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Naturally Derived Extracellular Matrix for Cellular and Tissue Biomanufacturing Qi Xing, Zichen Qian, Wenkai Jia, Avik Ghosh, Mitchell Tahtinen, and Feng Zhao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00235 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016

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ACS Biomaterials Science & Engineering

Natural Extracellular Matrix for Cellular and Tissue Biomanufacturing Qi Xing, Zichen Qian, Wenkai Jia, Avik Ghosh, Mitchell Tahtinen, Feng Zhao *

Department of Biomedical Engineering, Michigan Technological University, Houghton, MI 49931, USA

* Corresponding author: Feng Zhao Department of Biomedical Engineering Michigan Technological University Houghton, MI 49931 Email: [email protected]

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Abstract Natural extracellular matrices (ECM) derived from native tissues or cultured cells are extensively employed to fabricate biocompatible scaffolds or living tissue constructs for the application in cellular and tissue engineering. The composition and structure of ECM are not only heterogeneous, but also tissue or cell specific. Recapitulating the unique cell or tissue niche, ECM-based products are promising to quickly integrate with host tissues and accelerate restoration of tissue function. A variety of natural ECM-based scaffolds and tissue constructs have been biomanufactured using different approaches. Native tissue derived ECM is typically grounded into powders that can be further processed into hybrid composites in the form of hydrogels, foams, nanofibers, and 3D-printed complex constructs. Cell-derived ECM follows different biomanufacturing methods. Usually, cells are seeded on a scaffold to deposit ECM resulting in ECM-ornamented materials. The employment of resolvable scaffolds and cell sheet engineering technique enables production of complex 3D constructs exclusively composed of ECM with/without cells.

In order to enhance mechanical strength, in vivo stability, and

biological performance of ECM-based products, crosslinking reagents or bioactive factors are often used for modification. The major focus of this article is to provide an overview of current biomanufacturing approaches that utilize either native tissue or cell-derived natural ECM in the field of cellular and tissue engineering. Furthermore, the existing challenges for translational application of ECM-based products and the potential resolutions are discussed. Key

words: natural

extracellular matrix,

biomanufacturing, tissue engineering,

bioprinting, chemical modification

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1. Introduction Natural extracellular matrix (ECM) is widely used in cellular and tissue engineering in various forms after different biomanufacturing processes. A variety of ECM-based materials or tissue constructs have been fabricated with the intent to regenerate tissues such as abdominal wall 1, heart valves 2, muscle 3, blood vessel 3, and trachea 4. ECM is a complex network that determines the structure and function of every tissue within the body 5. It consists of collagen, elastin, fibronectin, proteoglycans (PGs), glycosaminoglycans (GAGs), and many other different molecules. Collagen and elastin offer structural support and resistance to mechanical loading 6. The PGs and GAGs retain growth factors and large amounts of water, aiding in cellular interactions with the surrounding environment 7. Adhesion proteins, such as laminin and fibronectin, not only link collagen, elastin, GAGs, and integrin receptors, but also dominate the cell attachment, migration, and phenotype. Due to the structural complexity and important roles of all the ECM components, natural ECM offer a more complicated and biomimetic microenvironment to cells and tissues compared to synthetic materials or natural polymers. The natural ECM can mediate biochemical and biophysical signaling properties that result in dynamic cellular response to surrounding microenvironment including growth factors, ECM stiffness, and component alterations 8. Utilizing the structural and functional support of the ECM has been a vital area to the field of tissue engineering and regenerative medicine. Constructive remodeling, described as site-appropriate functional regeneration 9, could be achieved with ECM-based scaffolds since ECM not only supports cell adhesion, but also promotes cell migration, differentiation, and proliferation 7. For example, the ECM-based vascular grafts showed excellent performance at certain sites of implantation (e.g. aorta, carotid, pulmonary arteries), exhibiting substantial host cell invasion, early capillary penetration, and

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complete endothelialization

10

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. Highly mimicking the physiological environment in vitro, ECM

can serve as a better model to study cell interactions with the surrounding microenvironment. One of the applications is in vitro drug screening models, which currently fail to replicate the physiological environment

11

. Involving natural ECM allows for simulation of cell-matrix

signaling driven by biochemical or mechanical inputs. Therefore, the multiple roles played by the natural ECM in tissue regeneration and in vitro tissue models make it an important and popular material in biomanufacturing. Natural ECM can be used alone to make completely biological scaffolds or living constructs when seeded with cells. The mechanical and biological instability caused by natural origin of ECM led to development of hybrid materials that combine synthetic materials and natural ECM. Compared to biological scaffolds, synthetic materials are easier to be processed into a variety of structures and can be modified to have different mechanical properties that fulfill the criteria of various injury sites 12. Moreover, the mass production of synthetic polymer scaffolds is more applicable and cost effective than biological scaffolds. However, synthetic materials usually lack the bioactivity to promote cell proliferation responses

14

13

and mediate inflammatory

. The hybrid composites of synthetic materials and natural ECM may retain the

advantages of both materials - reproducible mechanical properties and good biocompatibility. The natural ECM can be derived from either native tissues or cultured cells, and the biomanufacturing methods used for these two types of natural ECM are different. This paper provides an overview on the current biomanufacturing approaches of employing natural ECM (tissue-derived and cell-derived ECM) to fabricate biological scaffolds (completely biological and hybrid) or living constructs for the purpose of tissue repair and regeneration. The sources of

