Potential Synergistic Effects of Stem Cells and Extracellular Matrix

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Potential Synergistic Effects of Stem Cells and Extracellular Matrix Scaffolds Lewis Gaffney, Emily Ann Wrona, and Donald O. Freytes ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00083 • Publication Date (Web): 18 Jul 2017 Downloaded from http://pubs.acs.org on July 19, 2017

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ACS Biomaterials Science & Engineering

Potential Synergistic Effects of Stem Cells and Extracellular Matrix Scaffolds

Lewis Gaffney1,2, Emily A. Wrona1,2 and Donald O. Freytes Ph.D1,2,*

1) Joint Department of Biomedical Engineering, North Carolina State University/ University of North Carolina-Chapel Hill, Raleigh, NC 2) Comparative Medicine Institute, North Carolina State University, Raleigh, NC

Contact Author:

**Donald O. Freytes, Ph.D. Joint Department of Biomedical Engineering North Carolina State University University of North Carolina-Chapel Hill 4208D Engineering Building III Campus Box 7115 Raleigh, NC 27695 [email protected] [email protected] Office: 919-513-7933 Lewis Gaffney Joint Department of Biomedical Engineering North Carolina State University University of North Carolina-Chapel Hill 4208 Engineering Building III Raleigh, NC 27695 [email protected] Emily A. Wrona Joint Department of Biomedical Engineering North Carolina State University University of North Carolina-Chapel Hill 4208 Engineering Building III Raleigh, NC 27695 [email protected]

ACS Paragon Plus Environment

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ABSTRACT In recent years, extracellular matrix (ECM)-derived biomaterials have been used as scaffolds to help regenerate diseased or damaged tissues. These biomaterials are prepared by decellularization of a tissue of interest by chemical or physical removal of the cellular components. The goal of the decellularization process is to remove cells without disturbing tissue specific composition, growth factor content and, in some cases, the mechanical properties. As decellularization can be achieved without significantly affecting the native architecture of the tissue or organ of interest, it provides a scaffold material with native-like composition and structure. ECM scaffolds promote constructive remodeling through several mechanisms that include chemotactic properties, growth factor release and modulation of the immune response. Constructive remodeling by ECM scaffolds relies, in part, on the recruitment of neighboring or circulating cells to the wound site. However, this is a relatively lengthy process and the cells recruited may not be sufficient for complete tissue repair, suggesting that there might be applications that would benefit from the combination of a cellular component with an ECM biomaterial. Stem cells represent a potential therapeutic cell source with clinical evidence of potential beneficial effects. This review will describe how ECM scaffolds can help localize stem cells to the site of injury while directing stem cell differentiation and modulating the response towards tissue regeneration. As the field of tissue engineering takes its next steps, recellularized ECM scaffolds could become the next class of promising bioengineered constructs. This review covers the advantages of acellular ECM scaffolds, and the potential beneficial effect that seeding stem cells in the scaffold has on tissue regeneration.

Key words: Extracellular Matrix (ECM), Stem Cells, Pluripotent Stem Cells (PSCs), Wound Healing, Macrophage Polarization


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1. INTRODUCTION The mammalian wound healing response is a highly complex process involving orchestrated interactions between cells at the site of injury and the activation and recruitment of circulating cells with the eventual goal of promoting tissue repair while minimizing bleeding and infection. The ultimate goal from a tissue engineering or regenerative medicine perspective is to restore damaged or diseased tissue to a healthy, functional structure. Functional restoration can be achieved via two interconnected mechanisms: tissue repair and tissue regeneration. Adult tissue regeneration does not occur in some human tissue, but does occur more regularly in other species and during fetal wound healing1. Regeneration can be defined as the repair of a tissue to nearly identical function and structure of uninjured tissue through proliferation and activation of resident stem cells or progenitor cells and production of new matrix. Tissue repair is the coordinated mechanisms that restore tissue barriers and homeostasis. However, in the case of most wounds, this process results in unorganized matrix production and a higher density of cells, mostly fibroblasts. If the integrity of the extracellular matrix (ECM) is compromised, which occurs in wounds with large defects, cells produce unorganized matrix to close the wound quickly, resulting in the formation of scar tissue and a significant mismatch in morphology and appearance between the native tissue and the repaired tissue. In cases in which there is a pathological alteration of the wound healing response, there could be chronic deficiencies and inadequate healing that could lead to a poor quality of life. Around 6-7 million patients suffer from chronic wounds in the US with healthcare costs of managing such conditions totaling $25 billion2. Chronic or difficult to heal wounds have limited healing capacity that may be due to changes in the healthy cellular response that often leads to incomplete repair of the tissue and loss of function. Regardless of the type of wound, interventions through tissue engineering strategies aim to harness the inherent mammalian healing process to promote constructive tissue remodeling by providing one or a combination of: mechanical


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support, bioactive signals, and cellular components1. The ultimate goal is the restoration of the native composition and function through the enhancement of the local wound healing process3. In recent years, there has been exciting research focused on the regenerative properties of naturally derived biomaterials obtained from the ECM of tissues. We define ECM scaffolds in this review as biomaterials prepared via the decellularization of target tissues using a combination of detergents and solutions that remove the cellular components while maintaining as much of the native structure of the ECM as possible4-7. ECM scaffolds are also characterized by the retention of similar mechanical properties to the tissue of origin and the retention of bioactive components (e.g. growth factors, peptides, etc.) capable of tissue regeneration via the recruitment and/or activation of surrounding cells8. Retention of mechanical properties and bioactive components after decellularization allow these materials to be tissue specific, which could have effects on tissue specific cell proliferation and differentiation9. One of the most attractive properties of ECM scaffolds is their ability to degrade during the host tissue response, shifting the normal mammalian wound healing response towards constructive remodeling and helping to avoid scar tissue formation10 (Figure 1). During the degradation process, ECM scaffolds release bioactive proteins and peptides that help activate and recruit local and circulating cells, beginning with the inherent wound healing process that ultimately leads to the generation of more organized and functional tissue10-12. Recent research has also emphasized the role of macrophage polarization and T-cell modulation as a result of ECM scaffold implantation13-15. For these reasons, ECM scaffolds have been used in a variety of applications5, 16 and have been adopted in clinical settings, resulting in an increasing number of medical devices composed of decellularized ECMs (reviewed elsewhere)17-19. However, there are situations in which there is incomplete remodeling when using only an acellular ECM scaffold. For example, an early study using a tubular form of an ECM scaffold for esophageal reconstruction benefited from the retention of native muscle layers during implantation to avoid stricture and improve the remodeling response20.

