Decellularized Matrices As Cell-Instructive Scaffolds to Guide Tissue

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Decellularized matrices as cell-instructive scaffolds to guide tissue-specific regeneration Kevin P Robb, Arthi Shridhar, and Lauren Flynn ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00619 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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ACS Biomaterials Science & Engineering

Decellularized matrices as cell-instructive scaffolds to guide tissue-specific regeneration

Kevin P. Robb1, Arthi Shridhar2 and Lauren E. Flynn*2,3 1

Biomedical Engineering Graduate Program, Claudette MacKay Lassonde Pavilion, The University of Western Ontario, London, ON, Canada N6A 5B9

2

Department of Chemical and Biochemical Engineering, Thompson Engineering Building, The University of Western Ontario, London, Ontario, Canada, N6A 5B9 3

Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1

* Corresponding Author: Lauren E. Flynn, Ph.D., P.Eng. Department of Chemical and Biochemical Engineering Department of Anatomy and Cell Biology Western University London, ON, Canada N6A 5B9 Phone: 519-661-2111 x87226 Email: [email protected]

ACS Paragon Plus Environment


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Abstract Decellularized scaffolds are promising clinically-translational biomaterials that can be applied to direct cell responses and promote tissue regeneration. Bioscaffolds derived from the extracellular matrix (ECM) of decellularized tissues can naturally mimic the complex extracellular microenvironment through the retention of compositional, biomechanical, and structural properties specific to the native ECM. Increasingly, studies have investigated the use of ECM-derived scaffolds as instructive substrates to recapitulate properties of the stem cell niche and guide cell proliferation, paracrine factor production, and differentiation in a tissuespecific manner. Here, we review the application of decellularized tissue scaffolds as instructive matrices for stem or progenitor cells, with a focus on the mechanisms through which ECMderived scaffolds can mediate cell behaviour to promote tissue-specific regeneration. We conclude that while additional pre-clinical studies are required, ECM-derived scaffolds are a promising platform to guide cell behaviour and may have widespread clinical applications in the field of regenerative medicine.

Keywords: Decellularization, Extracellular Matrix (ECM), Cell-instructive Scaffolds, Stem Cells, Stem Cell Niche, Microenvironment.


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1.0 Introduction The extracellular matrix (ECM) is a complex network of proteins and polysaccharides with tissue-specific composition and architecture that is a critical component of the stem cell niche1. Recognizing the importance of the 3D microenvironment in mediating cell function both in vitro and in vivo, there has been growing interest in the design of cell-instructive biomaterials platforms to help direct the response of pro-regenerative stem or progenitor cell populations2-5. More specifically, the development of higher-fidelity culture models that more closely mimic the compositional and biomechanical properties of the complex cellular milieu would help to advance our understanding of the potential mechanisms of stem/progenitor cell-mediated regeneration6-7. Further, from a translational perspective, the extremely poor localization, retention and survival of cells delivered in suspension points to the need for rationally-designed cell delivery systems with properties tuned to the target site for regeneration8-10. As the complexity of the native ECM cannot be easily replicated using synthetic materials, a growing body of work has examined the use of scaffolds derived from decellularized tissues for stem/progenitor cell culture and delivery6, 11. Decellularization protocols have been developed for the majority of tissues in the body to generate off-the-shelf bioscaffolds enriched in structural ECM proteins that can promote in situ tissue regeneration12-15. Methods of tissue decellularization have been reviewed previously16-17, but commonly include chemical, enzymatic and mechanical steps to remove antigenic cellular material while preserving the complex structure and composition of the native ECM as much as possible. In addition to being applied in their intact form, a variety of modified ECM-derived biomaterial formats have been developed including sheets18, hydrogels19-21, microcarriers22, porous foams23-24, and 3D-printed scaffolds25. While decellularized tissues can stimulate a pro-regenerative response, there is an increasing



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body of evidence to support that pre-seeding ECM-derived scaffolds with stem/progenitor cells may enhance host tissue regeneration relative to unseeded controls. In the current review, we will focus on the use of decellularized tissues as complex ECMderived scaffolding sources for stem/progenitor cell delivery and tissue engineering, recognizing that some of these approaches may be adapted using ECM produced by cells in culture (reviewed in detail elsewhere26) or for use as in vitro platforms for stem/progenitor cell culture and expansion11, 22, 27-31.Given that the structural and compositional properties of the ECM are unique to each tissue source32-33, there is evidence to support the rationale of using tissue-specific ECM for bioscaffold fabrication. Thus, the focus of this review will be on the cell-instructive effects of ECM-derived scaffolds sourced from decellularized tissues on stem and progenitor cell populations, and their application in tissue-specific regenerative strategies. 2.0 Mechanisms of Stem/Progenitor Cell Modulation by ECM-derived Scaffolds The ECM consists of cell-secreted structural and functional molecules essential for providing physical support and modulating cell signalling within tissues34. Broadly, the ECM is comprised of collagens, elastin, cell-adhesive glycoproteins, proteoglycans, and a diverse array of growth factors and matricellular proteins; however, the proportion of these, their organization and/or the presence of specialized macromolecules varies between tissues and species32. This tissue-specificity is crucial for imparting unique properties that act in concert to regulate cell phenotype and function within the tissue. In the following subsections, the compositional, biomechanical, and structural features of decellularized scaffolds are discussed, with a focus on how these properties may influence stem or progenitor cell behaviour (summarized in Figure 1). In addition, the impact of decellularization on the properties of the native tissue ECM will be described, along with strategies that may be applied to more fully characterize ECM-derived


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scaffolds, as a next-step towards elucidating the mechanisms of stem/progenitor cell-mediated tissue regeneration within these engineered cellular microenvironments.

Figure 1. Proposed mechanisms by which compositional, biomechanical, and structural properties of ECM-derived scaffolds direct stem/progenitor cell behaviour and responses. A) Stem/progenitor cells may interact with the scaffold directly through receptor-ligand binding or indirectly through binding of growth factors and/or cryptic peptides released by proteases that degrade the ECM. These cell-ECM interactions may in turn influence paracrine factor secretion by the cells. B) Biomechanical properties of the scaffold influence mechanical forces transmitted via the cytoskeleton to alter cell responses through mechanotransduction pathways. C) Scaffold porosity governs the surface area available for cell-ECM contacts, as well as the degree of cellcell contact. Surface topography and molecular functionality influence host cell and protein binding. Vascular networks and basement membranes may be conserved after decellularization, and may play a role in cellular organization.



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2.1 Compositional properties Similar to the importance of ECM components in directing cellular processes within the native stem cell niche1, during regeneration35 and in pathological conditions36, the complex ECM composition within decellularized scaffolds likely plays a key role in influencing stem/progenitor cell behaviour. Indeed, the retention of structural and functional proteins in decellularized tissues has been demonstrated by a number of research groups12, 14, 37-38. Supporting that ECM-derived scaffold composition can regulate cell behaviour, our group has shown that decellularized adipose tissue (DAT) scaffolds promote the adipogenic differentiation of human adipose-derived stem/stromal cells (ASCs)39, and that these pro-adipogenic properties are conserved across a wide array of scaffold formats with varying structural and biomechanical properties including cryomilled ECM particles within hydrogel composites40-41, as well as microcarriers42-43 and foams23 fabricated from enzyme-digested DAT. Moreover, Rothrauff et al. have shown that the tissue-specific differentiation effects of urea-extracted decellularized bovine tendon and articular cartilage ECM were conserved when human bone marrow-derived mesenchymal stem/stromal cells (bmMSCs) were cultured on tissue culture polystyrene (TCPS) with media supplemented with soluble ECM, as well as in cell-seeded hydrogel composites and on aligned electrospun nanofibers incorporating the ECM from each tissue44. Interestingly, these tissue-specific effects were not observed when the cells were cultured with pepsin-digested ECM, a finding that may be attributed to compositional differences that were noted between the two preparations, with a greater fraction of low to moderate molecular weight proteins and growth factors observed in the urea-extracted groups44. A similar study using hydrogels incorporating ECM derived from ureaextracted decellularized meniscus showed enhanced chondrogenic differentiation of human


