Modular Strategies to Build Cell-Free and Cell-Laden Scaffolds towards Bioengineered Tissues and Organs

Abstract

Engineering three-dimensional (3D) scaffolds for functional tissue and organ regeneration is a major challenge of the tissue engineering (TE) community. Great progress has been made in developing scaffolds to support cells in 3D, and to date, several implantable scaffolds are available for treating damaged and dysfunctional tissues, such as bone, osteochondral, cardiac and nerve. However, recapitulating the complex extracellular matrix (ECM) functions of native tissues is far from being achieved in synthetic scaffolds. Modular TE is an intriguing approach that aims to design and fabricate ECM-mimicking scaffolds by the bottom-up assembly of building blocks with specific composition, morphology and structural properties. This review provides an overview of the main strategies to build synthetic TE scaffolds through bioactive modules assembly and classifies them into two distinct schemes based on microparticles (µPs) or patterned layers. The µPs-based processes section starts describing novel techniques for creating polymeric µPs with desired composition, morphology, size and shape. Later, the discussion focuses on µPs-based scaffolds design principles and processes. In particular, starting from random µPs assembly, we will move to advanced µPs structuring processes, focusing our attention on technological and engineering aspects related to cell-free and cell-laden strategies. The second part of this review article illustrates layer-by-layer modular scaffolds fabrication based on discontinuous, where layers' fabrication and assembly are split, and continuous processes.

Keywords: additive manufacturing; bioprinting; drug delivery; microparticles; scaffold; soft lithography; vascularization.

Figure 1

Microfluidic emulsion: Fabrication of microparticles (µPs) with advanced processes. (a) Co-flow and (b) flow-focusing pictures of fluidic emulsion devices. Effect of processing conditions on µPs morphology, composition and structure: (c) spherical monodisperse µPs, (d) Janus µPs, (e) core-shell µPs with dual and triple cores, (f) disks and (g) rods µPs obtained by controlling the dimension of the outlet channel, (h) highly porous polylactic-co-glycolic acid (PLGA) spherical µPs prepared by double emulsion. Flow-lithography: (i) Picture of the flow-lithography continuous process for making shape-controlled µPs by exposure of precursor solution to patterned ultraviolet (UV) light. Morphology of (j) triangles, (k) squares and (l) hexagons µPs prepared by the continuous flow-lithography process. Single-cell encapsulated within (m) square and (n) triangular µPs prepared by the stop-flow-lithography (SFL) process. Soft-lithography: (o) Schematic drawing of the soft-lithography and lift-out molding fabrication protocol of µPs: (p,q,r) effect of mold type on µPs shape. Morphology of µPs obtained by the StampEd Assembly of polymer Layers (SEAL) process before (s) and (t) after sealing. (u) Morphology of vascular endothelial growth factor (VEGF)-loaded PLGA microsphere after solvent vapor shaping process. c, f, g Reproduced with permission from Reference [55] (Xu, Angewandte Chemie International Edition; Published by John Wiley and Sons, 2005); d Reproduced with permission from Reference [45] (Cao, RCS. Advances; published by Royal Society of Chemistry, 2015); e, i Reproduced with permission from Reference [54] (Baah, Microfluid Nanofluid; published by Springer Nature, 2014); h Reproduced with permission from Reference [53] (Choi, Small; published by John Wiley and Sons, 2010); j, l Reproduced with permission from Reference [47] (Dendukuri, Nature Materials; published by Springer Nature, 2006); m, n Reproduced with permission from Reference [56] (Panda, Lab Chip; published by Royal Society of Chemistry, 2008); o Reproduced with permission from Reference [57] (Canelas, Nanomed Nanobiotechnol; published by John Wiley and Sons, 2009); p, q, r Reproduced with permission from Reference [59] (Guan, Biomaterials; published by Elsevier Ltd, 2006); s, t Reproduced with permission from Reference [60] (Kevin J. McHugh, Science; published by American Association for the Advancement of Science, 2017); u Reproduced with permission from Reference [46] (Renato de Alteriis, Scientific Reports; published by Springer Nature, 2015).

