Extracellular Matrix-derived Nanoparticles For Imaging And Immunomodulation

can travel.[9] Hyaluronan, a GAG not bound to a core protein, serves as an important matrixcomponent during embryonic development and in adult tissues, forms complexes withproteoglycans, and also generates empty spaces in which cells can migrate during woundhealing.[9] The elastic fiber components fibrillin and fibulin also mediate all sorts of cellularactivity, including normal lung and kidney development, matrix deposition, and activation ofTGF- and bone morphogenetic protein-7 in mice.[8]Table 1: List of common ECM components, their functions and where they can be found within the body.[5]FunctionExample LocationsFibrillar CollagensStructural, cell adhesion andBone, dermis, heart (I); cartilage (II);I, II, III, V, XIproliferation, mediates proteoglycangranulation tissue (III); basement membraneinteractions(V); articular cartilage, ear (XI)Nonfibrillar collagensStructural, angiogenesis,Kidney (IV); sclera and vasculature (VIII);IV, VIII, Xcompartmentalizes ECMhypertrophic cartilage (X)componentsAssociation collagensStructural, interaction with otherLiver (VI); basement membrane (VII);VI, VII, IX, XII, XIV, XIXECM components,cartilage (IX), skin (XII); blood vessels(XIV); muscle cells (XIX)Transmembrane collagensStructural, interacts with ECMSkin (XIII); cutaneous basement membraneXIII, XVIIcomponents, cell-matrix adhesion(XVII)MultiplexinsOrganizes ECM, inhibitsBasement membraneXV, XVIIIangiogenesis and tumor growthElastinStructural, provides elasticityLungs, blood vessels, dermisFibrillinsStructural, tissue homeostasisMicrofibrilsFibulinsStructural, interacts with ECMBasement membrane, blood vessels1, 2, 3, 4, 5, 6, 7components, modulates platelet1, 2adhesion, angiogenesis, formationof elastic fibersHyaluronanHyalectansAngiogenesis, cell motility, woundMost tissues; notably skin, cartilage, vitreoushealing, cell adhesionhumour, jointsStructural, interacts with ECMArticular cartilage (aggrecan), braincomponents, cell adhesion and(brevican, neurocan), blood vessels (versican)migrationFibronectinCellular adhesion, ECM assembly,Most tissuestissue injury and inflammation,angiogenesisLamininsCell adhesion, migration anddifferentiation3Basement membrane

These are just several examples in which extracellular matrix components regulatecellular activity, though there are many others. However, these brief examples illustrate theeffectiveness of ECM components to influence cell organization, proliferation, differentiation andmigration. The effect goes both ways. While the ECM provides physical and chemical cues thatdirect cell behavior, the cells also produce cytokines that remodel the ECM. This dynamicreciprocity is important in homeostasis and tissue repair, as will be discussed in followingsections.[29-31] With the ECM playing such a critical role in cell behavior, it makes sense toconsider this material in biomedical applications.1.2 ECM AS A BIOMATERIAL IN TISSUE REPAIRWith the extracellular matrix directing so many physiological phenomena, there has beenan increasing interest in isolating ECM material for use as a biomaterial in tissue repair. Upon adestructive stimulus, the wound repair process is triggered. Regardless of the type or location ofinjury, this process is highly directed by chemical signals, including numerous cytokines andgrowth factors, and generally involves three major overlapping steps: inflammation, cellproliferation and remodeling.[13][14] Immediately after injury, circulating platelets adhere toexposed collagen in the tissue. These platelets produce clotting factors that trigger the formationof a provisional fibrin matrix, as well as platelet-derived growth factor (PDGF) and transforminggrowth factor-beta (TGF- ) that signal the chemotaxis of neutrophils, macrophages, smoothmuscle cells and fibroblasts to the damaged region. TGF- also plays a role in stimulatingmacrophages to produce pro-inflammatory cytokines, including tumor-necrosis factor-alpha(TNF- ) and interleukin-1 (IL-1). Recruited neutrophils, as well as macrophages differentiatedfrom monocytes, actively remove dead tissue and foreign material through phagocytosis. Upontransition to the proliferation phase, the fibrin matrix is replaced with granulation tissue, andangiogenesis facilitated by vascular endothelial growth factor A (VEGFA) and fibroblast growthfactor 2 (bFGF) occurs. Macrophages can release soluble factors that trigger the differentiation of4