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bmMSCs in gels with ECM isolated from the inner versus outer meniscus, which they hypothesized was related to regional differences in ECM composition45. While scaffold composition may influence stem/progenitor cell behaviour via direct binding of the cells to bioactive ECM components, another potential mechanism could involve the action of ECM degradation products, termed matrikines46. In native tissues, matrikines are generated through the proteolytic action of matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinase) family members47, and are known to recruit stem/progenitor cells, as well as modulate cell adhesion, migration and differentiation48-50. Examples of these “cryptic peptides” include the Arg-Gly-Asp (RGD) peptide derived from fibronectin and collagen, which promotes cell adhesion47, as well as the hydrophobic sequences VGVPG and VGVAPG derived from elastin, which stimulate smooth muscle cell proliferation51 and fibroblast, monocyte, and macrophage chemotaxis52 respectively. Fragments of other ECM products such as hyaluronic acid generated during tissue injury and inflammation have been shown to stimulate angiogenesis and MMP production47, as well as modulate macrophage phenotype53. Further, retained glycosaminoglycans (GAGs) within the decellularized tissues can bind and sequester soluble growth factors, regulating their availability and activity54; MMP degradation of the ECM may trigger their release, allowing for the dynamic regulation of stem/progenitor cell behaviour1. As mentioned above, decellularization protocols typically involve a range of chemical, biological and/or physical processing steps that may impact protein structure and function, including treatment stages with detergents and/or enzymes55. While it is often specified that the goal in decellularization is to preserve the complex matrix composition, the processes applied will invariably alter the ECM to some degree55. For example, detergent treatment can cause



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marked reductions in more soluble ECM components such as GAGs56-57 and growth factors58. However, the extent to which individual matrix components are retained remains poorly understood and should be more fully assessed through detailed characterization of the tissues and processing solutions at varying stages of the decellularization process59. In addition, the bioactivity of the retained components remains a further question to be addressed. When developing and characterizing decellularized matrices, many studies apply standard biochemical assays to quantify total collagen and GAG content, as well as the levels of residual dsDNA12. Further, immunohistochemical, ELISA or western blotting techniques may be used to probe for the retention of specific proteins of interest, such as growth factors12, 14. While the majority of previous work examining decellularized ECM composition has focused on identifying a pre-selected subset of proteins within specific matrices, recent efforts have applied high-throughput mass spectrometry-based proteomics approaches to more fully characterize the diverse array of proteins present in these materials37, 60-63. Although greater emphasis has been placed historically on assessing retained growth factors and cytokines, other ECM-associated factors such as matricellular proteins64-65 and small leucine rich proteoglycans (SLRPs)66-67 warrant further investigation as potentially important modulators of cell function within decellularized tissue bioscaffolds. For example, our team recently developed processing methods using a purified collagenase to deplete highly-abundant collagen in protein extracts from human DAT and decellularized cancellous bone (DCB) to enable the enhanced detection of these lower abundance constituents using mass spectrometry-based screening63. Our studies demonstrated that the DAT and DCB extracts were compositionally distinct, identifying 804 and 1210 proteins in the collagenase-treated samples respectively, including a diverse range of non-structural bioactive ECM components. Further, we identified a variety of adipogenic proteins that were


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consistently more highly expressed in the DAT, including FGF2, FGF7, and WNT11, as well as osteogenic proteins that were more highly expressed in the DCB, including osteonectin, osteocalcin, and osteopontin, as well as transforming growth factor-beta (TGF-β) and bone morphogenetic protein (BMP) family members. Taken together, these findings could support that scaffold composition plays an important role in mediating the tissue-specific differentiation effects observed with these matrix sources. 2.2 Biomechanical properties The biomechanical properties of native ECM vary greatly across tissues68 and are known to influence cell adhesion, proliferation, migration and differentiation1, 69. Cells are able to sense mechanical forces through cell adhesion complexes that can cause activation of signalling cascades to influence cell behaviour70. For example, mechanotransduction through integrin binding to the ECM can activate downstream focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) pathways, which regulate stem cell self-renewal71-72. While the effects of ECMderived scaffold biomechanical properties on stem/progenitor cell function have not been wellstudied to date, previous work with synthetic hydrogel platforms suggests that mimicking the biomechanical properties of the native tissues may help to direct lineage specification. In 2D culture studies on collagen-containing polyacrylamide gels, human bmMSCs demonstrated enhanced expression of neurogenic markers on soft matrices simulating the stiffness of brain (0.1 – 1 kPa), myogenic markers on intermediate matrices simulating the stiffness of muscle (8 – 17 kPa), and osteogenic markers on the stiffest substrates studied (25 – 40 kPa)73. A number of studies have reported on the mechanical properties of decellularized scaffolds; however, the impact of decellularization protocols on these properties is highly variable. For example, one study demonstrated increased stiffness of rat right ventricle following



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anionic detergent perfusion74, while another documented a significant decrease in stiffness in porcine aortic valves decellularized using anionic detergent, non-ionic detergent, or enzymatic digestion, which was postulated to be due to the disruption of ECM components75. Additionally, a study comparing techniques to decellularize porcine pulmonary valves demonstrated that treatment with the anionic detergents sodium deoxycholate or sodium dodecylsulphate preserved several wall longitudinal tension parameters, while treatment with proteolytic trypsin/EDTA significantly altered the ECM properties76. The variability in these findings is likely associated with differential changes in the ECM composition and/or structure depending on the specific tissue source, including the tissue type and species, as well as the processing methods applied. As such, decellularization protocols refined to minimize compositional and structural changes should theoretically better preserve the biomechanical properties of the native ECM. Since mechanotransduction can greatly impact cell function, these aforementioned studies emphasize the importance of characterizing scaffold biomechanical properties to be able to assess their potential role in mediating the response of stem and progenitor cells. However, characterization of these properties can be challenging since these materials are often soft, amorphous and/or heterogeneous depending on the tissue type and scaffold format. As such, methods for mechanical testing must be selected based on the specific material and application of interest, taking into consideration that testing should be performed in a hydrated state, as well as the types and magnitudes of mechanical forces that the scaffold may be subjected to in vivo. For example, Petersen et al. assessed the tensile strength and elastic behaviour of decellularized rat lung by performing cyclic pre-stretching followed by measurement of the ultimate tensile strength by stretching the material until failure, showing that the decellularized tissue retained similar properties to the native tissue77. In heterogeneous tissues, micro- or nano-indentation


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techniques such as atomic force microscopy (AFM) may be required in order to assess regional variability in the scaffold. In addition to providing highly sensitive force measurements, AFM offers the additional advantage of high resolution imaging that can aid in characterizing the ultrastructural topography of decellularized scaffolds, as has been performed by some groups7879

. Recent efforts have also focused on developing scaffolds with tunable mechanical

properties to investigate the influence of baseline matrix stiffness. For example, Jang et al. has developed a strategy to mechanically tune 3D printed scaffold composites incorporating decellularized porcine left ventricle using sequential vitamin B2-induced UVA crosslinking and thermal gelation, and showed that scaffolds with similar mechanical properties to native cardiac tissue enhanced the cardiomyogenic differentiation of human cardiac progenitor cells80. Another group has developed a composite ECM-derived scaffold with tunable local and bulk stiffness while maintaining the 3D microstructure of the material by coating decellularized cancellous bone with varying proportions of collagen and hydroxyapatite, showing that the scaffolds supported the osteogenic differentiation of rat bmMSCs for bone tissue-engineering applications81. 2.3 Structural properties The molecular and three-dimensional architecture of decellularized scaffolds is likely another key mediator of stem/progenitor cell behaviour. Notably, Brown et al. showed that decellularized scaffolds derived from a variety of tissue types displayed distinct structural and morphological differences, demonstrating the tissue-specificity of these features82. Certain structural features, such as the basement membrane, which can be conserved following decellularization, have been shown to guide cell migration and growth patterns83. The developing