Another example is the use of an ECM


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scaffold to repair ventricular wall defects in which studies show promising results towards remodeling of the infarcted ventricular wall but could not restore significant cardiac muscle21-23. In the case of the ECM scaffolds alone, there appears to be a passive mechanical effect that improves regional contractility of the tissue24. However, once the material is degraded, there is limited mechanical assistance and the muscle component is not completely replenished. This is not surprising given the large number of cells lost (~1 billion) after myocardial infarction25. For cardiac applications, it might be necessary to use therapeutic cells such as cardiac progenitor cells or cardiomyocytes derived from pluripotent stem cell sources to help regain the active cellular mass lost. For example, myocardial ECM scaffolds combined with bone marrow-derived mesenchymal stem cells (MSCs) show increased vascularization and potential healing when compared to the ECM alone26. Clinical studies have also shown the efficacy of c-kit

+

cardiac

progenitor cells and bone marrow-derived mesenchymal stem cells at promoting cardiac healing. In both cases, the amount of scar tissue was reduced, neovascularization increased and ventricular function was improved in the presence of therapeutic cells at the site of myocardial infarction27, 28. These studies show the importance of other passive components that should be considered in the case of ventricular remodeling, such as passive mechanics provided by the scaffold, and cells that may not contribute active forces but modulate the development of new tissue. In addition to supplementing cells that support the active component of functional cardiac tissue, it may also be important to supplement the passive cell population, such as myocardial fibroblasts, which regulate matrix production and aid in tissue regeneration29. This highlights the beneficial effects of a cellular component while harnessing the favorable effects of the ECM material (mechanically and bioactively). At this time, it is unclear how much cellular component is needed to achieve the best possible reparative outcome or stimulate regeneration, but it is most likely dependent on the target tissue being replaced. Therefore, current research should also focus on determining the need and/or benefit


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of using a cellular component while weighing it against the safety and economic hurdles posed by such addition. While the use of multipotent stem cells has been established for years now, the targeted use of stem cells for tissue repair and regeneration have come into focus in the last decade as a potential cell treatment as multiple new multipotent cell types have been discovered and new technologies make the expansion of stem cells more efficient30-32. As previously stated, it has been thought that multipotent stem cells could help replenish some of the cellular components lost due to injury via in situ differentiation of recruited or delivered cells. However, most studies suggest that paracrine effects are the main mode of action by supporting angiogenesis and cell survival when using current therapeutic cells33, 34. Some studies have shown improvements in tissue function via the addition of specialized cells lost during injury that can only be obtained in sufficient numbers using pluripotent cell sources (i.e. myocardium)35-37. In addition, stem cells have been shown to promote a pro-healing macrophage phenotype by helping modulate macrophage activation, further supporting the notion of a regulatory function during wound healing38-42. Although stem cell therapy shows promise, there are still difficulties with its implementation and can benefit from further improvements on their retention and survival after implantation43. ECM scaffolds represent an ideal delivery biomaterial by providing a physiologically relevant substrate to the cells while helping modulate the site of healing, thus providing an ideal environment for stem cells to engraft and support reparative and regenerative processes. ECM scaffolds can also promote and aid in the differentiation of stem cells. In other words, therapies that include both stem cells and ECM components may have several advantages over acellular ECM scaffolds such as the ability to promote a pro-healing environment vital for tissue repair and regeneration5, 16, 44, 45. This review will provide a brief background on the composition and derivation of ECM scaffolds and their potential use as a stem cell delivery vehicle. We summarize the current efforts to harness this class of biomaterial and their inherent bioactivity to further develop new


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tissue engineering strategies in combination with stem cells that can result in superior tissue remodeling for applications that may necessitate a cellular component. 2. Production and Processing of ECM Scaffolds One unique aspect of ECM scaffolds as biomaterials is the fact that they retain important material properties (e.g. mechanical properties, porosity, etc.) and biochemical information (e.g. growth factors, peptides, structural proteins, etc.) from the source tissue4,

46, 47

. In addition to

material properties, ECM scaffolds often retain many bioactive peptides and growth factors that, when released during the normal degradation process, result in the modulation of the host tissue response by affecting key cellular players such as macrophages, T-cells and resident progenitor cells. Therefore, it is important to briefly review the most common components found in these ECM scaffolds and how they relate to the overall properties of the biomaterial.

2.1. Composition of ECM Scaffolds The native ECM is an organized, acellular, three-dimensional network that fills the intercellular space and provides the necessary support for cells in tissues throughout the body48. Specifically, the ECM is composed of a variety of macromolecules in tissue-specific amounts that include collagen, elastin, laminin, soluble growth factors, and glycosaminoglycans49. The main fibrous ECM proteins are collagens, elastins, fibronectins and laminins. Adhesive proteins, such as fibronectin and laminins, play important roles in organization of the matrix and promoting cell attachment. Depending on the tissue of origin, the ECM scaffold may also retain the basement membrane50, 51 which provides a supportive layer for cells and aids in cell growth, migration, and differentiation52. Collagen is the main structural protein component of the ECM and is mostly secreted by fibroblasts49. Collagen fibrils bundle together to form fibers that provide most of the mechanical strength within the tissue. There are at least 29 types of collagens 53 with collagen type IV being the most abundant in the basement membrane and collagen type I the most abundant in the


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interstitial matrix. Tissues such as tendons, ligaments, bone, skin, and cartilage consist largely of collagen. Due to collagen’s uniaxial mechanical properties, the orientation of collagen is extremely important in providing the overall and tissue-specific mechanical properties of ECM scaffolds54, 55. Other important structural components of the ECM include laminins, elastins, and glycosaminogylans.

Laminins are large glycoproteins found within the ECM and basement

membrane of ECM scaffolds51, 56, 57. Laminin is important for cellular adhesion and is often a desired component to be retained in the decellularized ECM. Elastin is another structural component that can improve elasticity of tissues. Elastin is especially abundant in skin and large elastic blood vessels, such as the aorta, and is key to the function of other mechanically active tissues such as: the lungs, the urinary bladder, and elastic cartilage. Glycosaminoglycans (GAGs) are long-chain polysaccharide based polymers and occur naturally in the ECM and connective tissue. GAGs swell in aqueous environments, providing shock absorption in reaction to compressive forces and act as lubricant in various soft tissues. GAGs also promote growth factor retention and modulate critical cell processes.58 Other components present in ECM that affect wound healing and tissue regeneration include: Hyaluronic Acid/Hyaluronan, Chondroitin Sulfate, Keratin Sulfate, Heparin Sulfate, and proteoglycans. The composition and role of all these components have been reviewed elsewhere5, 59, 60. However, given the diversity of ECM constituents among different tissues and their individual contribution to the inherent properties found within ECM scaffolds, it is important to understand the composition of each constituent and to determine how to maximize their retention during the derivation of ECM scaffolds to preserve tissue-specific signals. Another critical factor in the effectiveness of ECM-derived materials is the age of the animal from which the tissue for decellularization is obtained61-64. In vitro studies by Williams et al concluded that cardiac ECM derived from fetal rats had higher proliferation of neonatal cardiomyocytes when compared to ECM derived from neonatal and adult rats61. The possible