some fibroblasts into myofibroblasts, which work towards closing the wound and depositing newECM. In the last stage, the newly deposited matrix is remodeled by matrix metalloproteinases,which typically involves the conversion of type-III collagen to type-I, and the tissue achieveshomeostasis.This describes the typical wound-healing cascade in many tissues. However, injuriesinvolving high volumetric damage or non-regenerative tissues (i.e. cardiac) may result inalternative responses, including excessive or deficient healing.[14] Excessive healing, commonlyknown as fibrosis, leads to the deposition of too much matrix material that interferes with propertissue re-growth, resulting in an overall loss in tissue functionality. The build-up of this nonfunctional tissue is thought to lead to many diseases, including congestive heart failure, cirrhosisof the liver, transmission blockage following nerve injury and hypertrophic scarring. Deficienthealing can occur when infiltrating neutrophils degrade deposited matrix, for example throughcollagenase or elastase production, faster than it can accumulate. This is the main cause ofchronic ulcers that affect debilitated and elderly patients.[14]Treatment for both overhealing and underhealing in patients focuses on theadministration of key growth factors or healthy cells to the defective area in attempts toregenerate functional tissue. However, single-agent therapies have seen limited success.[13]Delivery of individual growth factors shows little impact due to the redundant nature of thewound healing response and short half-life of the agent at the wound site. Cells delivered bythemselves are difficult to isolate and deliver while maintaining viability, and they lack thephysical and chemical cues that help them organize and proliferate. Thus, there has been a recenttrend in integrating both growth factors and cells into carefully engineered, three-dimensionalscaffolds that help promote proper incorporated cell organization and mimic specific biologicalsignals. Synthetic scaffolds derived from polymers have been used in this manner. For example,surgical implantation of porous poly(lactide-co-glycolide) scaffolds, loaded with autologousmesenchymal stem cells, demonstrated cartilage regeneration in damaged sheep joints.[15] Aligned5

electrospun poly-caprolactone fiber meshes displayed improved cell proliferation, migration andorientation in neural regeneration.[16] These polymer scaffolds, extending but not limited topolystyrene, poly-l-lactic acid, polyglycolic acid and poly (acrylonitrile-co-methacrylate), havealso shown some success in repairing bone defects, nerves, liver, skin and blood vessels.[15-17]However, while these synthetic scaffolds are highly customizable, easily reproduced, and haveshown positive tissue regeneration effects, their structures are often too simplistic to properlymimic the in vivo cell environment. The extracellular matrix is a highly complex threedimensional combination of proteins and secreted molecules, each with a unique structurespecifically suited to their function. A simple porous or mesh scaffold cannot replicate theintricate features of numerous matrix proteins, such as the triple helix of collagen or the carefullycross-linked network of elastin. As a result, synthetic scaffolds lack the true bioactivity of nativematrix that promotes proper cell differentiation and behavior. Synthetic scaffolds have also beenshown to trigger the host immune response, typically resulting in fibrous encapsulation of thescaffold.[18][19]ECM-derived scaffolds show much more promise in tissue repair, as they address manyof the problems found in their synthetic counterparts. As discussed previously, the in vivo cellularenvironment consists of a highly dynamic and complex extracellular matrix. The structure andcomposition of this matrix is optimized to promote cell adhesion, organization, proliferation,differentiation and migration through a combination of physical and chemical cues. Thus, byderiving the scaffold material from tissue ECM itself, we can inherit the native structural andfunctional molecules, as well as the exact three-dimensional environment, that providebioactivity.[13] ECM-derived scaffolds have been successfully produced and demonstrateimproved tissue regeneration in a wide variety of applications. A decellularized equine cartilagematrix scaffold was introduced in a horse knee containing a critically sized osteochondral defect.After 8 weeks, significant bone and cartilage regeneration was observed.[20] Implantation ofporcine urinary bladder matrix (UBM) facilitated constructive healing of the esophageal wall in a6

