Applications of decellularized extracellular matrix in bone and cartilage tissue engineering

Abstract Regenerative therapies for bone and cartilage injuries are currently unable to replicate the complex microenvironment of native tissue. There are many tissue engineering approaches attempting to address this issue through the use of synthetic materials. Although synthetic materials can be modified to simulate the mechanical and biochemical properties of the cell microenvironment, they do not mimic in full the multitude of interactions that take place within tissue. Decellularized extracellular matrix (dECM) has been established as a biomaterial that preserves a tissue's native environment, promotes cell proliferation, and provides cues for cell differentiation. The potential of dECM as a therapeutic agent is rising, but there are many limitations of dECM restricting its use. This review discusses the recent progress in the utilization of bone and cartilage dECM through applications as scaffolds, particles, and supplementary factors in bone and cartilage tissue engineering.


| INTRODUCTION
Regenerative medicine offers the ability to repair injuries that the body fails to heal. Although there are many synthetically designed materials to support tissue regeneration, these materials fall short of fully replicating a tissue's microenvironment. 1 Looking to the function of this microenvironment for inspiration has provided insight into how materials used in tissue regeneration can be improved. One potential therapeutic material is the native extracellular matrix (ECM), which is the noncellular component of tissue that provides the structural support and biochemical cues for determining a cell's fate. 2 ECM is a natural material that encompasses both the cell microenvironment and biochemical factors for living cells. 3,4 Each tissue type has a specialized ECM structure and composition that modulates cell responses and benefits the survival of cells within that tissue. 2 ECM is composed of two major components, collagen and proteoglycans, which are secreted by cells and assembled in a manner specific to individual tissue types. It contains a reservoir of growth factors and cytokines; these send signals that regulate cell proliferation and migration as well as modulate differentiation and phenotypic expression of the cell. Due to its inherent compositional similarity and modulatory abilities of supporting tissue growth and differentiation, the use of tissuespecific ECM for tissue regeneration has gained popularity, including in the areas of bone and cartilage engineering.
Bone ECM consists of an organic and inorganic phase. The organic phase, mostly type I collagen, provides the tissue with flexibility, while the inorganic phase, mainly consisting of calcium phosphate, specifically hydroxyapatite (HA), 5 is the source of bone strength. 6 In addition, there are four cell types in bone tissue that contribute to osteogenesis: (a) undifferentiated osteoprogenitor cells, (b) matrix- † These authors contributed equally to this study.
Manuscript submitted for consideration and publication in the Bioengineering & Translational Medicine special issue in honor of Professors Robert Langer and Nicholas Peppas. depositing osteoblasts, (c) mature osteocytes that no longer deposit matrix, and (d) osteoclasts that resorb bone tissue. In natural maintenance of the tissue as well as in response to injury, these cell types work in conjunction to homeostatically build up and breakdown the matrix. 7 Bone tissue is one of the few tissues that can heal itself with little to no formation of scar tissue. However, there is a critical size limit of 2.5 cm for most bone, 8 above which regeneration will not occur. In these cases, it is necessary to induce and support osteogenesis to heal the defect.
Cartilage ECM is primarily a collagenous network, 3 with varying compositions and types of collagen depending on the cartilage type.
Hyaline cartilage is mainly type II collagen, while fibrous cartilage is a mixture of both type I and II collagens. 9,10 Another major component of these networks is proteoglycans. Proteoglycans consist of multiple chains of glycosaminoglycans (GAGs) branching off from a core protein. Aggrecan is the most abundant proteoglycan present in cartilage, of which chondroitin sulfate and keratan sulfate are the main GAG components. Aggrecan is highly anionic at physiological pH and attracts water molecules, which gives cartilage its elastic and swelling properties, allowing it to have high shock absorbance under compressive load. 3,11 Different cartilage types have varying collagen/GAG compositions, giving each type distinct mechanical properties. For instance, knee meniscus, a type of fibrocartilage, is predominantly composed of type I collagen, but it has lower GAG content compared to hyaline cartilages such as articular cartilage. This results in meniscus having a higher tensile modulus and lower compressive modulus compared to articular cartilage. 12 Cartilage ECM is maintained solely by chondrocytes, which comprise only 1-5% of total cartilage volume. 13 This low cell density contributes to cartilage tissue having low regeneration capabilities, which is also compounded by the avascular nature of the tissue. In cases when healing does occur, it often yields the formation of fibrous cartilage, which leads to stiffer tissue at the injury site and long-term performance issues. 3,11 To improve function, regenerative therapies promote the formation of native articular/hyaline cartilage rather than fibrous cartilage.
To process ECM for use in regenerative therapy, the excised tissue must first undergo decellularization. Decellularization refers to the process of treating a tissue with any combination of physical stress and chemical/enzymatic agents to remove cellular components, leaving behind only the noncellular ECM that can be used for therapeutic applications. The specific method of decellularization used depends on the tissue type; for instance, while cartilage tissue is able to undergo a relatively harsh treatment, lung tissue requires a more sensitive decellularization method to preserve its tissue composition. 14 The resulting decellularized extracellular matrix (dECM) can then be processed further for different tissue engineering applications. These applications are summarized in Table 1. The main benefit of dECM is that it retains components of the natural cell environment 2 ; with proper decellularization, the complex biomolecular and physical cues in the ECM are preserved and can support cell growth and viability.
Unlike in transplanted tissue, dECM has a lower risk for immune response because almost all the cellular DNA is removed. 15 However, the decellularization process does present challenges, the foremost of which is maximizing the removal of cellular material while limiting damage to the ECM. 15 Although synthetic materials have their benefits, such as tunability of physicochemical properties, they are unable to fully replicate the native microenvironment and structure of the tissue, even with modifications or the addition of bioactive factors. 1 Thus, incorporating dECM presents a promising method for creating an environment that better mimics that of native tissue and suits repair of the injury site.