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mechanism for this finding was described by the same group in a later study that examined the effect of partially solubilizing adult ECM. The partially solubilized ECM contained higher amounts of soluble matrix proteins, similar to fetal ECM, and led to the same increase in proliferation of neonatal cardiomyocytes62. Sicari et al also found that ECM materials derived from younger pigs (3 and 12 weeks old) showed a more constructive tissue response when compared to ECM derived from older pigs (26 and 52 weeks old), which was characterized by a lower collagen content after 180 days and lower M1/M2 macrophage ratio63. Tottey et al compared small intestine submucosa derived ECM (SIS-ECM) from different age source animals, and examined mechanical, compositional and structural characteristics of the derived scaffolds. This study found several differences, namely that stiffness of the ECM material was highest when the ECM was derived from 12 and 26 week old pigs. In addition, all groups showed chemotactic properties towards perivascular stem cells, with the highest migration in the scaffolds derived from 12 week old pigs. More proliferation was observed in the ECM of 52 week old pigs, and growth factor content was lower in scaffolds from 3 week old pigs, when compared to all of the other scaffold groups64. These studies highlight the importance of age and ECM composition on bioactivity and structure and re-enforces the need for further investigation to determine the optimal ECM source. While previous studies have shown that there may be differences in the mechanical properties of ECM based on the age of the donor animal64, these changes may not be as significant as other effects such as chemotactic properties and bioactivity, which are more inherent in ECM materials derived from younger sources.61, 62 The source will also be dictated by availability and cost which would make market weight animals more attractive as a tissue source. Furthermore, as mechanical and bioactive properties are direct functions of the decellularization process, the optimization of ECM material derivation should continue to be a topic of research. Ultimately, the most cost effective scaffolding material will most likely succeed in the clinical setting.


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Growth and chemotactic factors are important parts of the signaling mechanisms present during the wound healing response. An inherent advantage of using ECM scaffolds is the presence of growth factors and chemotactic agents following decellularization has been shown to modulate the healing response by avoiding scar tissue formation. Growth factors control several processes, such as monocyte recruitment, macrophage polarization, cell differentiation and matrix production. In tissue-specific ECM scaffolds, these growth factors can be harnessed to direct local cell recruitment and differentiation, ultimately improving the regenerative response. The retention of growth factors is highly dependent on the processing and decellularization protocol used65 and may be correlated to the type of tissue that is being decellularized. In addition, degradation products of ECM have been found to affect progenitor cells but not differentiated cells. Reing et al used physiologically relevant enzymes pepsin and papain to degrade a decellularized urinary bladder matrix (UBM) to determine the chemotactic effects on blastema cells, human aortic endothelial cells and human microvascular endothelial cells10. The blastema cells had a significantly stronger chemotactic response over a control with no degraded ECM. Depending on the intended application, it is crucial to assess whether these factors are necessary and have remained in a sufficiently active conformation after decellularization and further processing.

2.2. ECM Scaffold Preparation Decellularization is the removal of cellular content from a tissue, that otherwise may cause an immune response when implanted in vivo such as cellular wall debris and DNA content46,

66, 67

. Keane et al demonstrated the requisite of near complete cellular removal by

decellularizing SIS-ECM at varying degrees and found that decellularized ECM with lower DNA content was better for promoting a pro-healing response rather than an inflammatory response when compared to materials with higher DNA content68. The decellularization process is characterized by a series of steps that may include multiple water or buffer washes, sonication,


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electroporation, freeze/thaw cycles, enzymatic digestion, acid/base exposure or a combination of these4, 5, 9. A summary of decellularization processes is presented in Figure 2. A successful decellularization protocol will remove most the cellular content without damaging the native architecture of the ECM and can be performed on tissue sheets or on perfused organs9, 69-71. Methods for processing ECM scaffolds have been reviewed elsewhere 5, and include combining with polymers, lyophilized to make sheets, powdered, digested or made into hydrogels, (summarized in Figure 3). Overall, xenogenic ECM scaffolds are optimal due to their availability46 and reduced immunogenicity when properly decellularized46. Given the complexity of each tissue and its unique composition, decellularization protocols are customized and optimized for each type of tissue. For example, lungs can be best decellularized via the ionic detergent

3-((3-Cholamidopropyl)dimethylammonio)-1-propanesulfonate

(CHAPS),

urinary

bladders via peracetic acid, and vocal folds via a combination of enzymatic and ionic detergent treatments72-74. Cytotoxicity and cell proliferation assays such as DNA quantification and MTT assays are commonly used during the optimization of decellularization protocols to test for detrimental residues.

Ultimately, the objective is to determine which protocol adequately removes the

cellular material while maintaining the rest of the ECM intact so that the efficacy of the ECM material is maximized73-76. It has been suggested that sufficient decellularization is achieved when the DNA content found is no more than 200 base pairs and contains less than 50 ng of dsDNA per mg of dried ECM9. Histological staining can be used in combination with DNA quantification to determine the retention and distribution of common ECM components: Hematoxylin and Eosin (for nuclear and protein content), Movat’s Pentachrome (to determine the arrangement of collagen, elastic fibers, mucins, fibrin, and muscle fibers), and Alcian blue (for GAG content). During histological evaluation, decellularized ECM should have no visible nuclei present while showing visible retention of ECM components. Additional collagen


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quantification and GAG colorimetric assays and lipid quantification can further examine the retention of these important ECM components. Recently mass spectrometry and proteomics have been used as additional methods to assess ECM quality and content77, 78, and have been reviewed by other groups79, 80. Recently, Li et al used proteomics to analyze several different ECM derived materials: Rat Tail Collagen Type 1, Growth Factor Reduced Matrigel®, decellularized rat liver, and decellularized human lung. They also analyzed the decellularized rat liver at several points throughout the decellularization process. While the decellularization techniques used were adequate for removal of nucleic content and disruption of cellular membranes, several plasma membrane and intermediate filament proteins remained. While the effects of these proteins are unclear, proteomics may be necessary to fully characterize the cellular environment of ECM materials77. Welham et al. characterized vocal fold mucosa scaffolds with 2D electrophoresis, and mass spectrometry. While the presence of structural and organizational proteins was observed, cellular proteins that are known to be bioactive and possibly antigenic were also present, even though there was no evidence of cellular proteins after histological and immuno-histochemical analysis78. These examples highlight the need for new methodologies to further characterize ECM scaffolds, leading to less variability in the final products and more stringent quality control for clinical translation. Hill et al. used a new methodology for proteomics analysis that was targeted at identifying specific proteins that were present within the ECM scaffold rather than a discovery based proteomic approach. Using ECM specific labeled peptides and chemical digestion to include insoluble proteins, this method improves repeatability and accuracy during the characterization of ECM materials81. If proteomic analysis could be included during the initial characterization alongside bioactivity, it could help researchers place priorities on specific components that are needed for optimal ECM derivation. Furthermore, proteomic analysis coupled with chemotactic and bioactive studies may highlight mechanisms that make one source material more advantageous, which could direct the manufacturing process81.