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field of whole organ decellularization has also provided evidence of the importance of ECM structure in directing cell function. For example, using whole decellularized rat lungs, one group has shown that mixed populations of neonatal rat lung epithelial cells displayed site-specific attachment of various cell types to the appropriate anatomical regions, as evidenced by immunostaining for markers of Clara cells, airway epithelial progenitor basal cells, and type I and II alveolar cells84. Moreover, site-specific differentiation of murine embryonic stem cells (ESCs) seeded on whole decellularized rat lung scaffolds has also been reported85. Together, these studies suggest that the local ligand landscape and ultrastructural characteristics of ECMderived scaffolds may provide regional cues for cell attachment and differentiation. In addition to the importance of ultra- and microstructural features, the surface topography of ECM-derived scaffolds has been postulated to mediate the binding of host cells to the material, and may thereby influence the initial host response82. Other parameters such as scaffold porosity could impact the cellular response by facilitating nutrient and oxygen diffusion, as well as by modulating cell-cell and cell-ECM interactions that can influence cell adhesion, migration, proliferation, and differentiation86-87. In particular, cell density and shape are wellrecognized mediators of stem cell lineage commitment88, and may be important factors in modulating the response of stem/progenitor cells seeded on decellularized bioscaffolds. For example, in comparing the adipogenic differentiation of human ASCs in composite hydrogels incorporating DAT particles of varying sizes (278 ± 3 µm vs. 38 ± 6 µm), our group found that adipogenic differentiation was enhanced in the scaffolds containing small particles and a higher cell density, which we postulated was due to enhanced cell-cell interactions in the ASCs clustered around the smaller particles40.


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Through the process of decellularization, it is inevitable that structural changes will occur since the detergents and enzymes commonly used in these protocols can denature or cleave ECM proteins16. Strategies applied to investigate the structural properties of decellularized scaffolds include histological analysis, as well as the use of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to examine ultrastructure at both the macro and micro levels. Additionally, Hwang et al. recently reported on the use of a carboxyfluorescein-labeled collagen hybridizing peptide that binds specifically to denatured collagen, in order to visualize the effects of decellularization on collagen integrity in porcine ligament and urinary bladder89. In general, it is currently unclear at what level the ECM structure needs to be retained in order to preserve its bioactive effects. To begin to address this question, recent work has focused on investigating strategies to better conserve the structural integrity of the ECM following tissue decellularization. Fischer et al. showed that the ECM structural integrity of porcine kidneys was best preserved when detergent decellularization was performed at 4˚C as compared to either room temperature or 37˚C, as evidenced by histological staining and immunohistochemical (IHC) detection of laminin, fibronectin, and collagen IV90. Moreover, Mayorca-Guiliani et al. demonstrated improved preservation of ECM integrity for several organs in mice including the lung and liver through in situ decellularization of the tissues via perfusion of the solutions through the native vasculature91. 2.4 Challenges and future directions to understand mechanisms of stem/progenitor cell modulation by ECM-derived scaffolds In working with decellularized tissues, it is extremely challenging to decouple the individual effects of the compositional, biomechanical, and structural properties of the scaffold, as they are closely linked and the scaffolds have innate heterogeneity. Moreover, the strategies



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used to investigate these properties must be adapted based on the material and application of interest. In addition, there is a dynamic interrelationship between the stem /progenitor cells and these engineered cellular microenvironments. Seeded or infiltrating regenerative cell populations will sense and respond to the ECM within the decellularized tissues, and in turn remodel the scaffolds via the action of MMP and ADAM family members, as well as through the synthesis of new matrix components47. The properties of the remodelled ECM may be extensively altered through these cell-ECM interactions, which can subsequently impact the downstream cellular response92-93. As such, future studies should strive to monitor and model these changes over time in order to develop a deeper understanding of the mechanisms of stem/progenitor cell-mediated regeneration using decellularized tissue bioscaffolds. Another potential hurdle to the broad-scale clinical application of decellularized tissues is that tissue donor variability may result in bioscaffold heterogeneity, with factors such as sex, age, anatomical site, and health status potentially impacting the properties of the ECM 60, 94. As such, there is a need for larger-scale studies that more fully characterize the ECM from a range of donors and begin to systematically investigate the potential effects of each of these parameters on the cellular response to the engineered bioscaffolds. A limitation is that a substantial number of tissue samples would be required in order to obtain statistically and biologically meaningful data95. From this perspective, the further development of high-throughput proteomic techniques using mass spectrometry provides a more feasible approach to begin to compare the complex protein composition of decellularized matrices in a large number of donors63. Building from this, high-throughput cell-based assays such as tissue matrix microarrays represent another valuable advancement in the field that can be used to screen stem or progenitor cell responses to varying tissue-specific ECM compositions in both 2D and 3D formats96. In general, the influence of


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tissue-specific composition is more easily explored in emerging platforms that strive to control for the potentially confounding effects of scaffold biomechanics and/or structure. However, new tools and technologies are required to complement this knowledge by enabling the systematic exploration of the roles of these other key properties in directing cellular responses in these complex 3D biological systems. Improved characterization of the compositional, biomechanical, and structural properties of ECM-derived scaffolds will aid in understanding the cell-instructive mechanisms mediated by these materials to design optimized scaffolds that enhance tissuespecific regeneration, as well as assisting with their clinical translation. 3.0 Stem/progenitor cell-instructive effects of decellularized ECM In this review, we define “stem/progenitor cell-instructive effects” of ECM-derived scaffolds as any cellular response to the material that may ultimately aid in tissue regeneration. By this definition, decellularized ECM can instruct stem or progenitor cell behaviour through a variety of direct and indirect means, such as by mediating stem/progenitor cell adhesion, migration, proliferation, paracrine factor production, and/or differentiation (Figure 2). However, it should be noted that in studies investigating the use of these constructs within animal models, the overall in vivo response is likely dependent on a number of factors including the scaffold properties, local microenvironment, and whether the delivered cell populations are sourced autologously or allogeneically, as well as the selection of specific immune-competent or immune-compromised animal models. A common challenge that has been encountered in strategies attempting to deliver stem cells in suspension is that of poor long-term cell viability, which may be related to the loss of cell adhesion leading to anoikis, or to harsh conditions within the target microenvironment97-98. Consequently, ECM-derived scaffolds have been explored as potential cell delivery platforms to



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enhance stem/progenitor cell adhesion, viability, and proliferation. Indeed, Sharma et al., have shown in a baboon bladder regeneration model that autologous green fluorescent protein (GFP)labelled bmMSCs seeded on decellularized porcine small intestine submucosa (SIS) displayed high expression levels of the proliferation marker Ki-67 within the implant region at 10 weeks post-implantation99. Similarly, decellularized porcine SIS scaffolds were shown to significantly improve survival and proliferation relative to cell delivery alone, as evidenced by bioluminescence imaging of donor syngeneic luciferase+ murine ASCs seeded onto the scaffolds in a murine excisional wound model100. By promoting stem/progenitor cell retention and survival, ECM-derived scaffolds may allow these cells to have increased therapeutic efficacy, while providing an inductive substrate that could potentially direct differentiation and/or paracrine factor production, as discussed in detail below.


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Figure 2. Stem/progenitor cell-instructive effects mediated by ECM-derived scaffolds. ECM-derived scaffolds can influence stem/progenitor cell adhesion, migration, viability, proliferation, and differentiation along multiple lineages. Moreover, these scaffolds can influence stem/progenitor cell secretion of paracrine factors to promote angiogenesis, and modulate the immune response. VEGF: vascular endothelial growth factor; ANG-1: angiopoietin-1; PDGF: platelet derived growth factor; IL-6: interleukin 6; IL-10: interleukin 10; TNF-α: tumor necrosis factor-alpha.