| GENERAL METHODS OF DECELLULARIZATION
To retain as much of the tissue's bioactivity as possible while maximizing the removal of nuclear material, the decellularization process must minimize the loss of native ECM components. Implantation of decellularized tissue that has had its nucleic materials incompletely removed or degraded could result in host foreign body reaction, which leads to the formation of fibrous capsule surrounding the implant site. 16,17 This eventually can result in improper tissue remodeling and therefore limit the regenerative potential of the decellularized tissue. 18 Preserving the ECM ultrastructure is also important in applications where dECM is not further processed but used as a scaffold by itself. Specific decellularization procedures vary according to the tissue type and can involve a combination of (a) physical, (b) enzymatic, and (c) chemical processes. The most frequently used techniques are discussed below.

Physical decellularization
Introducing physical stresses such as freeze-thaw and osmotic pressure can result in cell lysis without significantly disrupting the ultrastructure of the tissue. Freeze-thawing is one of the most widely used physical decellularization methods, during which the formation of ice crystals puncture cell membranes. The cycle is repeated multiple times before the tissue sample can be processed further. Another option is osmotic lysis, during which tissues are placed in either a hypertonic 19 or hypotonic solution such as deionized water 20 that ruptures the plasma membrane via osmotic shock. Other common physical decellularization methods include hydrostatic pressure, 21 sonication, 22 and electroporation. 23 Tissues that undergo only physical decellularization, specifically freeze-thawing, are considered to be devitalized but not decellularized, as the cells have been lysed, but the cell debris and genetic material still remain within the processed tissue. These samples are most often processed into particles, during which the tissues go through a combination of freeze-thawing and lyophilization, and are then ground into powder using a freezer/mill. 19,[24][25][26][27] Devitalized tissue particles have been shown to have higher quantities of ECM components, such as GAGs, than those that have been additionally treated with chemical/ enzymatic decellularization methods. 19,25 However, there are safety concerns of possible immune responses that could result from residual cellular material. There have also been conflicting reports on whether the increase in ECM components leads to a better cellular response. 19 For instance, rat bone marrow-derived mesenchymal stem cells (MSCs) cultured with devitalized cartilage (DVC) particles had lower cell viability and chondrogenic gene expression levels than MSCs cultured with decellularized cartilage (DCC) particles. 19 This difference may be due to the increased quantity of GAGs in devitalized tissue causing the particles to be too dense for adequate cell infiltration. 19 Physical decellularization is the least disruptive decellularization method, with most of the ECM components and structure left intact after treatment. 28 However, physical decellularization alone cannot completely remove cellular debris from the tissue. Often it is used in conjunction with additional chemical or enzymatic methods. 29 Similarly, incubating a tissue sample in chemical or enzymatic agents without physical agitation does not result in an acceptable degree of decellularization due to limited diffusion into the tissue. Therefore, a combination of all three methods has a synergistic effect where physical agitation enhances the tissue penetration depth of chemical and enzymatic agents, thereby facilitating the removal of lysed cell material. 30,31