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Decellularization can be performed at the whole organ level or on dissected pieces of tissues. If the decellularization is performed via perfusion of an organ, the ECM scaffold can be maintained with the native three-dimensional structure of the target organ9, 82-84. Alternatively, the decellularization process can be performed on pre-sliced tissue sections resulting in 2-D sheets that can be sutured onto the site of injury. There are extra steps that can be taken after the decellularization step such as dehydration and terminal sterilization to prepare the ECM scaffold for cell seeding or for implantation85-87. In addition, ECM scaffolds can be processed in a way that produces a hydrogel that can be used to encapsulate cells and self-assemble in situ88. Hydrogels can create a 3D environment during the delivery of cells via minimally invasive methods, possibly improving cellular function and survival. The production of ECM hydrogels largely affects the native ultrastructure of ECM proteins due to the digestion step. While this affects the quaternary and tertiary structures of ECM proteins, which affects the tissue’s mechanical properties and topographical features, matricryptic sites that are exposed during degradation could maintain some of the inherent bioactivity89. For instance, while studying the metabolic activity of kidney stem cells cultured on different forms of kidney papilla ECM (sheets, hydrogels, and solubilized ECM), similar metabolic activity was observed across all three forms of the ECM scaffolds, suggesting that composition of the stem cell niche was sufficient to affect cell metabolism when compared to ECM derived from the medulla and cortex90. These results show that the versatility of ECM scaffold spans from organ level to a highly processed level such as hydrogels. Large decellularized organs have the added advantage that they can be recellularized via perfusion of a cell suspension using the same approach used during the decellularization process making it an ideal closed loop system

70, 91-93

. Two dimensional sheets

or smaller tissues can be surface seeded in traditional culture conditions and used for in vitro testing or implantation

83

. Figure 3 summarizes various methods for processing 2D ECM

materials to provide different functions.


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In summary, decellularized ECM materials have several advantages with respect to wound healing. Due to the removal of cellular content, there is almost no rejection of the material by the body’s immune system. As ECM scaffolds degrade, rather than altering the natural chemical content of the tissue, they have naturally occurring cryptic peptides and growth factors that enhance the overall host tissue response (Figure 1). This bioactivity can also be harnessed by combining with synthetic biomaterials to create bioactive hybrid scaffolds94.

3. ECM scaffolds and Tissue Healing There are a variety of commercial ECMs that are currently available for clinical use. Some of these examples include ECMs derived from the small intestine, urinary bladder (UBM), skin, pericardium and fasciae latae muscle, to name a few5. However, there is a lack of preclinical and clinical studies that address the necessity for the combination of an ECM scaffold with cells, let alone constructs with ECM and stem cells. The first tissue engineered construct with any living cells that was FDA approved for clinical use is Apligraf®, a “living skin equivalent” composed of a fibroblast dermal matrix layer supporting basal, spinous, granular and stratum corneum layers made of keratinocytes

95

. While other living tissue-engineered constructs have

been added to the market since then, the combination of extracellular matrix and stem cellbased therapies is relatively new and there are multiple efforts to harness the constructive remodeling found in ECM scaffolds with the therapeutic potential of stem cells. Obstacles to clinical translation of an ECM-stem cell product that should be considered include the safety and regulatory hurdles that will be encountered during FDA approval.

In addition, there are

important issues regarding safety and efficacy of the cellular component and the current cost of cell culture in a GMP environment that needs to be considered as well96. Although the manufacturing infrastructure is established for both components individually, combining the ECM and stem cell will pose new challenges that will need to be addressed and could influence its market readiness. The time required for the product to get to the market could also be


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affected by the form of ECM chosen (whole organ vs sheets vs hydrogels). While recognizing the manufacturing and regulatory difficulties that pose the use of cells, the combination of ECM scaffolds and stem cells could result in improved regeneration in specific applications.

3.1 ECM Scaffold-Macrophage Interactions Recent studies have highlighted the role of macrophage polarization as a predictor of constructive remodeling and wound healing as a result of ECM scaffold implantation13, 46, 47, 97, 98. These studies suggest that macrophages are responsible, in part, for orchestrating the wound healing response. There is a large amount of heterogeneity in macrophage phenotypes, but they can be generally described by their activating signals and can mostly be sorted into two broad categories: pro-inflammatory and pro-healing3, reviewed elsewhere

46, 98-101

. These activation states are

99, 100, 102-104

. M1-like or pro-inflammatory macrophages make up most of

the initial macrophage population at the wound site and can be activated by interferon-gamma (IFN-γ) and bacterial wall debris

13

. These cells typically release pro-inflammatory signals such

as interlukin-1β (IL-1β) and tumor necrosis factor- α (TNF-α) in humans and nitric oxide synthase (iNOS) in mice

105

. In addition to playing a crucial role during inflammation, M1-like

macrophages recruit other cells to the wound, and play a role during matrix degradation. Over time, the macrophage population changes to a predominantly M2-like or pro-wound healing macrophage phenotype. There are different subtypes of M2-like macrophages that are classified depending on their activation signal99, 106. For example, M(IL-4, IL-13) macrophages (also referred to as M2a) are activated in vitro by interleukin-4 (IL-4) and interleukin-13 (IL-13) while M(IL-10) macrophages (also referred to as M2c) are activated in vitro by interleukin-10 (IL10). M2-like macrophages promote proliferation and differentiation of progenitor cells, ameliorate inflammatory processes, and stimulate matrix production107. It should be noted that the role of macrophages is dependent on the local environment and is described more as a spectrum of activation rather than a binary state108. In wound healing, both populations are


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necessary and the timing of the transition is extremely important. For instance, while M1-like macrophages are necessary at the beginning of host tissue response, it is clear that with prolonged exposure there could be excess ECM degradation with impaired healing 109, 110 It is important to note, however, that there are exceptions to the biphasic appearances of M1-like and M2-like macrophages and that this is a general and highly simplified model

111, 112

.

ECM scaffolds have been reported to shift the macrophage polarization at the site of implantation towards an M2-like phenotype12,

113, 114

(Figure 1). The constructive remodeling

observed after the implantation of ECM scaffolds is in part attributed to this M2-like macrophage shift during pre-clinical studies46, 115. When ECM scaffolds are chemically cross-linked, resulting in diminished degradation and bioactivity when implanted, an M1-like macrophage population predominates, possibly leading to fibrous encapsulation and scar tissue formation116. The prohealing properties of minimally processed ECM scaffolds make them very attractive biomaterials and a focus of research using macrophages to screen ECM scaffold materials117. Synthetic materials, including biocompatible materials, often prolong the inflammatory process. This may be due to M1-like macrophages attempting to break down the material, which doesn’t occur as naturally as compared to ECM scaffold, and in some cases, results in encapsulation of the material