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natural ECM, the different biomanufacturing methods, and the modification approaches to enhance product performance are summarized. 2. Sources of Natural ECM The two major sources of natural ECM are native tissues and primary cells. Human and animal tissues (e.g. porcine or bovine) have been widely employed to manufacture natural ECM products 5. Each tissue has its own specific 3D environment of structural and functional ECM networks, which provides unique protein "footprints" for resident cells. For example, tendons have a much higher elastin content and an aligned orientation to resist the large tensile stresses experienced

15

; cartilage tissues have a high GAG content to retain more fluids for lubrication

and resist the compressive stresses 16. Thus, a wide variety of tissues have been decellularized to produce ECM for different applications. A lot of work has been done to remove the cellular components with minimal disturbance of the ECM functionality and mechanical strength. A comprehensive review on methods of antigen removal from animal tissue-derived ECM has been covered in a previous publication 17. Examples of ECM derived from different native tissues as well as their in vitro and in vivo biological performance are summarized in Table 1, which demonstrates diversity of tissue sources for ECM harvest and the excellent biocompatiblity of these ECM scaffolds. Although the ECM derived from native tissue shows important applications, problems may exist such as pathogen transfer, uncontrolled variability arising from age and health conditions of individual source

18

. The cell-derived ECM is an alternate source that may

overcome some of the limitations. It also provides a complex series of molecules that mimic the native stromal microenvironment

19

. Cell-derived ECM is more advanced than their tissue

derived counterpart in terms of pathogen exclusion and customization. Pathogen exclusion can 5



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be achieved by screening cultured cells for pathogens and then maintaining the cells in a pathogen-free condition for ECM harvesting. The customization of ECM products can be realized by choosing different cell sources, culture medium, and culture systems

19-20

. For

example, versican, a large chondroitin sulfate proteoglycan, is found in chondrocyte-derived ECM but not in fibroblast or mesenchymal stem cell (MSC) derived ECM

21

. The micro-

architecture of the cell-derived ECM scaffolds also can be modulated to achieve different spatial organizations by employing substrates with various patterns

18, 22

. However, there are some

obvious limitations involved in their in vivo applications such as long cell culture period, weak mechanical properties, and challenges in sterilization and storage. Examples of cell-derived ECM processed from different cell types and their in vitro and in vivo biological performance are summarized in Table 2. In most of the examples, ECM was derived from connective tissue cells and deposited on scaffolds for modification.

Table 1: Examples of common native tissue-derived ECM products and their functions. Tissue type

Tissue source

In vitro performance

In vivo performance

Small intestinal submucosa (SIS)

Porcine small intestine

Significantly enhance the release of several angiogenic factors of human MSCs (hMSCs) 23

Effectively work as body wall repair bioscaffold 25

Urinary bladder

Basement membrane and tunica propria of adult porcine urinary bladder

Support human epidermal cell differentiation and protein synthesis 24

Show rapid host tissue remodelling, degradation and elimination when used as temporary scaffold for urinary bladder repair 26

Incorporation of urinary bladder ECM into polymer increase smooth muscle cell adhesion and proliferation 27

Promote a significant acute endogenous repair response that could potentially be exploited to treat stroke 28 Show complete epithelialization of wound with limited formation of scar tissue 29

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Bone

Porcine femoral trabecular bone

Enhance fibroblast attachment and spreading, investigate the important role of bone phosphoprotein 30

Well-infiltrated with newly formed tissue and blood vessels 31

Biophasic ECM scaffolds seeded with MSCs integrate in osteochondral area and form new cartilage and bone interface with good mechanical strength 32

Cartilage

Porcine knee joints and nasal septal cartilage; Human cadaveric joint

Liver

Ischemic rats;

Rats

Form cartilage like tissue after reseed MSCs 34

Provide suitable 3D environment to support adhesion, proliferation and differentiation of MSCs 34 Significantly improve human hepatocyte attachment and survival rate, as well as maintain hepatocyte phenotype 35

Human adult

Kidney

Support human primary chondrocytes proliferation 33

Support embryonic stem cells proliferation and differentiation towards kidney-related pathway

Support liver-specific function 36

Repopulated kidney has excretory function after transplantation 38

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Skin

Human fetal skin from elective pregnancy termination;

Strong chemoattractant activity of ECM degradation products for keratinocyte progenitor cells 39

Human adult skin;

Support fibroblast proliferation 40

Accelerate full thickness wound healing 40

Porcine adult skin Nerve

Porcine brain;

Increase the number of cells expressing neurites and the neurite length 41

Rat brain; Porcine spinal cord; Human cadaveric nerve

Cardiac tissue

Porcine ventricular

Increase glial differentiation potential of MSCs 42

Prolong viability of cardiac fibroblasts, cardiomyocytes, and MSCs; promote mature and functional cardiomyocyte phenotype 45

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Capable of returning adequate sensation in nerve defects ranging from 0.5 to 3 cm without infection/rejection 43 Schwann cells re-cellularized nerve ECM increase functional regeneration to isograft level while nerve ECM does not require nerve source 44 Increase endogenous cardiomyocytes in the infarct area and maintains cardiac function without inducing arrhythmias 46