Figure 2

Overview of µPs applications in tissue engineering (TE) scaffold-based strategies classified by random (left column) and ordered (right column) assemblies, cell-free (first row) and cell-laden (second row) approaches. (a) morphology of µPs’ sintered polycaprolactone (PCL) scaffold obtained by thermal sintering. (b) Morphology of porous µPs’ sintered PLGA scaffold obtained by chemical sintering. (c) Effect of µPs’ diameter and thermal sintering time on mean pore size and compressive modulus of PLGA-sintered scaffolds. (d) Optical images of sintered scaffolds with homogeneous and heterogeneous spatial distribution of loaded µPs. (e) Release profiles of bone morphogenic protein (BMP)-2 and transforming growth factor (TGF) b1 from µPs’-sintered scaffolds for osteochondral interface TE. (f) Optical image of ordered scaffold obtained by selective laser sintering (SLS) and made of PCL µPs. (g,h) morphology of SLS scaffold evidencing the order and random structures, respectively. (i) Comparison of random and ordered PCL scaffolds on degree of vascularization in vivo. Results proved that the internal vascularization of the ordered scaffolds has significantly better vascularization in the inner core if compared to the random scaffold. (j) Culture device used to generate three-dimensional (3D) bone in vitro by cell-laden µPs’ assembly and morphological and optical visualization of corresponding tissue. (k) Assembly of cells and multifunctional poly-ethylene glycol (PEG) µPs to study cells migration in vitro as a function of scaffolds porosity and sphingosine 1-phosphate (S1P) release. (l,m) Porous scaffolds obtained by µPs’ printing for osteochondral and vascular tissues repair, respectively. (n) Schematic of assembly processes of cell-laden µ-scaffolds obtained by soft-lithography process and resulting cell-laden constructs. a Reproduced with permission from Reference [4] (Luciani, Biomaterials; published by Elsevier Ltd., 2008); b Reproduced with permission from Reference [62] (Qutachi, Acta Biomaterialia; published by Elsevier Ltd., 2014); c Reproduced with permission from Reference [68] (Borden, Biomaterials; published by Elsevier Science Ltd., 2002); d Reproduced with permission from Reference [71] (Jaklenec, Biomaterials; published by Elsevier Ltd., 2008); e Reproduced with permission from Reference [73] (Dormer, Annals of Biomedical Engineering; published by Springer Nature, 2010); f–h Reproduced with permission from Reference [77] (Du, Colloids and Surfaces B: Biointerfaces; published by Elsevier B.V, 2015); i Reproduced with permission from Reference [79] (Rossi, Journal of Materials Science Materials in Medicine; published by Springer Nature, 2016); j Reproduced with permission from Reference [87] (Chen, Biomaterials; published by Elsevier Ltd., 2011); k Reproduced with permission from Reference [88] (Scott, Acta Biomaterialia; published by Elsevier Ltd., 2009); l Reproduced with permission from Reference [40] (Levato, Biofabrication; published by Institute of Physics Publishing, 2014); m Reproduced with permission from Reference [89] (Tan, Scientific Reports; published by Springer Nature, 2016); n Reproduced with permission from Reference [90] (Xiao, Materials Letters; published by Elsevier Ltd., 2018).

Figure 3

Discontinuous processes overview scheme. Left side: two-dimensional (2D) layers’ fabrication processes. Right side: three-dimensional (3D) scaffolds’ assembly processes. APS: Poly (ester-amide),1:2 poly (1,3-diamino-2-hydroxypropane-co -polyol sebacate); ECM: Extracellular matrix; ES cells: Embryonic stem cells; hMSC: Human mesenchymal stem cell; HUVEC: Human umbilical vein endothelial cell; NIH3T3: Mouse embryo fibroblast cell line; PCGA: Poly (ε-caprolactone–co-glycolic acid); PCL Polycaprolactone; PGS: Poly (glycerol sebacate); PLGA: Polylactic-co-glycolic acid; PLLA: Poly(L-lactic acid); PLT32o: Poly (limonene thioether); Poly(NIPAAm-co-HEMAHex): Poly (N-isopropylacrylamide–co-2-hydroxyethylmethacrylate-6-hydroxyhexanoate); POMaC: Poly (octamethylene maleate (anhydride) citrate; RLC: Rat liver cells; SMC: Smooth muscle cell; SPCL: Starch-polycaprolactone; TE: Tissue engineering.

Figure 4

(a) Integrated tissue–organ printer (ITOP) system components and materials. (b) Photograph of the printed calvarial bone construct. (c) Histological image of the printed calvarial construct after in vivo implantation. (d) Image of the bioscaffolder + micro-tissue injection system (inset: working concept overview of the micro-tissue injection system) used for the preparation of the osteochondral joint resurfacing device. (e) Computer-aided design (CAD) images and (f) optical image of an assembled hemispherical construct. (g) Image of µ-tissues in 3D printed PCL fibers and (h) resulting 4′,6-diamidino-2-phenylindole (DAPI) (blue) and Aggrecan (purple) antibodies staining of the construct showing cells distribution and µ-tissues fusion at 35 days of in vitro chondrogenic culture. (i) Fused deposition modeling (FDM) machine overview and materials for the elastic cardiac patch fabrication. (j) Illustration of the scaffold implantation site after induced myocardial infarction in rats. (k) Representative Masson’s trichrome stained heart section four weeks after implantation. Black boxes denote higher magnification area of the left panel. Black arrows indicate the annular-shaped PGS-PCL scaffolds. Scale bars: 2.0 mm. (l) Microscale continuous projection printing (μCPP) system used to fabricate PEG–gelatin methacrylate scaffolds loaded with neural progenitor cells (NPCs) for nerve regeneration. (m) Spinal cord structure evidencing fascicles regions (motor systems are shown in green and sensory systems are shown in blue) and (n) corresponding scaffold. (o) Image of the NPCs-loaded scaffold after four weeks in vivo showing channels filled with green fluorescent protein (GFP)-expressing NPCs. (a–c) Reproduced with permission from Reference [110] (Kang, Nature Biotechnology; published by Springer Nature, 2016). (d–h) Reproduced with permission from Reference [113] (Mekhileri, Biofabrication; published by IOP Publishing, 2018). (i–k) Reproduced with permission from Reference [116] (Yang, Advanced Healthcare Materials; published by John Wiley and Sons, 2019). (l–o) Reproduced with permission from Reference [119] (Koffler, Nature Medicine; published by Springer Nature, 2019).