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3.1 Tissue-specific differentiation effects of decellularized ECM A wide array of studies have reported that donor stem/progenitor cells can enhance tissue regeneration in ECM-derived scaffolds and as highlighted in Table 1, there is a growing body of literature with a range of cell types that supports that seeded stem or progenitor cell populations may have the potential to directly contribute to regeneration through engraftment and differentiation. In particular, tissue-specific ECM has been widely postulated to promote lineagespecific differentiation in vitro. For example, our group demonstrated that DAT provided an adipo-inductive microenvironment for human ASCs, inducing the expression of the key transcription factors peroxisome proliferator-activated receptor-gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in the absence of exogenous differentiation factors39. Through the development of a variety of DAT-based bioscaffolds, we have shown that the adipose-derived ECM displays both adipo-conductive and adipo-inductive properties by enhancing the in vitro differentiation of human ASCs in adipogenic culture medium and by promoting adipogenic differentiation in proliferation medium that would normally inhibit adipogenesis23, 39-43. In one study, we found that human ASC adipogenesis was enhanced through culture on DAT-based microcarriers in comparison to gelatin microcarrier and TCPS controls, as evidenced by adipogenic gene expression, increased activity of the lipid biosynthetic enzyme glycerol 3-phosphate dehydrogenase (GPDH), and intracellular lipid accumulation42. Elevated adipogenic gene and protein expression, as well as low levels of intracellular lipid accumulation, were observed in the non-induced DAT microcarrier controls maintained in proliferation medium, demonstrating retention of the adipo-inductive properties in the pepsin-digested ECM42. Similar effects have been shown using stem or progenitor cells cultured on cartilage matrix. In vitro studies have demonstrated the chondro-inductive properties of decellularized and


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devitalized cartilage matrix through culturing MSCs in 3D aggregates incorporating ECM particles101, as well in hydrogel pastes102, showing augmented expression of chondrogenic genes such as collagen II and aggrecan. Culturing human ASCs on decellularized porcine articular cartilage has also been shown to increase sulphated GAG production, as well as selectively upregulate chondrogenic markers over time at both the transcript and protein levels in the absence of exogenous chondrogenic factors103. In terms of myocardial matrix, our laboratory has developed 3D porous foams derived from decellularized porcine left ventricle and demonstrated that these scaffolds enhanced gene and protein expression of cardiomyocyte markers including Nkx2.5, myosin heavy chain 6, and atrial natriuretic peptide in human pericardial fat ASCs as compared to collagen gel controls24. In another study, human induced pluripotent stem cell (iPSC)-derived cardiovascular progenitor cells displayed enhanced cardiomyocyte marker gene and protein expression when cultured on decellularized whole mouse heart, and after 20 days of perfusion with vascular endothelial growth factor (VEGF) and dickkopf-related protein 1 (DKK1) exhibited spontaneous contractions104. In a study by Rajabi-Zeleti et al., 3D macroporous scaffolds derived from enzyme-digested decellularized human pericardium were shown to enhance the migration, survival, proliferation, and differentiation of human Sca-1+ cardiac progenitor cells relative to collagen scaffold controls that had a similar porous structure, as well as intact decellularized pericardium105. These results translated in vivo, showing improved retention and staining suggestive of augmented myogenic differentiation of the donor progenitors in the 3D macroporous scaffolds following subcutaneous implantation in immunosuppressed rats, as detected by immunohistochemical staining for α-myosin heavy chain and the human marker hTRA-1-85 in separate tissue sections105.



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In studies comparing multiple ECM sources, one group used ECM-based coatings derived from skin, skeletal muscle, and liver to demonstrate that tissue-matched cell types showed enhanced proliferation and differentiation when cultured on ECM sourced from their tissue of origin106. In addition, murine ESCs grown on heart-derived ECM showed increased expression of cardiomyocyte differentiation markers as compared to cells grown on liver ECM107. Similarly, human ESCs cultured on kidney- or lung-derived ECM demonstrated upregulated lineage-specific gene expression, suggesting that the ECM source played a role in directing stem cell differentiation108. In a study by O’Neill et al.109, ECM-derived scaffolds were developed from the renal cortex, medulla, and papilla of porcine kidneys, each of which exhibited distinct ECM characteristics. Interestingly, the group found that kidney stem cells displayed differential cell proliferation, morphology and structure formation according to the regional specificity of the ECM and pointed toward a key role for ECM composition since the cell-instructive effects were observed with three different ECM-derived scaffold formats: intact sheets, ECM hydrogels, and pepsin-digested ECM delivered in suspension. Taken together, these results demonstrate that differences in the ECM properties can modulate stem/progenitor cell lineage specification and support the rationale for further investigating tissue-specific ECM as a cell-instructive component to guide stem/progenitor cell differentiation in regenerative platforms. 3.2 Effects of decellularized ECM on paracrine factor production While stem/progenitor cells have the potential to contribute to new tissue development through differentiation, there is evidence to suggest that the predominant mechanism of regeneration for cell types such as MSCs is indirect through the secretion of beneficial paracrine factors that promote the establishment of a more conducive milieu for host tissue regeneration110111

. Table 2 summarizes the main findings of key studies demonstrating the pro-angiogenic and


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immunomodulatory functions of MSC populations seeded within decellularized tissue bioscaffolds. In all cases, it is important to note that the overall response of stem/progenitor cells to ECM-derived scaffolds in vivo will also be mediated by reciprocal signalling and action of host cells that infiltrate the scaffolds following implantation, including immune cells such as macrophages, and host-derived progenitors. MSC populations, including those derived from bone marrow and adipose tissue, secrete a variety of angiogenic, immunomodulatory, and cytoprotective factors, and these can play a central role in mediating tissue regeneration110-111; however, little is known about the impact of ECM-derived scaffolds on the stem/progenitor cell secretory profile. In terms of angiogenic factor production, Nie et al. demonstrated that rat ASCs cultured on decellularized human dermal ECM sheets (AlloDerm®) secreted significant quantities of VEGF, hepatocyte growth factor (HGF), TGF-β, and basic fibroblast growth factor (bFGF) in vitro112. Using a diabetic rat excisional wound model, the same study reported increased CD31+ blood vessel density and augmented protein levels of the same four angiogenic factors within seeded scaffolds as compared to unseeded controls. A number of other groups have also provided histological and immunohistochemical evidence that seeding decellularized scaffolds with MSCs improves neovascularization in vivo as compared to unseeded controls113-118. The immune response to decellularized materials has emerged as an area of significant research interest. In particular, several groups have demonstrated that ECM-derived scaffolds may promote a favourable “M2-like” macrophage phenotype more conducive to tissue regeneration119-122. However, few studies have examined the response to these scaffolds in tandem with stem/progenitor cells. In a rat bladder reconstruction model, immunohistochemical analysis revealed differential cytokine production in the implant region of decellularized rat



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bladder matrix seeded with allogeneic bmMSCs as compared to unseeded implants or MSCs alone, with qualitatively increased anti-inflammatory cytokine production observed in parallel with improved tissue regeneration118. Using a rat subcutaneous implantation model, our laboratory demonstrated that seeding human DAT with allogeneic rat ASCs modulated the macrophage phenotype, increasing the relative fraction of infiltrating macrophages that expressed the pro-regenerative M2 macrophage marker CD163, with enhanced angiogenesis and host-derived adipose tissue formation observed within the seeded bioscaffolds113. Other groups have reported that seeding decellularized porcine SIS123 or decellularized rat spinal cord124 with allogeneic bmMSCs decreased macrophage and/or T cell infiltration in comparison to unseeded controls in a porcine epicardial patch model and rat spinal cord injury model respectively. 3.3 Challenges and future directions to understand stem/progenitor cell-instructive effects of decellularized ECM While a number of studies have investigated tissue-specific differentiation of stem/progenitor cells on decellularized scaffolds, a notable limitation is that very few studies to date have systematically investigated the role of the ECM source to be able to more fully assess the benefits of a tissue-specific approach. In addition, the relative importance of compositional, biomechanical, and structural features is not well understood, since as stated, these properties are inherently interrelated and seeded or infiltrating cells can remodel the scaffolds, changing the properties over time. As such, an improved understanding of these features and the dynamic changes that may occur with scaffold remodelling may aid in elucidating the predominant mechanisms guiding tissue-specific differentiation on decellularized ECM. Paracrine factor production by donor stem/progenitor cells as a regenerative mechanism is a relatively recent area of interest in tissue engineering. The majority of studies that have