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Table 2: Examples of common cell-derived ECM products and their functions. Cell type

Tissue source of cells

In vitro performance

In vivo performance

Fibroblasts

Human cardiac tissue;

Stimulate proliferation and migration of cardiac primitive cells

Promote dermal regeneration and epithelial migration 50

Human foreskin;

47

Perivascular connective tissue of human saphenous vein

Maintain pluripotency and differentiation ability of human pluripotent cells 48 Provide better mechanical and contractile properties for TEBV, accelerate production efficiency 49

Chondrocytes

Femoral condyle area of young calves

Enhance chondrogenic differentiation of rabbit and hMSCs 51

Mesenchymal stem cells (MSCs)

Human bone marrow; Human fetal umbilical cord; Rat bone marrow; Rat tibiae and femora

Enhance the osteogenic differentiation of MSCs 53 Slow the loading and release kinetics of growth factors; increase alkaline phosphatase (ALP)expression and calcium deposition of MSCs 54

Support chondrocytes with favourable in vivo environment and form hyaline-like tissue 52 Inhibit cancer progression and serve as innovative approach to study stem cell environment on cancer cells without complex cell-cell interaction 58

Promote MSCs proliferation and preserve better osteogenic differentiation capacity 55 and chondrogenic potential 56 Promote adult MSC expansion without changing phenotype 57 Endothelial cells (ECs)

Human umbilical vein

Promote osteogenic differentiation of MSCs 59

No records

ECM-covered titanium greatly enhances EC adhesion and proliferation, decreases platelets adhesion 60 Osteoblasts

Subchondral bone from normal and

ECM from OA osteoblasts decreases integrin β1 expression of

8


No records

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osteoarthritis (OA) patients

osteocytic cell and affect its attachment and functions 61

Glial cells

Rat glioma

Regulate γ-GTP activity in ECs 62

No records

Schwann cells

Rat Schwann cells

No records

Schwann cell ECM modified scaffold used to bridge 10mm rat nerve gap works as well as nerve graft 63

3. Natural ECM Biomanufacturing Methods Biomanufacturing, defined as “the use of biological systems or the products of biological systems to generate new materials and devices, with a view towards scalability and industrialization”, has become a significant area to translate research findings regarding tissue engineering towards production of biological products in a large scale for the medical industry 64. In the case of ECM-based biomanufacturing, the specific ECM of tissues or cells will be derived, and served as building blocks to re-fabricate bioactive products for tissue regeneration or cell delivery. Through specialized techniques, the re-fabricated products could resemble the composition, structure, and functions of the targeted tissues. The processing methods of natural ECM derived from native tissues and from in vitro cultured cells are quite different. 3.1 Processing methods for ECM-derived from native tissues The tissue-derived ECM has a long biomanufacturing history for tissue regeneration applications. Usually, the native tissues are separated from unwanted tissues, decellularized, disinfected, dehydrated (sometimes), and sterilized to obtain ECM scaffolds. The direct applications for the conventional form of two-dimensional (2D) ECM sheets have limitations, such as dense and heterogeneous structure or mechanical instability 12. Thus, the tissue-derived ECM is often processed into particles or powders for more versatile applications and greater

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control of physical properties. The suspension of ECM powder can be directly injected into a targeted site for tissue repair performance

66

65

; however, rapid in vivo degradation significantly weakens its

. The powdered form of ECM also loses its original ultrastructure and 3D

architecture, along with its mechanical support function 67. Therefore, ECM powders are usually mixed with other materials or being processed into other forms with structures. ECM powders can be mixed with synthetic or natural polymers to enhance their biocompatibility. For example, 3D composites of poly(D,L-lactide-co-glycolide) (PLGA) and small intestinal submucosa (SIS) powder showed increased cell attachment with increasing SIS content, and the in vivo implantation demonstrated that the PLGA-SIS composites were more osteoinductive than control PLGA scaffolds

68

. Another example, urinary bladder matrix powder was mixed in alginate

microparticles, which were subsequently employed to encapsulate neonatal porcine Sertoli cells. The trapped ECM powder promoted retention of cell viability (44% increase than control at day 14) and function (5 fold increase of phenotype maintenance gene expression than control at day 14)

69

. The ECM powder can be further processed into hydrogels, porous foams, electrospun

nanofibers, or 3D bioprinted constructs

70

. The employment of tissue-derived ECM for

biomanufacturing is illustrated in Figure 1. 3.1.1 ECM hydrogels Hydrogels, especially in situ polymerized hydrogel under physiological conditions, provide a minimally invasive way to deliver materials, drugs, bioactive molecules, and cells to the site of interest for tissue repair. The hydrogels employed in tissue engineering have been predominantly focused on ECM derivatives such as collagen, gelatin, and hyaluronic acid

71

.