Chemical decellularization
Chemical methods of decellularization can largely be divided into two subcategories where tissue samples can be treated with either (a) acidic or basic conditions or (b) detergents. Treating tissues with acids or bases results in cell degradation and the removal of cellular components such as nucleic acids. The degree of successful decellularization will vary according to the type and concentration of the acid/ base being used, processing time, and the type of tissue being treated.
Bases are considered the harsher option of the two and can result in significant loss of GAGs. 32,33 Preservation of GAGs during decellularization is important to maintain tissue mechanical properties (e.g.,tensile, viscoelastic properties) 33,34 and to retain growth factors in the tissue, 35,36 the latter of which have been linked with enhanced biocompatibility in vitro. 37 Except for cases where reduction of GAGs is a desired outcome, 20 alkaline treatment is rarely used as an option for decellularization of bone and cartilage tissue. Peracetic acid is frequently used to decellularize thin tissues such as small intestine submucosa (SIS). For more dense tissues such as menisci, formic acid is considered the best choice for removing both collagen and GAGs. 38 Chemical decellularization can also be performed through the use of detergents. Three main types of detergents are used: nonionic, ionic, and zwitterionic. Nonionic detergents such as Triton X-100 lyse cells through insertion into the lipid bilayer, disrupting the cellular membrane. While disrupting lipid interactions, these largely preserve protein-protein interactions. 39 Proteins are solubilized, but their native structure mostly remains intact. 18 Ionic detergents such as sodium dodecyl sulfate (SDS) are known as denaturing detergents; they disrupt cell membranes and also completely denature proteins. Generally, ionic detergents are considered harsher than nonionic detergents, and they are more detrimental to the ECM structure. For instance, MSCs seeded on tendons treated with SDS had a lower viability and distribution throughout the tissue than cells seeded on tissues treated with Triton X-100. 40 Zwitterionic detergents such as 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulfonate (CHAPS) have a net zero charge and show characteristics of both ionic and nonionic detergents. 15 Although zwitterionic detergents result in less denaturing of proteins compared to ionic detergents, they also tend to remove less cellular material than ionic detergents. 41,42

Enzymatic decellularization
Enzymatic decellularization is most often used directly after chemical decellularization to further facilitate the cell degradation and the removal of residual nuclear material from the tissue. Nucleases and proteases are the most widely used enzymes for enzymatic decellularization. Nucleases, such as deoxyribonuclease and ribonuclease, act directly on DNA and RNA chains, respectively, to hydrolyze phosphodiester bonds. Proteases, such as trypsin, act on proteins by hydrolyzing peptide bonds. Trypsin is a serine protease that cleaves the carbonyl side of lysine or arginine residues. Because of its specific activity on peptides, trypsin treatment can severely disrupt ECM proteins such as elastin and collagen. 15 Enzymatic methods are frequently used in conjunction with chelating agents such as ethylenediaminetetraacetic acid (EDTA), which disrupt cell adhesion to ECM proteins by sequestering metallic ions such as calcium. 15

Evaluation of the degree of decellularization
Measuring the amount of double-stranded DNA (dsDNA) in dECM is the current gold standard for evaluating the degree of successful decellularization. By comparing the amount of DNA in tissue samples before and after decellularization using quantitative assays such as PicoGreen, 43  3 | POSTDECELLULARIZATION PROCESSING METHODS

| Decellularized ECM as a scaffold
One of the simplest methods of using dECM is as a scaffold that maintains its original geometry. The biggest advantage of this method is that, compared to other processing methods that completely pulverize the dECM, using it as an unprocessed scaffold suggests that the tissue retains a large portion of its original ECM architecture. dECM can be prepared from various tissue types to accommodate different compositions, topographies, and mechanical properties. 49

| Bone
The development of a decellularized bone scaffold has been motivated by the need to improve the biocompatibility of allograft bone 50 and the benefit of preserving the bone's native structure. 45 46 The authors reported that bone from an old donor (≥70 years age) was more porous and less dense than that from a young donor (≤50 years age), but the tissues otherwise had similar composition (e.g.,mineral density, calcium/phosphate ratio). MSCs seeded on decellularized bones from older donors expressed higher levels of osteogenic markers than those seeded on decellularized bones from young donors, which the authors attributed to enhanced porosity. Decellularized bone has also been subjected to further modifications, such as collagen/HA coating. 51 When type I collagen solutions were applied to the surface of decellularized porcine cancellous bone, the coating modulated the stiffness of the matrix.
Higher collagen concentration led to higher matrix stiffness compared to uncoated matrices, which in turn guided more robust differentiation of seeded MSCs into osteogenic lineages.

| Cartilage
Due to its dense ECM, decellularizing cartilage and seeding cells after-  60 The authors also created channels through the tissue, which was then overlaid with cell suspension and centrifuged (to pull the cells deeper into the tissue). These treatments, although successful in enhancing decellularization, did not improve recellularization rates. Tyler et al. conducted an in vivo study using the ovine osteochondral defect model, during which a decellularized osteochondral allograft was implanted and studied after 12 weeks. 61 The constructs were remodeled by infiltrating cells, but the cell density was still lower than that of healthy cartilage, resulting in a low GAG concentration within the decellularized implant.
Although maintaining the native architecture during decellularization has its benefits, dECM scaffolds are limited to certain geometries and cannot easily be scaled. To circumvent this, decellularized tissue can be pulverized and freeze-dried into particles that are packed into molds, making it possible to fabricate highly porous dECM scaffolds of varying geometry. 59,[62][63][64][65] Architectural attributes like the size of pores within these scaffolds can impact the behavior of seeded cells.
Almeida et al. prepared coarse and fine dECM particles by processing porcine cartilage using two different methods (homogenizer and cryomill, respectively). 59 By changing the concentration of dECM particle slurry, the authors were able to prepare scaffolds with pore sizes ranging from 32 AE12 to 65 AE20 μm. Scaffolds with larger pore sizes

