophilicity. e) ECs and VSMCs co‐culture: ECs were seeded on the inner layer in which nanofibers are oriented along a axial direction whereas VSMCs were seeded on the outer layer in which nanofibers are oriented with a circumferential direction. Adapted under the terms of the Creative Commons License.^{[ 36 ]} Copyright 2021, the Authors. Published by the Royal Society of Chemistry.

Figure 4

Schematic illustration of fabrication process set up by Gupta et al. a) Fabrication methodology of bilayered small vascular graft using silk‐based scaffolds. It is composed of an inner porous layer prepared by molding and freeze‐drying, followed by coating with electrospun outer nanofibrous layer. b) Graphical description of the bilayered scaffold and in vivo implantation in rat. Adapted with permission.^{[ 44 ]} Copyright 2020, American Chemical Society.

Figure 5

a) Entire view of the bilayered tubular scaffold composed of PGS/PCL electrospun nanofibers. b) SEM image of PGS/PCL nanofibers layer. c) SEM image of PCL layers. d) Cross section. e) SEM image of the interface between outer and inner layers. Reproduced with permission.^{[ 46 ]} Copyright 2018, John Wiley & Sons Ltd.

Figure 6

a) Scheme of the two‐steps thermal‐induced phase separation technique to fabricate heterogeneous porous bilayered nanofibrous vascular grafts. The bilayered scaffold is PLLA‐based: a PLLA/PLCL microporous as an inner layer, and a PLLA/PCL macroporous as an outer layer. b) Reproduced with permission.^{[ 49 ]} Copyright 2018, Elsevier.

Figure 7

HDFas cell seeding onto the chitosan/gelatin‐based bilayered scaffold at days 1, 5, 10, 15, and 20 after culture in proliferation medium. Reproduced with permission.^{[ 60 ]} Copyright 2016, Elsevier.

Figure 8

Osteochondral tissue structure. Cross section of a long bone and a schematic presentation of the osteochondral unit. Reproduced with permission.^{[ 66 ]} Copyright 2020, Springer Nature.

Figure 9

a,b) General view of the bilayered PGLA scaffold structure with the cartilage and the subchondral layer. c–e) SEM micrographs of the three groups of bilayered scaffolds porosities. The dashed line indicates the border of the two layers. Reproduced under the terms of the Creative Commons License.^{[ 68 ]} Copyright 2015, the Author(s). Published by Oxford University Press.

Figure 10

Schematic representation of the preparation of the bilayered gene activated composite osteochondral scaffold: the hyaline cartilage layer was made with a mix of chitosan‐gelatin (CG) whereas HAP/chitosan‐gelatin (HCG) were used for the subchondral bone layer. pTGF‐ β1: plasmid TGF‐ β1; pBMP‐2: plasmid BMP‐2; MSC: mesenchymal stem cell; CG: chitosan‐gelatin; HCG: hydroxyapatite/chitosan‐gelatin. Reproduced with permission.^{[ 75 ]} Copyright 2011, Elsevier.

Figure 11

a) Schematic representation of hypothetic cell migration in the random and aligned structures randomly structure compared to the aligned structure. b) Observation of migrating cell morphology on BCP‐based bilayered scaffold through R‐270 (Random structure‐270 µm pore size) and A‐270 (Aligned structure‐270 µm pore size) after 7 d of seeding. The yellow line indicates the top surface of the bilayered scaffold. The yellow line represented the top surface of the BCP scaffold. Adapted with permission.^{[ 83 ]} Copyright 2017, Wiley‐VCH GmbH.

Figure 12

Macroscopic evaluation of defect site of the control group (left in blank without any processing), single cartilage layer (HLC‐HA) and bilayered scaffold (HLC‐HA cartilage layer and HLC‐HA‐HAP subchondral) after 8 and 12 weeks after surgery. Reproduced with permission.^{[ 87 ]} Copyright 2020, Science China Press and Springer‐Verlag.

Figure 13

Histological examination of PVA/Gel/V‐n‐HA/PA6 scaffolds at 12 weeks. a,b,d,e,g,h) Refer to Masson's trichrome stain, whereas c,f,i) to safranin‐O stain. a) Group A—cell‐seeded bilayered scaffold (×20). b,c) Detail of the PVA/gelatin/vanillin zone (×40). d) Group B—bilayered scaffold (×20). e,f) Detail of the PVA/gelatin/vanillin zone (×40). g) Group C—control group (×20). h,i) Detail of the defect zone (×40). The triangle symbol refers to the nHA/PA6 scaffold and the star symbol refers to the PVA/gelatin/vanillin scaffold. Reproduced with permission.^{[ 97 ]} Copyright 2015, John Wiley & Sons.

Figure 14

Anatomical structure of the skin. a) Cross‐section through the skin. b) Layers of skin. Adapted with permission.^{[ 112 ]} Copyright 2019, EMAP Publishing Ltd.

Figure 15

Schematic representation of the bilayered epidermal‐dermal scaffold. a) In a clinical context, the dermal component (hydrogel), containing dermal human fibroblasts, would be injected directly into the lesion. It would instantly cross‐link in situ and adapt to the shape of lesion. b) Then, the epidermal component, pre‐seeded with keratinocytes is applied on top of the dermal layer. c) Fibroblast‐containing dermal matrix (blue) and keratinocyte‐containing epidermal membrane (pink) are linked together by covalent imine bonding (amine‐aldehyde interactions). PLL and HAX abbreviations refer to poly‐L‐lysine and cross‐linked hyaluronic acid, respectively. Reproduced with permission.^{[ 116 ]} Copyright 2014, Elsevier.

Figure 16

Petri‐dish showing the antibacterial activity of gentamicin after 1 d of culture with: a) Serratia marcescens test group (bilayered dermal scaffold with gentamicin‐loaded PLGA microspheres); b) Serratia marcescens positive control group (collagen/chitosan/gentamicin complex); c) Serratia marcescens negative control group (collagen/chitosan scaffold). d) Diameters of inhibition zone with the culture time. s.a refers to Staphylococcus aureus; s.m refers to Serratia marcescens; s.a‐c and s.m‐c refer to their positive control group (referring to (collagen/chitosan/gentamicin complex). Adapted with permission.^{[ 128 ]} Copyright 2015, Elsevier.