Actually, the decellularized tissues can be directly processed into hydrogels by being partially digested with pepsin, solubilized, and polymerized in situ

10

70a

(Figure 1A). A series of studies


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have demonstrated that ECM hydrogels can be processed from a variety of tissues including heart 72, dermis 73, bone 74, brain 41, fat 75, meniscus 76, lung 77, and umbilical cord 78. Compared to synthetic or natural polymer hydrogels, ECM hydrogels are composed of a more complex set of ECM molecules that may offer additional biological motifs. Specific tissue-derived ECM hydrogels may have a more favorable response on the same type of tissues 79. Most of the tissuederived ECM hydrogels mentioned above have been proven to promote growth of the regarding cell types in vitro and enhance in vivo tissue regeneration

73-75

. Furthermore, the ECM hydrogel

can be mixed with other materials to achieve desired rheological properties similar to the tissue that is being repaired. For example, poly(ethylene glycol) (PEG) was conjugated with the myocardial matrix-derived hydrogel. This hybrid hydrogel maintained the ECM nanofibrous network and biocompatibility with a significantly enhanced ability to resist enzymatic degradation 80. ECM hydrogels can be fabricated into porous 3D foam by the phase separation method (Figure 1A). Porous scaffolds provide interconnected networks for tissue vascularization and neo-tissue formation during the regeneration process 81. Phase separation is a delicate method to create porous 3D scaffolds from natural ECM hydrogels without damaging the integrity of the ECM components 82. After lyophilization, the water content is removed from the hydrogel while the protein fibers and other ECM molecules are left. For example, decellularized porcine left ventricle was processed to form hydrogel that was subsequently freeze-dried to obtain ECM foam. The in vitro study showed that the ECM foam provided a more supportive microenvironment for inducing human pericardial fat adipose-derived stem/stromal cell cardiomyogenesis than type I collagen gel 83. In another example, the porous foam was prepared from decellularized human adipose tissue (DAT). The DAT foam promoted the in vitro

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adipogenic differentiation of human adipose-derived stem cells and strongly supported in vivo angiogenesis and adipogenesis 84. 3.1.2 ECM blended electrospun nanofibers ECM powders can be further fabricated into nanofibers by an electrospinning method (Figure 1B), which can generate fibrous and porous matrices with great cellular response controlling ability by varying fiber and pore sizes or fiber alignment. Recently, research on electrospun scaffolds have focused on utilizing tissue-derived ECM as the base material, or blended with other synthetic or natural polymers to assign tissue specific bioactivity towards the electrospun nanofibers

85

. Direct electrospinning of demineralized bone matrix (DBM) into

nanofibers (diameter range: 100-350 nm) has been realized by dissolving DBM (concentration: 5-14 g dL-1) in hexafluoro-2-propanol (HFIP)/trifluoroacetic acid (TFA). Live/dead staining has shown that fibroblasts exhibited good cytocompatibility on the DBM nanofibers

86

. Cartilage

derived ECM powder was dissolved in HFIP and mixed with poly-ε-caprolactone (PCL) solution at a ratio 1:1 to electrospin nanofibers. The incorporation of ECM stimulated sulfated GAG synthesis and chondrogenesis-related gene expression of seeded adipose-derived stem cells

70c

.

Baiguera et al. reported a nanofibrous scaffold combining gelatin and rat decellularized brain extracellular matrix (dBECM) for neural engineering applications. Lyophilized dBECM powder was mixed with gelatin powder (1% w/w), dissolved in acetic acid/deionized water into a solution, and electrospun into nanofibers with average diameter 320 ± 70 nm. The presence of the brain matrix induced an initial stromal cell differentiation towards neural precursor cells 87. 3.1.3 ECM constructs generated from 3D bioprinting

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With the emerging of 3D printing technology under the principle of rapid prototyping in the past decade, construction of complex 3D functional living tissues or artificial organs is not only feasible, but also more time and cost effective (Figure 1C)

88

. The 3D bioprinted ECM

constructs represent one of the future directions for ECM biomanufacturing. Successful 3D printing is dependent on the fabrication of appropriate hydrogel based bioink, which is a basic carrier of cells to protect them from external mechanical stress during the printing process 89. To meet the criteria of both 3D bioprinting process and the maintenance of printed tissues, the bioink should also allow mass transportation of nutrients, oxygen, and wastes disposal. Hydrogels including synthetic and natural polymer hydrogels are widely adapted as bioinks for their excellent biocompatibility and mechanical properties that maintain the integrity of cells during printing

89-90

. Using decellularized ECM hydrgels as bioink allows for 3D printing of

constructs with the resemblance of native tissue components (Figure 2A)

70d

. For example,

decellularized adipose, cartilage, and heart tissues were processed and dissolved to make ECM bioink that was printed into a PCL framework to make a 3D construct 70d. The presence of tissue specific components in the construct facilitated survival and differentiation of stem cells. Human adipose derived stem cells encapsulated in 3D printed adipose ECM constructs showed increased expression of adipogenesis-related genes

70d, 91

. Rat myoblasts embedded in 3D printed heart

ECM constructs also demonstrated enhanced expression of cardiogenesis-related genes

70d, 92

.A

newly developed method allows for using scaffold free "tissue strands" as bioink for bioprinting (Figure 2B) 93. In this work, a tubular semi-permeable alginate capsule was extruded and injected with chondrocytes pellets. After 7 days of culture, alginate capsule was dissolved and the resulting tissue strand had acceptable cohesiveness to be loaded into nozzles for 3D printing 93.