46

. This prolonged inflammatory response has an inhibitory effect

on tissue healing and regeneration. ECM scaffolds on the other hand, typically break down via secreted enzymes facilitating the transition from M1-like to M2-like macrophages and therefore improving the wound healing response. Sicari et al showed that solubilized SIS-ECM combined with perivascular stem cells (PVSCs) affected the chemotactic response of macrophages115. The study found that the macrophages cultured within the ECM hydrogel were biased towards the M2-like phenotype and the ECM scaffold could promote the migration and myogenesis of the PVSCs. Another study demonstrated that polypropylene mesh coated with porcine dermis and UBM hydrogels significantly reduced the M1 response in a rodent model when compared to polypropylene alone98. Dearth et al recently described a possible explanation of this dominant


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M2-like response with ECM when exploring the effect of Aspirin, a common cyclooxygenase 1 and 2 (COX1/2) inhibitor118. The application of the COX 2 inhibitor to UBM hydrogel-treated in a rat model of skeletal muscle injury caused an increase in M1-like macrophage presence. This suggests that the COX2 pathway promotes a M2-like response when using an ECM scaffold. Interestingly, Dziki et al examined the response of bone marrow-derived macrophages towards ECM derived from various sources119 and found that macrophages exposed to dermal ECM were dominated by a M1-like phenotype, while brain, bladder, colon, small intestine and esophagus ECM had a M2-like phenotype. These studies further highlight the diversity of ECM compositions of different organs and tissues and the need to select appropriate scaffolds depending on the needs of the site of damaged tissue. Other immune cells are also affected by the components and/or degradation products of ECM. For instance, Bayrak et. al. investigated the gene expression of T-cells in response to various biomaterials. Native porcine tissue had the highest increase in cytotoxic T-cell proliferation and activation, while porcine ECMs with elastin still present had a significantly lower T-cell activation14. When comparing xenogenic tissue implants in mice with SIS implants, Thelper cells had more of a regenerative phenotype (Th-2) than mice who received other implants120. The modulation of T-helper cells by biomaterials has been reviewed elsewhere121. In models of volumetric muscle loss, ECM biomaterials skewed the ratio of CD4+:CD8+ towards the CD4+ regenerative phenotype when compared to control groups. In addition to skewing of the T-cell population, the ECM biomaterial also promoted release of IL-4 which is implicated with Th-2 and M2 macrophage responses. Sadtler et al concluded that the ECM promoted the T- Helper cell regenerative phenotype which further plays a role in the immune microenvironment, supporting the M2-like regenerative macrophage phenotype.15 This suggests that multiple mechanisms exist by which ECM materials promote a regenerative immune microenvironment.


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3.2. Stem Cell-Macrophage Interactions An understanding of the inflammatory environment and how therapeutic cells interact with such environment is critical for a successful implantation of tissue-engineered construct comprised of an ECM scaffold and stem cells122. One potential therapeutic cell source are mesenchymal stromal cells or MSCs.

These cells can be harvested in a clinical setting,

commonly from the bone marrow or adipose tissue, and have been shown to have beneficial effects in clinical trials123,

124

. The immunomodulatory properties of MSCs has been reviewed

elsewhere123. However, in vivo and in vitro experiments using cells of the immune system and other repair cells remain necessary to gain a complete understanding of the potential interactions between a tissue-engineered construct and the inflammatory environment after injury. As previously stated, adult stem cells have been shown to modulate macrophage polarization by promoting more pro-healing phenotypes125-127. Recruitment of macrophage cells is one mechanism by which MSCs promote tissue regeneration. In vitro and in vivo studies showed that MSC pre-conditioned media had a higher chemotactic effect on macrophage cells, resulting in a larger population at the wound site, as well as promoting angiogenesis128. In response to the wound environment, MSCs can secrete cytokines that promote either pro-inflammatory or pro-healing macrophages.

For example,

when exposed to low concentrations of interleukin-6 (IL-6), MSCs produce interferon-gamma (IFN-γ) and interleukin-1 (IL-1) which promotes classically activated macrophages123. However, when MSCs are exposed to an environment with a high concentration of inflammatory cytokines, there is an increase in expression of pro-healing factors. Nemeth et. al. observed that MSCs promote a pro-healing response in vivo and in vitro through E2 prosteaglandin secretion which binds to receptors on macrophages to promote production of IL-10, cyclooxygenase 2, and TNF-α in a sepsis model126. MSCs are also implicated in polarization of macrophages to M2-like phenotypes125. Another study showed that macrophage associated cytokines (IFN-γ, TNFα, IL-1β and IL-6 for M1-like macrophages and IL10, TGFβ, VEGF for M2-like


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macrophages) had different effect on the survival and viability of MSCs in vitro

41

. M2-like

macrophages associated cytokines were able to support MSC growth while M1-like macrophages and cytokines inhibited growth. When delivered to the site of myocardial infarct, MSCs promote an M2-like phenotype and the secretion of pro-healing cytokines40. In summary, while ECM scaffolds have immune modulating effects, stem cells also have similar effects on their own, re-enforcing the premise for stem cell seeded scaffolds. Another potential source for therapeutic cells are induced pluripotent stem cells (iPSCs). iPSCs are derived from somatic cells that have been reprogrammed to a pluripotent stem cell state via the expression of key transcription factors and have been reviewed elsewhere129. iPSCs could be derived from each patient making them an autologous therapy and thus avoiding immune-rejection (although this is still a subject of debate)130. Alternatively, iPSCs or human embryonic stem cells (hESCs) could be manufactured and stored in haplobanks and closely matched to the recipient131.

Regardless, the interactions between these potential

therapeutic cells and the host tissue is relatively unknown and a subject that should be explored. Recently, Pallotta et al investigated the effect of polarized human blood-derived macrophages on hESC-derived cardiomyocytes (hESC-CMs)132. The study showed that in a 3D invasion assay, hESC-CM conditioned media recruited pro-inflammatory M1-like macrophages due, in part, to hESC-CM-derived bone morphologic protein 4 (BMP4). Furthermore, the proinflammatory M1-like macrophages affected a subset of hESC-CM progenitor-like cells’ proliferation and maturation due to, in part, to macrophage-derived BMP proteins. These results highlight the potential to harness stem cells and inflammation to improve the host tissue response via macrophage modulation and tissue regeneration via stem cell differentiation. This then can be coupled with the beneficial effects of extracellular matrix scaffolds to create superior tissue engineered constructs.