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reported paracrine effects in stem/progenitor cell-seeded decellularized scaffolds have done so using in vivo models. Given the dynamic interplay between donor cells, the ECM, and infiltrating host cell populations within animal models, in vitro culture studies may aid in providing greater mechanistic insight into the observed effects. For example, mass spectrometrybased proteomics approaches to characterize the stem/progenitor cell secretory profile 125-126 could be adapted as a high-throughput method to investigate the influence of ECM-derived culture substrates on the production of paracrine factors. 4.0 Applications of decellularized scaffolds for stem or progenitor cell-driven tissue-specific regeneration Regardless of the mechanisms, decellularized bioscaffolds have the potential to enhance the regenerative capacity of stem or progenitor cell populations to address a broad spectrum of clinical issues. ECM-derived scaffolds sourced from both xenogeneic and cadaveric human tissues have been applied in the clinic showing safety and efficacy for specific tissue-engineering applications including integumentary, urogenital and musculoskeletal reconstruction127-128. In terms of stem/progenitor cell sourcing for scaffold pre-seeding, a number of factors must be taken into account including cell availability and whether the strategy is targeting direct versus indirect regenerative mechanisms, as well as the use of allogeneic versus autologous sources, as has been reviewed previously129-131. In the following section, we highlight some specific examples involving stem/progenitor cell delivery using decellularized tissue scaffolds for the regeneration of cartilage and bone, as well as for wound healing applications and treatment of damaged cardiac tissue following myocardial infarction. In particular, studies incorporating clinically-relevant animal models will be discussed.



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Cartilage regeneration strategies are critically needed to treat diseases such as rheumatoid and osteoarthritis, and several groups are investigating decellularized cartilage matrix as a regenerative platform. MSC-seeded decellularized cartilage scaffolds implanted into rabbit knees have shown improved repair of cartilage defects as compared to unseeded controls based on histological scoring132. Using autologous ASCs in a similar rabbit knee cartilage defect model, Kang et al. showed that seeded decellularized human cartilage scaffolds improved histological scores, collagen II and GAG production, as well as biomechanical properties as compared to unseeded and sham controls133. Stem/progenitor cell-seeded ECM-derived scaffolds have also been investigated for the regeneration of damaged bone. A recent study reported that autologous ASCs incorporated in a bovine trabecular bone matrix scaffold improved scaffold remodelling and maintenance of anatomical structure as compared to unseeded scaffolds within a minipig model of craniofacial reconstruction134. Another group demonstrated that decellularized rat calvaria bone scaffolds supported in vitro osteogenic differentiation of rat bmMSCs based on histological staining and gene expression analysis of alkaline phosphatase and osteocalcin135. In vivo, the same study showed that MSC-seeded scaffolds improved bone formation in a rat calvarial defect model135. Additionally, Hung et al. have shown that 3D printed polycaprolactone (PCL) scaffolds incorporating micronized decellularized bovine trabecular bone augmented the expression of osteogenic genes (RUNX2, SPARC, and BGLAP) in human ASCs as compared to PCL scaffolds alone, and improved bone regeneration in a murine calvarial defect model136. There has been growing interest in developing strategies to guide the wound healing process in order to prevent tissue fibrosis and scarring in large wounds, or as a therapeutic for chronic wounds. While decellularized dermal scaffolds such as AlloDerm® are currently used in


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the clinic, recent evidence has demonstrated that seeding of decellularized dermal scaffolds with human ASCs (mouse ECM scaffold)137 or rat bmMSCs (human ECM scaffold)138 can enhance the wound healing process in full thickness defect models in nude mice and immunocompetent rats respectively. In this context, ASCs and MSCs may aid in the wound healing process through indirect paracrine mechanisms, and/or by directly contributing to newly formed vasculature, and possibly by displaying epidermal cell-like characteristics137, 139-140. Importantly, treatment of full thickness wounds in mice using allogeneic murine ASCs alone has been shown to result in poor cell viability, whereas ASCs seeded onto decellularized porcine SIS scaffolds promoted host cell proliferation and survival in vivo100, indicating that the combination of stem/progenitor cells and decellularized ECM may be a promising therapeutic. In the treatment of myocardial infarction, decellularized scaffolds have also been explored as a stem/progenitor cell delivery platform, with the main strategies involving either a cardiac patch composed of cells and ECM, or the use of thermosensitive hydrogels incorporating decellularized tissue8. One group has developed a bioengineered cardiac patch consisting of decellularized bovine pericardium and multilayered sheets of rat bmMSCs, showing that implantation resulted in improved cardiac function and angiogenesis with donor MSC retention over 12 weeks in a syngeneic rat myocardial infarction model141-142. Moreover, Gálvez-Montón et al. developed a graft consisting of decellularized human pericardium, allogeneic porcine ASCs, and an electrical impedance spectroscopy system to noninvasively monitor scar remodelling in a porcine myocardial infarction model, showing improved healing as well as cardiac function in grafts containing donor cells as compared to unseeded controls143. Using injectable decellularized porcine ventricular hydrogels, work from the Christman lab has shown increased recruitment of cKit+ progenitor cells and endogenous Troponin+ cardiomyocytes as



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compared to saline-injected controls in a rat ischemia-reperfusion model of myocardial infarction144-145. This group subsequently demonstrated that human cardiac progenitor cells encapsulated within the hydrogels showed enhanced gene expression of cardiac markers including GATA-4 and MLC2v, as well as increased cell proliferation and survival as compared to collagen controls, indicating a possible regenerative mechanism for the in vivo application146. 4.1 Challenges and future directions in the application of decellularized scaffolds for stem/progenitor cell-driven tissue-specific regeneration Both decellularized ECM products128 and stem/progenitor cell therapies147 are currently in clinical use, and the accumulating evidence described here suggests that the combination of these approaches may serve as a more effective strategy for some tissue-engineering applications. However, the specific scaffold format and properties need to be optimized for each target application, and careful consideration must be given to the cell source selected in terms of both safety and efficacy. The seeding methods and density applied should be refined to ensure the delivery of a predictable dose of cells with well-characterized functionality. From this perspective, strategies that require extensive in vitro pre-culturing may present substantial barriers from both cost and regulatory perspectives that could limit broad-scale application. In order to bridge the gap toward further clinical translation of decellularized tissue products, there is a critical need to first develop standardized decellularization protocols that preserve key tissue-specific features and generate scaffolds that have known cell-instructive effects. Further, the potential impact of the decellularized tissue source and scaffold format on the cellular response should be systematically explored, with a focus on developing a deeper understanding of the mechanisms of stem/progenitor cell-mediated tissue regeneration. These investigations could include assessing the potential effects of scaffold donor variability in the


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case of allogeneic matrices, as previously described, as well as comparing the regenerative response in allogeneic versus xenogeneic matrices to determine how scaffold bioactivity is altered when non-human ECM sources are applied. Regardless of the source, standard characterization methods and release criteria in terms of the acceptable amount of residual cellular materials need to be established to ensure safe use of these scaffolds in the clinic. Moreover, rigorous testing using relevant animal models is required to accurately assess the safety and efficacy of these therapies. Several studies using ECM-derived scaffolds have made use of immune-compromised mouse models to investigate the in vivo response to matrices seeded with human donor stem/progenitor cells. While these models can provide important insight, the role of the host response and immune cells in mediating tissue regeneration is becoming increasingly evident and merits further investigation in immune-competent models12, 148. From this perspective, a recent study by Wang et al. demonstrated the utility of humanized mouse models to investigate the immune response to hydrogels incorporating xenogeneic versus allogeneic myocardial matrix, reporting reduced CD4+ T cell infiltration into scaffolds with ECM derived from human cadavers as compared to porcine ECM, although both materials induced a pro-remodelling Th2 phenotype, as well as the expression of pro-regenerative M2 macrophage markers149. Similar use of these models with stem/progenitor cell-seeded ECM-derived scaffolds could help to more accurately predict the immune response to these constructs in humans, and could possibly serve as a pertinent in vivo system to study the immunomodulatory functions and paracrine factor production of allogeneic human donor stem/progenitor cells. While a number of studies report enhanced tissue regeneration in decellularized bioscaffolds seeded with stem or progenitor cell populations, detailed studies focused on the