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This study provides a novel approach for bioprinting with cells only, which leads to a construct exclusively composed of cells without any supporting scaffold. The next level of 3D bioprinting should be focused on reconstructing the whole organ with proper structure, function, and mechanical properties. To achieve this goal, recent study by Hinton T. et al demonstrated a proof-of-concept new bioprinting system that can print structures based on femurs, branched coronary arteries, trabeculated embryonic hearts, and human brains with appropriate anatomical architecture and mechanical strength 94. Murphy & Atala suggested that the ECM protein structure should be completely mapped out to extend the basis of increasing the bioprinting resolution on micro/nano scale to better mimic the microstructure of complex organs

95

. Based on current technologies, bioprinting large organs with clinically

relevant size and structural integrity is very challenging. In order to support a large construct while printing a complex organ, it is necessary to use a sacrificial scaffold which provides initial framework but dissolves after the construct is rigid enough to maintain its shape 96. In addition, organ printing is very time consuming; therefore, maintaining the metabolism of the printed parts by immersing them inside an automatic medium-filling reservoir could benefit the cellular activity in the printed organs. Other problems should be considered such as supplying nutrients to the artificial organ core, supporting its macro/micro structure, and promoting its function performance after implantation. Vascular networks could be integrated into the printed organs to make it self-sustainable before transplantation. For example, 3D printed rigid carbohydrate glass filament network could be used as sacrificial template in engineered tissue for vasculature formation

97

. Creating a lattice of microchannels inside a printed porous constructs facilitated

nutrient and oxygen diffusion as well as enhanced in vitro tissue formation 96. 3.2 Processing methods for ECM derived from cell culture

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Cell-derived ECM provides an alternative source for natural ECM. Different from tissuederived ECM, cell-derived ECM is obtained by culturing cells on supporting substrates for a certain period to allow deposition of ECM. The recent advancement of tissue engineering techniques enables the production of various in vitro grown tissue-like constructs from cells

98

.

The subsequent decellularization led to a construct composed of cell-laid ECM with or without the original supporting substrates 55, 99. The composition of the ECM can be adjusted by selecting different types of cells, and the mechanical properties of the constructs can be tuned by mechanical pre-conditioning

20

. Compared to tissue-derived ECM, cell-derived ECM has

relatively simple structures and components, which may make it easier to be decellularized yet negatively affect the mechanical strength. 3.2.1 ECM decoration on synthetic materials Direct deposition of ECM by cells on synthetic materials is the most common and simplest method for utilizing cell-derived ECM to improve substrate bioactivity. The cells are seeded on a synthetic scaffold followed by devitalization, leaving behind a cell-laid matrix (Figure 3A). The materials after ECM decoration exhibited enhanced in vitro cell attachment, proliferation and differentiation, as well as in vivo biological performance. For example, 3Dprinted scaffolds (made from a composite of PCL, PLGA, and β-tricalcium phosphate (β-TCP)) coated with MSC-deposited ECM significantly increased the calcium deposition compared to bare 3D-printed scaffolds. The osteogenic differentiation of re-seeded MSCs were also improved due to the ECM inclusion

100

. The expression of osteogenic genes (ex. alkaline phosphatase

(ALP), osteopontin, osteocalcin, and osteonectin) in MSCs cultured on ECM-ornamented titanium fiber mesh were drastically up-regulated

53a

. In another example, fibroblasts and pre-

osteoblasts were cultured on a PLGA/hydroxyapatite/β-TCP composite followed by

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decellularization. The ECM-coated scaffolds provided a favorable template for promoting both in vitro and in vivo osteogenesis of MSCs, which was evidenced by increased ALP activity and mineralization compared to their counterparts grown on uncoated scaffolds

101

. When compared

to collagen I-coated PDMS substrates, the fibroblast ECM-deposited PDMS substrates preserved much higher amounts of growth factors which may be accounted for stimulation of hMSC osteogenic differentiation 102. However, biomanufacturing ECM constructs using cell deposition/decellularization could be limited by the batch-to-batch variation, high cost of cell culture, and the storage of the product. Moreover, the ECM is not suitable for scaling-up production of surface modified scaffolds due to the complexity of the extracted components as well as the diminishment of cell-binding domains during the processing steps 103. For cost effective mass production of surface modified substrates, using ECM derived components, small fragments of proteins or shorter peptides derived from the ECM would provide better homogeneity and integrity to the modified surfaces 104. 3.2.2 ECM constructs with resolvable scaffolds A completely biological 3D construct can be created bottom-up from cells and resolvable scaffolds that usually serve as an initial backbone to guide cell attachment and growth and gradually dissolve or biodegrade at a later stage

105

(Figure 3B). For example, an engineered

graft was obtained by seeding dermal fibroblasts on fibrin gel in a tubular template for 5 weeks followed by decellularization. The fibrin gel was completely remodeled at this time point and the resulting 3D constructs were mainly composed of cell secreted ECM after decellularization

106

.

The tubular graft demonstrated similar burst strength and compliances to ovine femoral artery, as well as extensive recellularization and complete endothelialization after 24 weeks in vivo implantation. Another study on tissue engineered heart valve leaflets using a similar approach 16


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has shown that artificial leaflets developed mechanical anisotropy and tensile strength comparable to an ovine pulmonary valve leaflet and displayed better in vitro recellularization potential by hMSCs 107. Other than fibrin gel, rapidly degradable synthetic polymers can be used as resolvable scaffolds as well. Several studies have reported fabrication of tissue engineered blood vessel or heart valves by seeding fibroblasts or smooth muscle cells (SMCs) on PLGA templates and culturing them for several weeks to allow deposition of enough natural ECM

108

.