| Solubilized dECM as a hydrogel
Solubilized dECM is created when dECM is further digested using pepsin, creating a homogeneous solution that can undergo thermal gelation at physiological temperature and pH. As the tissue is homog-

| Cartilage
For cartilage regeneration, the same coating techniques can be applied. However, there has also been substantial work in applying these techniques in tissue culture flasks to promote ECM deposition and study its effects in cell culture. 95 was observed in vivo, where dECM scaffold groups not only contained more GAGs, but also had a higher compressive modulus than the control group. 97 The scaffold was also used with bone marrow stimulation (BMS) technique in a rabbit model, which further enhanced the degree of tissue regeneration compared to BMS only treatment. 98 ECM ornamentation directly addresses many of the limitations of other tissue-engineered dECM applications. As cells are seeded onto prefabricated scaffolds, complex geometries and tunable mechanical, physical, and biological properties can still be achieved through processes such as 3D printing of a synthetic scaffold while still utilizing the biological cues of natural ECM. 85 It can limit or prevent the disadvantages of other techniques, such as the effect of high pressure extrusion on cells during 3D printing, the dimensional mismatch between defect site and fabricated scaffold in implantable hydrogels, and the lack of structural support in dECM alone. 4 In addition, celldirected ECM-ornamenting offers a more physiologically relevant microenvironment formation than a simple, manual coating with dECM, as coating can yield fragmented ECM components that do not accurately represent native ECM. 4 Thus, by employing ECM ornamentation, scaffolds can be fabricated from tunable synthetic materials in complex geometric and synergistically incorporate natural ECM proteins to better recapitulate the ECM environment. 4

| Bioink
Another interesting application of dECM is its use as a 3D printable bioink. 3D printing, or additive manufacturing, is the creation of threedimensional structures layer-by-layer. The most common method of 3D printing bioinks is extrusion printing, during which the material is deposited via a mechanically controlled syringe into a desired geometry. 99 This technique is capable of creating complex architectures by depositing multiple materials with high spatial control. These complex geometries can be obtained through the design of a computer model to match a defect site identified via medical imaging modalities such  102 By utilizing two crosslinkers and two separate polymerization steps, the authors were able to fabricate a bioink that has a low enough viscosity to be printed and, following printing, increase the elastic modulus of the scaffold through a secondary polymerization step. dECM from different tissues (e.g.,liver, cardiac, skeletal) have been used with this tunable hydrogel system to formulate bioinks that replicate not only the physical but also the biological properties of the representative tissue. 102 Utilizing dECM on its own as a bioink for 3D printing is challenging due to its low viscosity and mechanical instability. 101,103 Bulk hydrogels formed using solubilized dECM only reach stiffnesses similar to or slightly better than that of pure collagen gels 71 and have very slow gelation times, ranging from 30 minutes to an hour. 104,105 Although increasing the weight percent of a dECM hydrogel can improve its stiffness, 106 using such methods for 3D printing is limited as the stiffness of the dECM bioink must be low enough to achieve a viscosity that enables dECM to be extruded through the needle. As such, multiple efforts have been devoted to enhancing the printability of dECM bioinks and the mechanical stability of printed scaffolds by combining them with secondary polymer frameworks, 101 mixing them with synthetic polymers, 102

| Particle aggregates
Cell-seeded dECM particles can be delivered into the defect site as a particle aggregate. 123  is best for a specific application. Additional guidelines that not only discuss the removal of nuclear material but also the retention of ECM molecules would contribute greatly to standardized decellularization procedures. Balancing the constraints between removing enough cellular material so as not to elicit an immune response while maintaining ECM composition to preserve bioactivity is a challenge that requires further investigation. Furthermore, tissue sources and storage conditions before being processed for decellularization also influence the quality of dECM, resulting in batch-to-batch differences even within the same tissue type. Universally accepted quality control measures for tissue sourcing and storage of dECM would prove beneficial in creating a more reliable and repeatable system. General decellularization guidelines that could widely be agreed upon by investigators will pave the way for a more standardized field of tissue decellularization. In addition, the development of methods to enhance the physicochemical properties of dECM while harnessing its native regenerative capacities will be the key to providing viable therapeutic applications for bone and cartilage tissue regeneration.

ACKNOWLEDGMENTS
We acknowledge support in bone and cartilage tissue engineering by the National Institutes of Health (P41 EB023833 and R01 AR068073). MM also acknowledges the National Science Foundation Graduate Research Fellowship Program.