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identification of retained donor cells are required to be able to determine whether the newlyforming tissues are host- versus graft-derived. Not only would this provide insight into the regenerative mechanisms, but could allow for long-term monitoring of the donor cells within the graft. In particular, donor cell tracking would be valuable for approaches that make use of cells derived from ESCs or iPSCs, where the potential for teratoma formation remains a longstanding concern that limits their clinical application150-151. Current methods to identify donor cells include the use of fluorescent dyes to label the cytoplasm or membranes (e.g. PKH) of the cells prior to implantation to enable histological detection; however these markers are not stably expressed and are sensitive to photo-bleaching152-153. Further, lipophilic dyes such as PKH can be transferred to other cell populations via exchange of membrane microdomains or consumption by macrophages154-155. Fluorescent in situ hybridization (FISH) allows for identification of male donor cells via the Y chromosome but this method is costly and is not applicable to autologous cell populations. Moving beyond histological analysis, imaging technologies have the benefit of allowing for longitudinal tracking of donor cells in living animals. Iron oxide and/or fluorine contrast agents can be used to pre-label donor stem/progenitor cells for in vivo detection through magnetic resonance imaging (MRI), and can also be administered intravenously to allow for uptake by host macrophages as a dual labelling technique156. Bioluminescence imaging using cells transduced with luciferase provides insight into donor cell viability, in addition to allowing sensitive, quantitative tracking of the population in living animals157. Further, the plasmids can be engineered to have a dual reporter system with a stably-expressed fluorescent marker, such as enhanced green fluorescent protein (eGFP), allowing for cell localization in complementary IHC analyses158. However, the possible alteration of the cells through transduction, which requires plasmid delivery via lentiviral vectors, as well as the costs and scalability of the process to


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generate sufficient cell numbers for large-scale studies are potential challenges with this approach159. 5.0 Conclusions and Future Outlook ECM-derived scaffolds sourced from decellularized tissues hold considerable promise in the field of regenerative medicine as they can mimic the native tissue microenvironment and stem cell niche. Emerging evidence suggests that tissue-specific ECM-derived scaffolds may have improved efficacy as compared to non-tissue matched ECM sources for regenerative applications due to the unique biochemical, biomechanical, and biophysical properties of the ECM in every tissue. However, further research is required to more fully assess this paradigm and determine whether some ECM sources may have broader utility, as the capacity to expand beyond a tissue-specific approach could be advantageous when the availability of healthy tissues for ECM derivation is limited. The complex biological milieu found within ECM-derived bioscaffolds may regulate the response of seeded stem/progenitor cells and infiltrating host cell populations to enhance tissue regeneration by promoting viability, cell retention, paracrine factor production, constructive tissue remodelling, and/or lineage-specific differentiation. Future studies should focus on developing a deeper understanding of the mechanisms of stem/progenitor cell-mediated regeneration within these tissue-derived bioscaffolds in order to design more effective and clinically-translational cell-instructive delivery platforms, including systematically assessing the benefits of delivering undifferentiated versus pre-differentiated cell populations, as well as investigating the roles of host immune cell and progenitor populations in greater depth. While both stem/progenitor cell therapies and decellularized ECM products are in clinical use, further pre-clinical studies are required to progress towards the clinical implementation of stem/progenitor cell-seeded ECM-derived scaffolds as therapeutics. However, a growing body of



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data suggests that the hybrid of these therapies may be more effective than either of its predecessors, and could have widespread applications in the treatment of injury and disease. Acknowledgements The authors would like to acknowledge funding from the Canadian Institutes of Health Research (CIHR) (FRN 119394), as well as scholarship support for Arthi Shridhar from the CONNECT! NSERC CREATE Program and the Collaborative Graduate Training Program in Musculoskeletal Health Research (CMHR) at Western University.


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Table 1. Direct Effects – Tissue-specific Differentiation* Tissue type

Scaffold format

Stem cell source

In vitro

Adipose

Human DAT39, 113, Micronized

Human ASCs23, 39-42,

human DAT in hydrogel

Allogeneic rat ASCs41,

GPDH enzyme activity and intracellular lipid accumulation at 7

composites40-41, Porous foams

113

and 14 d; Adipo-instructive effects observed in both adipogenic

√

In vivo √

Cell instructive effects In vitro: Adipogenic gene expression (PPARγ, C/EBPα, LPL),

from α-amylase-digested human

medium and proliferation medium controls

DAT23, Microcarriers

In vivo: Visualization of mature adipocytes in DAT foams and

incorporating pepsin-digested

scaffolds (8 & 12 wks) and hydrogel composites (12 wks) in a

human DAT42

subcutaneous Wistar rat model; Enhanced host-derived adipocyte formation in allogeneic ASC-seeded DAT vs unseeded controls at 8 & 12 wks in a subcutaneous Wistar rat model

Micronized human DAT160

Human ASCs

√

Mature donor-derived (HuNu+) adipocyte formation at 4 and 8 wks in an athymic rat model

Bone

Porous scaffold from milled

Autologous porcine

decellularized bovine trabecular

ASCs

√

bone134

CT and µCT showing bone volume and condyle regeneration vs unseeded controls and condylectomy, with sites of enchochondral ossification at 6 months in a porcine ramus condyle unit model; BSP and collagen I expression by IHC; Peak force during bending similar in magnitude to native bone (~30 N)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Particles of decellularized rat

Allogeneic rat MSCs

√

√

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In vitro: osteogenic gene expression (BSP, ALP, OCN) within

cortical calvaria (~2 mm

7 d in proliferation media; matrix mineralization seen by

diameter)135

alizarin red S staining at 10 d and 17 d vs TCPS controls In vivo: bone density and volume in seeded vs unseeded scaffolds observed through µCT and histomorphometry at 12 wks in a rat calvaria critical-sized defect model

Polycaprolactone (PCL) scaffolds

Human ASCs

√

√

In vitro: expression of osteogenic genes (RUNX2, OCN, ON)

incorporating micronized

in the absence of exogenous osteoinductive factors at 3 wks

decellularized bovine trabecular

In vivo: regenerated bone volume by µCT at 6 and 12 wks vs

bone136

PCL scaffolds without decellularized ECM; osteoid and mineralized tissue formation by histological staining at 12 wks in an athymic mouse calvaria critical-sized defect model

Decellularized and micronized

√

Allogeneic rat MSCs

bovine cortical bone161

µCT showed calcified tissue and bone volume in MSC-seeded decellularized scaffolds at 1 and 3 months in a subcutaneous rat or cranial critical-sized defect rat model vs unseeded and Cytodex controls

Cartilage

3D cell aggregates incorporating

Rat bmMSCs

√

Coll II gene expression in the absence of growth factors at 1

micronized decellularized porcine

and 3 d vs aggregates without ECM induced with TGF-β;

articular cartilage101

AGG and SOX9 gene expression in the absence of growth factors at 1 and 7 d vs aggregates without ECM


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Porous scaffold from micronized

Human ASCs

√

AGG and Coll II gene expression at 14 d in the absence of

decellularized porcine articular

exogenous growth factors; Coll I and X gene expression at 14

cartilage103

d (i.e. reduced fibrocartilage and hypertrophy markers); Coll I and X protein expression by IHC at 42 d; Sulphated GAG and Coll II expression by histology and IHC at 42 d; CAM of remodeled cartilage ~ 150 kPa at 42 d, more similar to native articular cartilage (500–900 kPa) vs unseeded controls (~50 kPa)