After decellularization, the in vitro grown vessel was seeded with autologous endothelial cells (ECs) and the in vivo implantation showed that the graft resisted both clotting and intimal hyperplasia. The decellularized tissue engineered heart valve was implanted in vivo and functioned similar to decellularized human native heart valve

108b

. These studies demonstrated

that completely biological engineered connective tissues could be grown from banked cells, rendered acellular, and then used for tissue regeneration in vivo. However, this method is extremely time consuming in order to accumulate enough ECM and achieve the target mechanical strength. Bioreactors with mechanical strain conditioning are usually applied to strengthen the constructs. 3.2.3 ECM constructs assembled from cell sheets Cell sheet engineering techniques are often used to fabricate 3D complex structures, such as tissue engineered blood vessels (TEBV) (Figure 3C). The earliest attempt to engineer TEBV, using fibroblast and SMC sheets, faced the problems of long-culture time and storage issue

109

.

The following study obtained an acellular TEBV by devitalizing the tubular construct and storing the TEBV in a -80 ⁰C freezer for several months before clinical trial. The graft with or without lined ECs was implanted as an atrioventricular shunt in the patient and remained patent 8 weeks postoperatively

110

. The fibroblast cell sheet can be decellularized first and then seeded with

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other cells to fabricate ECM based constructs. In addition, the aligned topographical cue could be created on the ECM fibers by seeding fibroblasts on nanopatterned Polydimethylsiloxane (PDMS), which was used to guide the cell orientation 22. For fabrication of TEBV, SMCs were seeded on a decellularized cell sheet, which was then rolled around a tubular mandrel for maturation. The resulting vessel had similar mechanical strength to native saphenous vessels. This strategy allows for the production of a vascular media using SMCs isolated from any patient, regardless of their capacity to synthesize ECM 111. The production time was shortened by 2 weeks compared to the first cell sheet-assembled TEBV. The perfusion bioreactors, or bioreactors with pulsatile flow, were often employed to condition the 3D constructs for better alignment and mechanical strength

106

. The results from those studies demonstrated the

possibility to produce a completely devitalized allogeneic ECM-based TEBV. The application of cell sheet engineering technique and resolvable scaffolds offers the potential for manufacturing "off-the-shelf" TEBV after devitalization without seeding ECs. However, the minimum production time is still around 4 weeks, and the in vivo performance without ECs lining needs more investigation. The biomanufacturing methods for cell-derived ECM are mostly in the laboratory setting scale. The quantity of ECM obtained through cell culture is still limited and highly dependent on cell type and culture time. Although fibroblasts can deposit large amount of ECM through extended period of culture, the long cell culture time will increase the risk of contamination and the cost of production. Cell-derived ECM generally has weaker mechanical properties compared to tissue derived ECM; however, bioreactor culture can significantly increase the strength of cellderived ECM based constructs by 2-3 fold 112. The cell-derived ECM allows for usage of human

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cells and stem cells, which is desirable for fabrication of in vitro models for mechanism studies or drug screening. 4. Modification of ECM to Improve Performance ECM contains a variety of proteins that have free amine or carboxyl groups, thus it is easy to crosslink ECM or graft bioactive molecules on the polypeptide chain in order to improve its mechanical or biological performance

113

. The natural ECM-based products were always

stabilized before implantation in the human body, aiming to increase their resistance against enzymatic degradation, improve mechanical strength, or reduce its antigenicity

113-114

. In

addition, incorporation of different molecules or nanomaterials in ECM scaffolds or ECMderived products can further enhance their application potential. 4.1 Strategies to enhance mechanical strength and stability The most extensively used method to improve mechanical strength and stability is chemical crosslinking. The chemical reagents form covalent bonds between amino or carboxyl groups of different polypeptide chains in the ECM. Crosslinking can effectively enhance the mechanical strength and stability of ECM; however, other biological properties are also altered including biocompatibility and hemocompatibility 115. The most commonly used crosslinkers are glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and genipin. The crosslinking mechanism of these three chemicals is illustrated in Figure 4A. GTA is the most commonly used reagent to crosslink biological tissues clinically. It is able to generate predominantly intermolecular bonds between amino groups, forming a tightly crosslinked network

116

. Due to the ability to self-polymerize, GTA is very efficient in crosslinking distant

protein molecules

117

. The ECM crosslinked with GTA showed significantly improved tensile

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strength, pliability, and resistance against degradation

118

Page 20 of 43

. However, the application of GTA-

crosslinked ECM is limited by the toxicity and calcification induced by GTA

119

. EDC and

genipin have been widely used as alternatives to GTA because they are more cytocompatible and less immunogenic

114

. EDC mainly activates carboxyl groups, which react with free amino

groups to form amide bonds. Genipin reacts with primary amine groups on ECM molecules and forms mono- up to tetramer crosslinks

120

. Both EDC and genipin crosslinking can significantly

improve physical properties of ECM such as tensile strength, thermal shrinkage temperature, and resistance to enzyme degradation

121

. Furthermore, compared to GTA-crosslinked ECM, the

EDC-crosslinked ECM shows better biocompatibility - more cell attachment, survival, and less pro-inflammatory cytokine release