3.3 ECM Scaffolds as a Delivery Vehicle for Stem Cells


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Although ECM scaffolds have shown positive effects on the host tissue response when used as an implantable biomaterial on its own, there are applications that may result in superior tissue repair and regeneration if combined with a stem cell or stem cell derived-cell. Extracellular matrix components can have numerous effects on the differentiation of stem cells (see Table 1 for examples). Conversely, stem cells can also influence matrix production at the site of wound healing, making them both equally important for the goal of modulating the host tissue response. In chronic and difficult to heal wounds, several factors inhibit normal tissue regeneration, including: increased activity of matrix metallo-proteinases (MMPs) that break down the ECM, inhibition of ECM production, and modulation of activity in cells such as fibroblasts, keratinocytes, and leukocytes16. In addition, the components of the ECM are crucial in maintaining the stem cell niche in vivo, being an important target in directing stem cell recruitment3, 10. Integrin binding is highly specific to cell types and components of ECM, varies between tissue and helps to direct cell migration and differentiation. Minimally processed ECM materials can maintain these integrin binding properties and thus preserve their biological effects on the cells at the wound site58. If ECM scaffolds are used in combination with stem cells, there are several advantages over acellular materials such as the initial delivery of a cellular component that can help modulate the local wound healing environment without the need to wait for the recruitment of cells from neighboring tissues or from circulation. If designed properly, there can be a synergistic effect between the bioactivity inherent within the ECM scaffold and the modulating effects of the delivered stem cell. The choice of stem cell to use is still up for debate and will likely be dictated by safety and applicability concerns. Currently, MSCs derived from various sources and progenitor cells are popular choices since, for the most part, they can be harvested under clinical conditions and can often be used in an autologous fashion. For example, MSCs have shown modest improvements in some clinical studies mostly related to positive effects on the wound and host tissue response123. However, these adult stem cells fail to replenish the


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musculature lost after a myocardial infarction. Pluripotent stem cells on the other hand have been shown to efficiently produce significant numbers of cells such as cardiomyocytes but are met with safety and feasibility limitations. Delivery of pluripotent stem cell-derived cells may benefit from the combination with extracellular matrix scaffold as a vehicle to improve their survival and maintain their differentiation state after implantation. Ultimately, the most efficient and safest cell should be used. ECM can also be used as a cell delivery system to provide structural support and to maintain the cells at the site of injection. Choi et al injected fluorescently labeled rabbit bone marrow-derived MSCs combined with SIS in a digested form as a delivery medium into rabbits with scars on the right vocal fold

133

. After 8 weeks, it was found that the MSCs injected within

the decellularized ECM showed higher engraftment and the vocal folds that received the MSC/ECM gels resulted in improved vibratory motions when compared to injections of MSCs or ECM alone. In this example, the MSCs were delivered to the injury site with a gelled ECM and were found to stay at the site and exhibit regenerative effects of the damaged vocal fold tissue. There have been several interesting studies where ECM scaffolds have been seeded with stem cells or progenitor cells. As discussed earlier, the composition and structure of ECMs varies from tissue to tissue, so studies have focused on these differences and on any impact on cell behavior. Heart ECM hydrogels, for example, have been shown to support the differentiation of hESC-CMs. Duan et al decellullarized porcine heart tissue and transformed decellularized myocardial slices into a hydrogel composed of digested myocardium and collagen type I

134

. hESCs were encapsulated within these hydrogels as embryoid bodies and

differentiated with or without supplemental growth factors. It was found that the cardiac ECM hydrogel without growth factors was sufficient to direct the differentiation of hESC-derived cardiac progenitor cells into hESC-CMs. This concept was expanded with the use of decellularized fetal and adult porcine heart ECM to aid in the maturation of iPSC-derived cardiomyocytes. ECM derived from cardiac tissue led to a higher expression in calcium pathway


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genes, and promoted the proliferation of the cardiomyocytes derived from pluripotent stem cells135. Current methods to derive cardiomyocytes from iPSC tend to make CMs that are characteristically more fetal than adult. However, these studies are a promising step towards using ECM scaffolds to help maintain the differentiation of cells during the tissue healing process. In vitro applications for skeletal tissue have also shown benefits from using ECM materials for differentiation of progenitor and stem cells. Alom et al studied demineralized bovine tibias using 1:1 chloroform and methanol and subsequently decellularized via a 24 hour treatment of 0.05% Trypsin, 0.02% ethylenediaminetetraacetic acid (EDTA) and 1% pencillin/streptomycin in PBS136. The bone ECM (bECM) was digested, gelled and seeded with C2C12 mouse myoblasts. After 7 days of in vitro culture, there was a significant difference in the expression of osteogenic markers such as osteopontin and osteocalcin. The results suggested that bECM hydrogels have osteogenic-inducing properties. Ning et al decellularized canine Achilles tendons using repetitive freeze-thaw cycles and enzymatic digestions with RNase and DNase137. The study used two types of cells to test the ECM: rat bone marrow-derived stem cells (brMSCs) and rat tendon-derived stem cells (TDSCs). Both cell types were seeded onto lyophilized slices of tendon for 3, 7 and 14 days and assessed for cell viability, proliferation and possible differentiation. BMSCs seeded on tendons demonstrated an increase in the gene expression of tendon-specific genes such as scleraxis (SCX), tenomodulin (TNMD) and thrombospondin-4 TBS4 when compared to BMSCs seeded on tissue culture plastic. TDSCs seeded on tendons showed an increase in gene expression of TNMD, TBS4 and collagen type 1 (COL1) when compared to TDSCs seeded on tissue culture plastic further supporting the tissue specific effect of ECM scaffolds. As an additional example, Yuan et al used tissue specific hydrogels to inject therapeutic hMSCs into rat models of meniscus injury. Bovine meniscus tissue was minced, lyophilized and washed in SDS for decellularization, then digested to form a gel. The gels supported hMSC integration and the formation of fibrocartilaginous tissue138.


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Decellularized tendon was altered using ultrasound to create a random-aligned-random structure to mimic a gradient found in native, healthy ligament. The biomimetic tendon material promoted the healing of the bone ligament junction which was characterized by higher expression of osteogenic and chondrogenic markers, and higher osteoinductivity with rabbit bone marrow stromal cells compared to the control139. Repair of skeletal tissue is a broad field where ECM scaffolds have great function due to these tissues consisting mostly of ECM in their mature form. Other organs have shown similar trends, such as liver and kidney. Studies by O’Neill et al90 and Batchelder et al140 highlight the potential influence of ECM scaffold on the phenotype of stem cells in renal tissue engineering. Since the kidney is comprised of three regions (cortex, medulla and papilla), O’Neill et al studied the effects of porcine kidney ECM derived from these three regions on kidney stem cell growth and behavior. This group showed that the ECM derived from these three regions affected cell growth and metabolic activity of mouse kidney stem cells (KSCs) and mouse MSCs. After decellularizing all three regions of the kidney, KSCs and MSCs were seeded on 2D sheets or on hydrogels. It was found that the papilla ECM, the location where KSCs normally reside, had a distinct effect on the metabolism and growth of KSCs but not on MSCs. Batchelder et al obtained monkey kidneys and used perfused solutions of heparin, PBS and 1% sodium dodecyl sulfate (SDS) for several days to the remove cellular content

140

. The sections of decellularized kidneys, as well as whole organs, were then

reseeded using hESCs in endothelial growth media and compared hESCs grown on embryoid bodies. Early renal developmental markers such as Wilms tumor 1 (WT1) and protein box 2 (PAX2) were expressed significantly higher on hESCs cultured on the renal ECM then on embryoid bodies, suggesting that renal ECM encourages hESCs to express early renal lineage markers. Additional studies have used iPSC-derived cells have also been coupled with ECM scaffolds. Wang et al