Porous scaffold derived from

Autologous rabbit

micronized, chemically

ASCs

√

Coll II staining in seeded vs unseeded controls by IHC at 3 and 6 months in a rabbit knee model; Coll II and GAG content of

crosslinked human articular

neocartilage reached ~ 70% and 90% respectively of native

cartilage133

cartilage levels by 6 months; Neocartilage stiffness reached 83% of native cartilage control at 6 months

Porous scaffold derived from

Allogeneic rabbit

micronized decellularized bovine

bmMSCs

√

tissue indicated by histology in seeded vs unseeded controls at 6

articular cartilage132 Dermal

Human decellularized dermal

De novo hyaline cartilage formation and integration with host

and 12 wks in a rabbit knee model Human GFP+ ASCs

√

tissue (Alloderm®)162

GFP+HSP47+ (fibroblast marker) and GFP+CK19+ (epidermal epithelial marker) cells observed by IHC at 2 and 4 wks respectively suggestive of in situ differentiation of donor ASCs in an athymic mouse cutaneous wound model



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Sections of decellularized murine

√

Human ASCs

dermal tissue137

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granulation tissue formation and epithelialization at 9 and 14 d respectively, in seeded vs unseeded scaffolds in a BALB/c nude mouse cutaneous wound model

Sections of decellularized porcine

Allogeneic GFP+

dermal tissue163

murine ASCs

√

wound closure and re-epithelialization in seeded vs unseeded scaffolds and chondroitin sulphate hydrogels in a C57BL/6 mouse cutaneous wound model at 7 d

Lung

Sectioned decellularized monkey

Human ESCs

√

lung108

expression of lung-associated genes (respiratory epithelial cells) (BPI, SP-B, MUC5B) at 2 d; IHC staining indicated a spatial organization of cells lining alveolar spaces with thin networks of surfactant protein B and C expressing cells (pneumocytes) at 8 d

Decellularized whole rat lungs85

Murine ESCs

√

CK18+ epithelial cells and pro-surfactant protein C+ pneumocytes by flow cytometry in digested decellularized scaffolds vs Matrigel and collagen controls at 7 d; IHC showing CK18+ & PDGFR-α + (single labelled) cells indicating presence of an epithelial cell phenotype at 14 d; Sheets of CK18+ cells lining upper trachea and CC10+ Clara cells in lower trachea, mimicking the native tissue distribution at 21 d; surfactant protein A secretion by SDS-PAGE at 21 d


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Decellularized whole mouse

Murine bmMSCs

√

gene expression of markers of lung epithelium (CCSP, AQP5)

lungs164

and lung tissue remodeling (FAP, OP) at 14 d

Myocardium/

Sections of decellularized human

Human iPSC-derived

Pericardium

ventricular tissue165

cardiomyocytes

√

Expression of NKX2.5 transcription factor and CX43 (gap junction protein) by IHC at 10 d; Expression of cardiomyocytespecific proteins (ASA, CTNT, α-MHC) by IHC at 10 d

√

expression of cardiac-specific genes (α-MHC, β-MHC, CTNT)

Decellularized whole mouse

Human iPSC-derived

heart104

cardiovascular

at 26 d; Cardiomyocyte markers (α-actinin and CTNT) and CX43

progenitor cells

by IHC at 20 d; Formation of vessel-like structures and spontaneous contractions at 20 d of perfusion with VEGF and DKK1

Composite hydrogels from

Human ESCs

√

CTNT gene expression at 8 and 12 d in scaffolds incorporating

pepsin-digested decellularized

ECM vs collagen controls in non-inductive media; CX43+ cell

porcine myocardium combined

distribution and organization by IHC in scaffolds incorporating

with collagen166

ECM in non-inductive media vs collagen controls in inductive media at 8 and 12 d

Porous scaffold from pepsin-

Human Sca-1+ cardiac

digested human decellularized

progenitor cells

√

√

pericardium105

In vitro: cardiac gene expression (NKX2.5, GATA4, MEF2c, CTNT, α-MHC) vs collagen I controls at 7 d In vivo: α-MHC+ cells visualized by IHC at 1 month in seeded ECM-derived scaffolds vs collagen I controls in a subcutaneous rat model with cell alignment and myocyte-like organization



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Decellularized human

Allogeneic GFP+

pericardium167

porcine ASCs

√

Page 46 of 54

MRI showed enhanced left ventricular ejection fraction and stroke volume in seeded vs unseeded scaffolds at 1 month in a porcine myocardial infarction model; GFP+ cells within the graft and infarct area positive for cardiac biomarkers (NKX2.5, cKit, MEF2, CTNI, and CTNT) by IHC at 1 month; Picosirius red staining showed collagen I/III ratio in seeded vs unseeded scaffolds suggestive of improved healing at 1 month

Composite hydrogel scaffold

Human c-kit + cardiac

from pepsin digested

progenitor cells and

scaffolds vs controls at 8 wks in a Fischer 344 rat myocardial

decellularized porcine left

human turbinate MSCs

infarction model; LV wall thickness and fibrosis in scaffolds

√

ventricle combined with PCL168

Improved ejection fraction and fractional shortening in seeded

seeded with both cell types vs unseeded and single cell type controls at 8 wks

Valve

Decellularized human and

Human bmMSCs

√

Endothelial (vWF, CD31), fibroblast (vimentin) and SMC (α-

porcine pulmonary valves169

SMA, SM22, vimentin) markers observed by IHC at 30 d, with site-specific differentiation; TEM showed monolayers of flat endothelial-like cells expressing mature tight junctions close to basal lamina

Decellularized ovine aortic

Autologous ovine

√


endothelial-like cells aligned in direction of blood flow at 18

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valve170

months in an autologous ovine model; desmin+ and cytokeratin+

bmMSCs

cells by IHC in seeded samples at 18 months, as well as α-SMA+ staining suggestive of repopulation with myofibroblasts Vessels

Decellularized ovine carotid

Autologous ovine

artery171

bmMSCs

√

PKH26+ donor cells detected up to 2 months in an autologous carotid ovine model; vWF+ cells on luminal side with EC-like morphology and α-SMA+ on medial side with SMC-like morphology by IHC at 2 months; SEM showed similar morphology of regenerated endothelium vs native tissues at 5 months

√

α-SMA+ SMCs detected at intimal layer by IHC and vWF+ ECs

Decellularized porcine iliac

Autologous ovine

arteries172

endothelial progenitor

on luminal surface of seeded scaffolds in a carotid interposition

cells

ovine model at 130 d; Seeded scaffolds developed into neovessels exhibiting vasomotor responsiveness at 130 d

*

AGG: Aggrecan; ALP: Alkaline phosphatase; α-MHC: Alpha-myosin heavy chain; AQP5: Aquaporin 5; ASCs: Adipose-derived stem/stromal cells; ASA: Alpha-sarcomeric actinin α-SMA: Alpha-smooth muscle actin; β-MHC: Beta-myosin heavy chain; bmMSCs: Bone marrow-derived mesenchymal stem/stromal cells; BPI: bactericidal/permeability-increasing protein; BSP: Bone sialoprotein; CAM: Compressive aggregate moduli; CCSP: Clara cell secretory protein; C/EBPα: CCAAT-enhancer-binding protein-alpha; CK18: Cytokeratin 18; CK19: Cytokeratin 19; Coll: Collagen; CT: computerized tomography; CTNI: Circulating troponin I; CTNT: Cardiac troponin T; CX43: Connexin 43; DAT: Decellularized adipose tissue; ECM: Extracellular matrix; ECs: Endothelial cells; ESCs: Embryonic stem cells; FAP: Fibroblast activation protein; GAG: Glycosaminoglycan; GFP: Green fluorescent protein; GPDH: Glycerol-3-phosphate dehydrogenase; HSP47: Heat shock protein 47; HuNu: Human cell nuclei; IHC: Immunohistochemistry; iPSCs: Induced pluripotent stem cells; LPL: Lipoprotein lipase; µCT: Micro-computed tomography; MEF2c: Myocyte enhancer factor 2C; OCN: Osteocalcin; ON: Osteonectin; OP: Osteopontin; PDGFR-α: Platelet-derived growth factor receptor-alpha; PPARγ: Peroxisome proliferator-activated receptor gamma; RUNX2: Runt-related transcription factor 2; SEM: Scanning electron microscope; SMCs: Smooth muscle cells; SP-B: Surfactant protein B; TEM: Transmission electron microscopy; TGF-β: Transforming growth factor-beta; VEGF: Vascular endothelial growth factor; vWF: von Willebrand factor; WB: Western blotting