122

. The genipin-crosslinked ECM does not affect cell

proliferation and differentiation as reported on GTA-crosslinked ECM

123

. However, the EDC-

crosslinked material is not as strong as the GTA crosslinked materials because GTA can crosslink distant collagen molecules, but EDC cannot. A recent study demonstrated that the combination of different crosslinkers (carbodiimide, neomycin trisulfate, and pentagalloyl glucose) can achieve high biomechanics as well as better biocompatibility and less calcification 115a

. It was reported that chemical crosslinking does not stabilize important ECM components

- elastin and GAGs. The ECM components lost by enzymatic degradation may compromise the mechanical strength and function

124

. Incorporation of polymers or nanoparticles is another

approach to improve mechanical stability. For example, decellularized rat artery was coated with several types of biodegradable and biostable medical-grade polymers to increase the mechanical strength. It was found that poly(ether urethane) (PEU) 1074A could adhere well to the vascular grafts and promote cellular repopulation following engraftment in the rat abdominal aorta

20


125

.

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The polymer-silica-ECM composite material showed equivalent viscoelastic response to densely-crosslinked matrix, and equal macrophage adhesion and in vitro bioactivity to noncrosslinked ECM scaffold 126. 4.2 Incorporating ECM with bioactive factors Incorporation of ECM with bioactive factors brings new functions and enhances biological performance of ECM. A variety of growth factors, metal ions, biopolymers, and nanomaterials can be either adsorbed or covalently bonded to ECM, as illustrated in Figure 4B. For example, epidermal growth factor (EGF) were directly adsorbed on ECM. The release of growth factors significantly enhanced the wound healing rate 127, as demonstrated in Figure 5A. Vascular endothelial growth factor (VEGF) loaded multi-walled carbon nanotubes were absorbed with ECM-based scaffold to enhance in vivo tissue regeneration

128

. Decellularized

valve scaffold conjugated with transforming growth factor-β1 (TGF-β1) loaded PEG nanoparticles showed improved biocompatibility and biomechanical property

129

. The copper

ions modified SIS promoted re-epithelialization, re-vascularization, and muscular regeneration 130

. Nitric oxide (NO) donor S-Nitroso-N-acetyl-D-penicillamine (SNAP)-conjugated ECM can

release NO to mimic the in vivo wound healing environment

131

, as shown in Figure 5B. These

studies show that there is great potential to modify ECM in different ways to enhance its function and applications. For cell-derived ECM, the incorporation of bioactive factors could be realized by changing the cell culture medium. For example, the ECM deposited by MSCs cultured in osteogenic induction medium contained large amounts of calcium. This mineralized ECM can significantly enhance the osteogenic potential of the subsequently seeded MSCs

53a

. The ECM

secreted by MSCs cultured with supplement ascorbic acid (aaECM) contained significantly 21



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higher amounts of sulphated GAGs and collagen than the ECM secreted by osteogenic-induced MSCs (osteoECM). In addition, aaECM bound greater amounts of growth factors (bFGF, VEGF, hepatocyte growth factor (HGF); interleukin-8 (IL-8)) compared to osteoECM

19

. The different

components in the aaECM supported higher proliferation rates, greater osteogenic and adipogenic differentiation efficiencies and increased secretion of growth factors (angiopoietin 1; stromal cell-derived factor; IL-8) in cultured MSCs and hematopoietic stem/progenitor cells. The aaECM better mimicked the bone marrow niche environment to improve the bone marrow stem cells expansion. 5. Concluding Remarks and Translational Challenges A variety of natural ECM, derived from either native tissues or cultured cells, has been successfully biomanufactured into different scaffolds or tissue constructs for cellular and tissue engineering applications. These biomimetic ECM-based products have shown promising potential

to

accelerate

tissue

regeneration.

The

tissue-derived

ECM

has

different

biomanufacturing methods than cell-derived ECM, and both have their own advantages and disadvantages. The synthetic or natural polymer scaffolds integrated with natural ECM demonstrated great biocompatibility as well as excellent and tunable physical properties and reproducibility. The biomanufactured ECM products exhibited promising biological performance in both in vitro and in vivo studies; however, the current in vivo studies are mainly based at small animal scales and displaying short-term in vivo function. Thus, a series of hurdles must be addressed before translation into clinical usage. 1) Standardize the protocols to obtain stable and reproducible products. Since the ECM is derived from native tissue or cultured cells, the uncontrollable variability may exist due to the age and health conditions of donors as well as the cell status and sources

132

. Due to the batch-to-batch variation, standardization of ECM

22


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processing protocols should be created in order to validate the quality of derived ECM for both research and industrial applications. For cell-derived ECM, genetically engineered cells may help to stabilize the ECM secretion ability of cells. For example, fibroblasts can be engineered to express more matrix protein - elastin to preserve the elastic structure of ECM. The engineered ECM may also help to improve elasticity and cardiac function when used for cardiac regeneration 133. 2) Shorten the fabrication time course and reduce the manufacturing cost. Many acellular ECM-based products are "off-the shelf" through careful control of storage condition in order to avoid contamination and degradation, whereas ECM-based living constructs need longterm culture (e.g. the average TEBV culture time is 4 weeks). Thus, the cost to fabricate these living constructs is relatively high. Growth factors supplemented cell culture medium is promising to promote rapid ECM deposition which not only reduces fabrication time but also maintains ECM strength