141

examined the effect of liver-derived ECM on the maturation of iPSC-

derived hepatocytes. Hepatocytes cultured in ECM were shown to have higher mRNA


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expression of P450 and increased metabolic enzyme activity when compared to cells cultured on a 3D printed poly-L-lactic acid (PLLA) scaffolds infused with collagen I and cells cultured on collagen I alone. Both scaffolds were biocompatible and could promote bile canaliculi-like structures. These studies highlight how ECM structure can influence cell growth and behavior and the importance of selection the appropriate ECM depending on the applications. There are a few, but valuable studies that use iPSC-derived cells and ECM in an in vivo setting. Porcine iPSC-derived hepatocytes have been supplemented with decellularized porcine liver ECM containing liver-derived growth factors and transplanted into rats142. The cells attached well to the grafts and expressed hepatocyte markers such as albumin (ALB) and αfetoprotein (AFP). iPSC-derived lung epithelial progenitor cells have also been recently investigated in vivo. Decellularized whole human and rat lungs were seeded with lung epithelial progenitor cells generated from hiPSCs143. Cells cultured on decellularized lungs proliferated and expressed NK2 homeobox 1 (NKX2.1) when cultured in a bioreactor. Left lung transplants were performed and showed red blood cell perfusion throughout the alveolar capillary network with some hemorrhages. The same group used iPSC-derived endothelial cells and pericytes and efficiently repopulated the vasculature in decellularized lung ECM144. While there are not many studies that have explored use of iPSCs and ECM delivery vehicles, it remains a promising direction for the field. These studies will be more feasible as technologies advance and the culture of iPSCs becomes more efficient. Another technique for cell delivery with ECM scaffolds is 3D printing. Advances in 3D printing technology have allowed for the development of bio-inks that contain materials that form hydrogels in the right environment, and cells specific to the intended application. The use of ECM-derived materials in bio-inks has the inherent advantages of using tissue specific ECM and the ability to form 3D structures145. For instance, bioprinting of cardiac progenitor cells and MSCs in a bio-ink made from decellularized porcine heart supported improved cardiac function and tissue repair when compared to bio-ink made from collagen. Moreover, the ability to


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engineer channels that promote vascularization in the patch also had beneficial effects for the delivered cells146. Structural control of the printed construct has also been used in the printing of skeletal muscle tissue using a bio-ink derived from decellularized muscle tissue147. Future directions and recent developments with 3D printing and ECM bio-inks has been reviewed elsewhere, and offers an promising area of research in the area of delivery of therapeutic cells145. Current challenges of using ECM scaffolds in combination with seeded stem cells include perfusion, especially with larger and whole organ scaffolds, and adjusting material stiffness to direct stem cell lineage. Whole organ perfusion and its challenges have been reviewed elsewhere9. For example, human hearts have been recently decellularized via perfusion and recellularized using iPSC-derived cardiomyocytes148. Material stiffness and composition is known to play a role in stem cell differentiation149 and how it applies to decellularized tissue remains to be investigated. In addition to the studies mentioned above, which focus on the use of ECM materials derived from decellularized tissue to deliver stem cells or progenitor cells, there are studies that use ECM to deliver and support primary cell lines such as cardiomyocytes150 and hepatocytes151, 152. In addition, studies have also been conducted that focus on the differentiation and the use of pluripotent cells with materials that mimic ECM, like fibrin cardiac patches153,

154

, hyaluronic acid materials150,

151, 155

and nanofiber scaffolds that

incorporate gradients156 . Future research should focus on optimizing these delivery vehicles and should be designed to drive tissue healing at the target site using the optimal combination of scaffolding material, therapeutic cells (if needed) and assembly paradigm.

4.0 Conclusions and Future Directions The advances in tissue engineering constructs using ECM scaffolds and stem cells remains promising, even at the face of the difficulties that hinder the progress of a combined ECM/cell-based regenerative approach to wound repair. Adult stem cells remain a viable clinical


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choice as therapeutic cells. With regards to pluripotent stem cells, a major hurdle has been overcome in recent years by the discovery of iPSCs, which avoids ethical concerns raised with the use of hESCs and cells derived from somatic cell nuclear transfer. However, our limited understanding of their differentiation potential and cellular abnormalities as a result of the reprogramming step and prolonged culture times still represent a challenge. Recent publications have highlighted the potential to generate these cells under GMP conditions, making them clinically viable96. However, long term safety studies are still needed to understand the risks associated with pluripotent stem cells and to determine quality control strategies that would ensure the removal of non-differentiated cells that could form teratomas and ectopic tissues. Nevertheless, the recent use of pluripotent stem cells during clinical trials of macular degeneration and other diseases show promising results on their safety (reviewed elsewhere157). Other challenges faced by the clinical use of stem cells include immature cell differentiation, poor cell engraftment, incorporation of the construct with native tissues, and vascularization of the construct before and after implantation. For example, pluripotent stem cells can be differentiated very efficiently to cardiomyocytes and delivered to the infarct. However, these cells are still immature when compared to adult cardiomyocytes and the cells do not seem to mature as expected when injected into the heart25. Interestingly, beta cells derived from pluripotent stem cell sources were previously only matured in vivo can now be derived in vitro157. The limitation of each cell type and how ECM can be harnessed, will need to be determined to address these limitations. In addition, it is imperative to test biocompatibility of the scaffold material and it is necessary for any tissue-engineered construct to be tested in an in vivo environment to ensure no negative host tissue response. The inclusion of ECM with stem cells is a promising approach to tissue repair and regeneration. Progress has been made in regard to processing technologies and the methods are becoming more efficient. As a rapidly evolving and defining field, tissue engineering and the


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constructs created with tissue engineering fundamentals, will advance the field of regenerative medicine through the enhancement of the local wound healing response.

5.0 Acknowledgements We thank our current funding sources: UNC/NCSU Joint Department of Biomedical Engineering, the Comparative Medicine Institute and NC TraCS 550KR141616. We also acknowledge our undergraduate research assistants, Andrew Baldwin and Brady Trevisan, for their help and contributions during the preparation of this review.


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FIGURE CAPTIONS

A. Chemotaxis

B. Growth Factors

C. Cryptic Peptides Interactions with Cells

VEGF TGF-ß

Th-1 Inflammatory Lympohocytes

Th-2 Pro-regenerative Lympohocytes

D. Lymphocyte Response

M1-Like, Pro-inflammatory M2-Like, Pro-healing Macrophages Macrophages

E. Macrophage Response

Figure 1: Diagrammatic representation of how ECM Scaffolds promote tissue regeneration through several mechanisms. A) ECM scaffolds have chemotactic effect on progenitor cells. B) Growth Factors such as Transforming Growth Factor- ß and Vascular Endothelial Growth Factor are released from ECM scaffolds during degradation. These growth factors promote matrix production and angiogenesis, respectively. C) Cryptic, bioactive peptides of proteoglycans contained in the ECM scaffold are released as the scaffold degrades and can promote tissue regeneration processes. D) Lymphocytes are shifted to T-Helper 2 Lympohocytes which have more of a regenerative phenotype. E) ECM scaffolds promote a transition of macrophages from pro-inflammatory phenotype to the pro-healing phenotype. The pro-healing phenotype promotes cell differentiation and matrix production. Art adapted from Servier Medical Arts.