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Table 2. Indirect Effects – Paracrine Factor Production* Mechanism

ECM Source

Scaffold format

Stem cell

In vitro

In vivo

Cell instructive effects

source Angiogenesis

Adipose

Human DAT113

Allogeneic rat

√

ASCs

CD31+VEGF+ cells by IHC at 4 wks, as well as vessel density and diameter at 8 & 12 weeks, in seeded scaffolds vs unseeded controls in an immunocompetent Wistar rat model

Enzyme-digested human

Human ASCs

√

DAT hydrogels114

host-derived CD31+ and α-SMA+ cells by IHC in seeded scaffolds vs unseeded controls in a subcutaneous athymic mouse model at 4 wks, with host-derived adipogenesis near new blood vessels

Dermal

Human decellularized

Human GFP+

dermal tissue

ASCs

(Alloderm®)162

√

GFP+vWF+ and GFP+α-SMA+ cells detected by IHC within seeded scaffolds indicating direct contributions to the developing vasculature at 2 and 4 wks in an athymic cutaneous mouse wound model


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Human decellularized

Allogeneic rat

dermal tissue

ASCs

√

√

In vitro: Angiogenic factor secretion (VEGF, HGF, bFGF, TGF-β) in ASC-ECM conditioned media vs

(Alloderm®)112

unseeded ECM conditioned media by ELISA at 2 d In vivo: neo-vascularization and CD31+ cells at 7 d by histology and IHC, and VEGF, HGF, bFGF, and TGF-β by western blots in seeded vs unseeded scaffolds and untreated controls in a cutaneous diabetic rat wound model

Sections of decellularized

Human ASCs

√

murine dermal tissue137

blood vessel density by transillumination analysis in seeded vs unseeded scaffolds and silicon scaffolds at 7 d in a cutaneous athymic mouse wound model; VEGF+ and vWF+ DiI-labeled human ASCs detected by IHC at 14 d indicating possible indirect and direct contribution to angiogenesis

Muscle

Decellularized rat

Allogeneic rat

gastrocnemius muscle115

bmMSCs

√

blood vessel density by histology and IHC in seeded vs unseeded scaffolds at 42 d in a Lewis rat model for volumetric muscle loss

Sheets of decellularized

Human MSCs

√

factor VIII+ (blood clotting protein) and α-SMA+ cells

human myocardium within

by IHC indicating enhanced angiogenesis in seeded vs

a fibrin hydrogel116

unseeded scaffolds at 4 wks in an athymic rat myocardial infarction model



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Bladder

bFGF-loaded scaffold from

Allogeneic rat

enzyme-digested

bmMSCs

√

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micro-vessel density in seeded scaffolds vs unseeded controls, with BrdU+ donor cells present in the

decellularized bovine

microvasculature at 12 wks in a Lewis rat model

pericardium117

(surgically created transmural ventricle defect)

Decellularized rat bladder

Allogeneic rat

matrix118

bmMSCs

√

capillary density in seeded vs unseeded scaffolds and a bmMSC-only group based on histology at 3 months in a Wistar rat bladder model

SIS

CD31+ cells by IHC in MSC-seeded scaffolds vs islet-

√

Decellularized porcine

Co-culture of rat

SIS173

bmMSCs and

only scaffold controls at 14 d; angiogenic factor

rat islet cells

(VEGFA, EGF, HGF) secretion by ELISA at 7 d in MSCseeded scaffolds vs islet-only scaffold controls

Immunomodulation

Adipose

Human DAT113

Allogeneic rat

√

ASCs

fraction of CD163+ M2 macrophages by IHC in seeded scaffolds vs unseeded controls at 12 wks in an immunocompetent Wistar rat model, with no difference in CD68+ cell (macrophage marker) recruitment between groups; IL-10+ cells by IHC in seeded vs unseeded scaffolds at 1 and 4 wks; CD163+ adiponectin+ cells by IHC at 8 & 12 wks in seeded vs unseeded scaffolds

Bladder

Decellularized rat bladder

Allogeneic rat

matrix118

bmMSCs


√

IL-4+, IL-10+ and TGF-β+ cells by IHC in seeded vs unseeded scaffolds at 3 months in a Wistar rat bladder

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reconstruction model SIS

Decellularized porcine

Co-culture of rat

SIS173

bmMSCs and

√

TNF-α secretion by ELISA in MSC-seeded scaffolds vs islet-only scaffold controls at 7 d

rat islet cells

√

IHC showed CD3+ cells (T cell marker) with no effect

Disks made of

Allogeneic

decellularized porcine

porcine

on CD79a+ cell (B cell marker) infiltration in seeded vs

SIS123

bmMSCs

unseeded scaffolds at 2 wks in a porcine epicardial patch model

Spinal cord

Decellularized rat spinal

Allogeneic rat

cord124

bmMSCs

√

CD68+ cells (macrophage marker) and CD5+ cells (T cell marker) in seeded vs unseeded scaffolds at 2 wks in an acute hemisected rat model of spinal cord injury

Pericardium

√

IHC indicated a significantly CD25+ cells (T cell

Decellularized human

Allogeneic

pericardium143

GFP+ porcine

marker) and CD25+ to CD3+ cell ratio in seeded

ASCs

scaffolds vs unseeded scaffolds at 1 month in a porcine myocardial infarction model

*

ASCs: Adipose-derived stem/stromal cells; α-SMA: Alpha-smooth muscle actin; bFGF: Basic fibroblast growth factor; bmMSCs: Bone marrowderived mesenchymal stem/stromal cells; DAT: Decellularized adipose tissue; EGF: Epidermal growth factor; ECM: Extracellular matrix; GFP: Green fluorescent protein; HGF: Hepatocyte growth factor; IL-4: Interleukin 4; IL-10: Interleukin 10; FvSIS: Small intestinal submucosa; TGF-β: Transforming growth factor-beta; TNF-α: Tumor necrosis factor-alpha; VEGF: Vascular endothelial growth factor; vWF: von Willebrand factor.



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For Table of Contents Use Only. Decellularized matrices as cell-instructive scaffolds to guide tissue-specific regeneration. Kevin P. Robb, Arthi Shridhar and Lauren E. Flynn.


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Figure 1. Proposed mechanisms by which compositional, biomechanical, and structural properties of ECMderived scaffolds direct stem cell behaviour and responses. A) Stem cells may interact with the scaffold directly through receptor-ligand binding or indirectly through binding of growth factors and/or cryptic peptides released by proteases that degrade the ECM. These cell-ECM interactions may in turn influence paracrine factor secretion by the stem cells. B) Biomechanical properties of the scaffold influence mechanical forces transmitted via the cytoskeleton to alter cell responses through mechanotransduction pathways. C) Scaffold porosity governs the surface area available for cell-ECM contacts, as well as the degree of cell-cell contact. Surface topography influences host molecule binding. Vascular networks and basement membranes may be conserved after decellularization, and may play a role in cellular organization. 279x215mm (300 x 300 DPI)



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Figure 2. Stem cell-instructive effects mediated by ECM-derived scaffolds. ECM-derived scaffolds can influence stem cell adhesion, migration, viability, proliferation, and differentiation along multiple lineages. Moreover, these scaffolds can influence stem cell secretion of paracrine factors to promote angiogenesis, and modulate the immune response. VEGF: vascular endothelial growth factor; ANG-1: angiopoietin-1; PDGF: platelet derived growth factor; IL-6: interleukin 6; IL-10: interleukin 10; TNF-α: tumor necrosis factor-alpha. 215x279mm (300 x 300 DPI)


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Decellularized Matrices As Cell-Instructive Scaffolds to Guide Tissue

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