134

. 3) Perform a series of standard biocompatibility tests and in vivo

functionality assays before clinical usage. Although there have been a wide diversity of FDA approved and clinically used acellular ECM-based products, their safety and regulation need serious consideration for translational application. Most of the work only addressed simple in vitro cytotoxicity or in vivo short-term regeneration results. Other assays such as sensitization, irritation, intracutaneous reactivity, carcinogenicity, and genotoxicity are necessary especially when novel materials or stem cells are involved 135. 4) Bring the ECM-based products to clinical quality and scale. Most of the work is performed in the laboratory setting and only requires relatively low cell number or small dimensional samples. In order to generate tissues of clinical size, rapid expansion of a large quantity of cells and longer culture period may be required. The bioprinting technology of assembling ECM and cells together is promising to significantly

23



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increase the product dimension, enhance the manufacturing speed, and increase the reproducibility of complex constructs 136.

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Acknowledgements This study was supported by the National Institutes of Health (1R15CA202656 and 1R15HL115521-01A1) and the Portage Health Foundation Research Excellence Funds (PHFREF) to F.Z.

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Captions Figure 1. Biomanufacturing strategies of tissue-derived ECM for cell culture or tissue regeneration applications. Various tissues (soft and hard) are processed into ECM sheet, which can be subsequently processed into ECM powder. ECM powder can be mixed with synthetic or natural polymer to make hybrid composites. In addition, ECM powder can be used to fabricate (A) hydrogel/lyophilized porous foam; (B) electrospun nanofibers; (C) 3D printed scaffolds/ living constructs. Figure 2. Applications of biomanufacturing with 3D bioprinting technology. (A)3D bioprinting with decellularized ECM bioink on polycaprolactone scaffold for biomanufacturing cardiac, cartilage, and adipose tissues 70d. Scale bar 10 mm. (B) Scaffold free "tissue strand" (containing chondrocyte pellets) used as bioink for 3D printing cartilage-like tissue 93. Figure 3. Biomanufacturing strategies of cell-derived ECM for cell culture or tissue regeneration applications. Different types of cells are cultured (A) on substrates surface followed by decellularization; (B) on resolvable scaffolds for extended period followed by decellularization; (C) to form cell sheets which are employed to form 3D complex constructs. Figure 4. Schematic illustration of the ECM modification mechanism. (A) Crosslinking of ECM with different crosslinkers glutaraldehyde (GTA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and genipin through covalent bonding of amine or carboxylic acid groups; (B) Incorporating functional materials into ECM through adsorption or grafting with the amine groups on ECM . Figure 5. Incorporation of bioactive factors in ECM scaffolds. (A) Morphology of decellularized pig peritoneums scaffolds; wound healing rate of scaffolds with different epidermal growth factor (EGF) amount in rabbit skin wound model on different day post-surgery 127. (B) Morphology and nitric oxide (NO) release profile of S-Nitroso-N-acetyl-DL-penicillamine (SNAP) conjugated fibroblast cell sheet derived ECM 131.

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Figure 1. Biomanufacturing strategies of tissue-derived ECM for cell culture or tissue regeneration applications. Various tissues (soft and hard) are processed into ECM sheet, which can be subsequently processed into ECM powder. ECM powder can be mixed with synthetic or natural polymer to make hybrid composites. In addition, ECM powder can be used to fabricate (A) hydrogel/lyophilized porous foam; (B) electrospun nanofibers; (C) 3D printed scaffolds/ living constructs. 80x55mm (300 x 300 DPI)


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Figure 2. Applications of biomanufacturing with 3D bioprinting technology. (A)3D bioprinting with decellularized ECM bioink on polycaprolactone scaffold for biomanufacturing cardiac, cartilage, and adipose tissues 101. Scale bar 10 mm. (B) Scaffold free "tissue strand" (containing chondrocyte pellets) used as bioink for 3D printing cartilage-like tissue 104. 75x40mm (300 x 300 DPI)



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Figure 3. Biomanufacturing strategies of cell-derived ECM for cell culture or tissue regeneration applications. Different types of cells are cultured (A) on substrates surface followed by decellularization; (B) on resolvable scaffolds for extended period followed by decellularization; (C) to form cell sheets which are employed to form 3D complex constructs. 77x41mm (300 x 300 DPI)


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Figure 4. Schematic illustration of the ECM modification mechanism. (A) Crosslinking of ECM with different crosslinkers glutaraldehyde (GTA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and genipin through covalent bonding of amine or carboxylic acid groups; (B) Incorporating functional materials into ECM through adsorption or grafting with the amine groups on ECM . 140x97mm (300 x 300 DPI)



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Figure 5. Incorporation of bioactive factors in ECM scaffolds. (A) Morphology of decellularized pig peritoneums scaffolds; wound healing rate of scaffolds with different epidermal growth factor amount in rabbit skin wound model on different day post-surgery 140. (B) Morphology and nitric oxide (NO) release profile of S-Nitroso-N-acetyl-DL-penicillamine (SNAP) conjugated fibroblast cell sheet derived ECM 143. 52x42mm (300 x 300 DPI)


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Table of Content Graphic. Table of Content Graphic 181x64mm (300 x 300 DPI)


Natural Extracellular Matrix for Cellular and Tissue ... - ACS Publications

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