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- 80°C +2 5 °C

+2 5 °C

- 80°C

Electroporation

Freeze/Thaw Cycles

Decellularization

Sterilization and washes

Enzymatic treatment

Sonication

Hypotonic or hypertonic solution Ionic and nonionic detergents

Figure 2: The decellularization process is characterized by a series of steps that may include multiple water or buffer washes, sonication, electroporation, freeze/thaw cycles, enzymatic digestion, acid/base exposure. These methods are usually used in combination for an effective decellularization.


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Hydrated Scaffold Cell seeding, structural support, implantation

Lyophilized Scaffold Cell Seeding, implantation

Hydrogel Cell seeding, encapsulation, implantation

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Lyophilized Powder Media supplementation

Digested ECM Vehicle for injection

Figure 3: ECM processing methods allow for these materials to be used in several applications. Hydrated scaffolds are the first product after decellularization of a 2D sheet and are used for cell seeding and implantation. After lyophilization, scaffolds have a much longer shelf-life and can be used similarly as hydrated scaffolds. Grinding the lyophilized sheet yields an ECM powder that can be used for media supplementation. Powder can also be digested and used for injection or in a mixture that gels at physiologic temperature creating a 3D microenvironment for cell seeding.


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Table 1: Examples of studies that used ECM scaffolds with stem cells and showed differentiation specific to the tissue.


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Potential Synergistic Effects of Stem Cells and Extracellular Matrix Scaffolds

Lewis Gaffney, Emily A. Wrona and Donald O. Freytes Ph.D

FOR TOC USE ONLY:

Synergistic Interactions

Combination of stem cells and extracellular matrix scaffold

GOAL: Enhanced Tissue Repair - Improved cell engraftment - Constructive remodeling

Host tissue response

- Direction of stem cell differentiation

Macrophages T Lymphocytes


42

A. Chemotaxis

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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

B. Growth Factors


C. Cryptic Peptides Interactions with Cells

VEGF TGF-ß

Th-1 Inflammatory Lympohocytes

Th-2 Pro-regenerative Lympohocytes

D. Lymphocyte Response

M1-Like, Pro-inflammatory M2-Like, Pro-healing Macrophages Macrophages

E. Macrophage Response ACS Paragon Plus Environment


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

-80°C

+25°C

Page 44 of 47

+25°C

-80°C

Electroporation

Freeze/Thaw Cycles Decellularization

Sterilization and washes

Enzymatic treatment

Sonication

Hypotonic or hypertonic solution Ionic and nonionic detergents


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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


Hydrated Scaffold Cell seeding, structural support, implantation

Lyophilized Scaffold Cell Seeding, implantation

Lyophilized Powder Media supplementation

Hydrogel Digested ECM Cell seeding, encapsulation, Vehicle for injection ACS Paragon Plus Environment implantation

Tissue 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 49 50 51 52 53 54 55 56 57 58 59 60

ECM Scaffold

Stem Cell Source

Outcome

REF

Duan et al 2011


Page 46 of 47

Heart

Decellularized Porcine Heart Tissue was sliced and digested to make a hydrogel

Human Embryonic Stem Cells/Induced pluripotent stem cells

Differentiated with and without supplements in media

Heart

Decellularized fetal and adulte Porcine heart tissue

Induced Pluripotent Stem cells

Promoted the expression Fong et al of Calcium signalling and 2016 promoted proliferation of iPSC derived Cardiomyocytes

Heart

Bio-ink derived from decellularized cardiac tissue

Cardiac Progenitor cells and MSCs

Compared to Collagen, bio-ink derived from cardiac tissue supported more functional cardiac regeneration

Jang et al 2017

Liver

Liver ECM

Induced Pluripotent Stem cells

Higher expression of P450 mRNA, increased metabolic enzyme activity

Wang et al 2016

Liver

Porcine liver ECM containing liver derived growth factors

Induced Pluripotent Stem Cells

Higher Expression of Hepatocyte Markers

Park et al. 2016

Kidney

Kidney ECM manufactured into Mouse kidney stem 2D sheets or hydrogels cells, mouse Mesenchymal Stem Cells

Stem cells had different O’neill et al. expression depending on 2013 which region was used for the ECM

Kidney

Decellularized monkey kidneys Human Embryonic using perfusion of heparin, PBS Stem Cells and 1%SDS

Renal Development markers were significantly higher in renal ECM than cells cultured in embryoid bodies

Vocal Fold

Small Intestine Submucosa in a Mesenchymal Stem digested form Cells

There was higher Choi et al. engraftment of cells, and 2014 improved vibratory motions compared to groups that had MSCs or ECM alone

Lung

Decellularized whole rat and human lung

Lung epithelial cells Higher proliferation and generated from expression of lung human induced markers Nkx2.1 Pluripotent Stem Cells

Gilpin, 2014

Lung

Decellularized whole rat and human lung

iPSC derived endothelial cells and pericytes

Resulted in complete vascularization in rat model and in scale up model for a human lung

Ren et al, 2015

Bone

Demineralized Bovine Tibia, digested and gelled

C2C12 Mouse Myoblasts

After 7 Days in culture, muscle progenitor cells expressed osteogenic markers

Alom et al, 2017

Tendon

Decellularized Canine Achilles tendons

Rat bone marrow derived stem cells, rat tendon derived stem cells

BMSCs expressed tendon Ning et al specific genes, TDSCs had 2015 higher expression of tendon specific markers compared to cells cultured on tissue culture plastic.

Tendon

Decellularized tendon, modified with ultrasound to form a random-alignedrandom structure

Rabbit Bone Marrow stromal cells

Modified ECM enhanced Osteogenic and Chonrogenic gene expression, resulting in higher osteoinductivity.

Meniscus

Minced, Decellularized Bovine Meniscus Hydrogels

Human MSCs

Hydrogels supported Yuan et al integration and formation 2017 of fibrocartilagenous tissue in rat models of meniscus injury


Batchelder et al 2015

Liu et al 2017

Synergistic Interactions Page ACS 47 Biomaterials of 47 Science & Engineering

Combination of stem cells and extracellular matrix scaffold

1 2 3 4 5 6 7

GOAL: Enhanced Tissue Repair - Improved cell engraftment - Constructive remodeling

Host tissue response

- Direction of stem cell differentiation

ACS Paragon Plus Environment Macrophages T Lymphocytes

Potential Synergistic Effects of Stem Cells and Extracellular Matrix

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