dog model, evidenced by growth of functional and innervated neotissue.[21] Porcine UBM wasalso capable of inducing reconstruction of the temporomandibular disk in vivo.[22] Tube-shapedECM scaffolds from porcine small intestine were implanted into patients suffering from highgrade dysplasia of the esophagus, showing rapid remodeling in the form of new epithelium andsubmucosal layer, and porcine small intestine submucosa (SIS) scaffolds implanted in a caninevolumetric muscle loss model showed formation of vascularized, functionally innervated skeletalmuscle.[21][23][24]Several FDA approved, commercially available ECM products are highlighted in Table2.[21][25] The production of these scaffolds requires careful washing of whole organs or tissuesections with a combination of detergents, acids, enzymatic solutions and other solvents. Thesesolutions work towards removing unwanted cellular material and solulizable proteins whilemaintaining the physical structure and key functional components of the organ or tissue. Ideally,decellularized ECM scaffolds retain all of the structural and functional proteins andpolysaccharides of native tissue, including collagens, GAGs, fibronectin and laminin ligands thatpromote cell adhesion and growth in a natural, organized three-dimensional space. These ECMcomponents are largely conserved across and within species and are relatively non-immunogenic.By removing the cellular content susceptible to immune recognition, decellularized ECMscaffolds can circumvent immunological complications witnessed by synthetic scaffolds.Tissues within the body vary greatly in morphology, cellular density, protein ratios andcomposition. The source of the ECM also dictates physical properties such as rigidity, porosityand topography. As a result, the optimal decellularization process may differ between each tissuetype. Most processes utilize a combination of the chemical decellularization agents mentionedabove, and physical disruption of the tissues to achieve acceptable decellularization. However, itis important to consider the impact of these treatments on the mechanical and physiologicalproperties of tissues. The physical architecture of proteoglycans, collagen fibers and otherproteins within tissues contributes greatly to its mechanical properties, and they also provide the7

matrix with functionality. Destruction of any component through too harsh a decellularizationprocess can lead to diminishing performance of the decellularized material. Badylak et al.provides a comprehensive summary of potential effects of many decellularization agents andprocesses.[26] Striking a balance between optimal cell removal and preservation of critical ECMcomponents is necessary for producing functional ECM materials.Table 2: Examples of commercially available scaffolds composed of ECM [21][25]ProductMaterialFormUseCompanyAlloDermHuman skinDry sheetAbdominal wall, breast, head andneck reconstruction, graftingLifeCellCollamendPorcine dermisDry sheetSoft tissue repairBardCuffPatchPorcine SISHydrated sheetSoft tissue reinforcementArthrotekMatristemPorcine UBMDry sheet;powderSoft tissue repairACellOasisPorcine SISDry sheetPartial and full-thickness burnsHealthpointPermacolPorcine skinHydrated sheetSoft connective tissue repairTissue d sheetSoft tissue repairSynovis SurgicalXenformFetal bovine skinDry sheetColon, rectal, urethral repairTEI BiosciencesZimmerCollagen PatchPorcine dermisDry sheetOrthopedic applicationsTissue ScienceLaboratoriesThis section served to highlight one of the prominent uses of extracellular matrix-derivedbiomaterials. Widely considered for tissue regeneration applications, ECM scaffolds showpromise in their ability to organize cells and produce functional tissue patches for wound repair.However, the extracellular matrix contributes to many more physiological phenomena within thebody, with host immunological response of particular interest.1.3 ECM IN IMMUNOLOGYHinted at earlier, immune cells play a critical role in every step of the wound healingprocess, with major players including neutrophils, eosinophils, basophils, dendritic cells,monocytes and macrophages, T cells and B cells. The recruitment of these cells and their8

behavior at the injury site are heavily influenced by local and systemic cascades of compleximmune events.[27] These events are often triggered and controlled by secreted cytokines or othersoluble mediators within the local tissue microenvironment, among which include numerousextracellular matrix components. For example, mindin is a secreted ECM protein that serves as anopsonin for macrophage phagocytosis of bacteria, and biglycan found commonly in bone,cartilage and tendon matrix can stimulate production of pro-inflammatory cytokines inmacrophages.[28][29]ECM remodeling plays a particularly important role in regulating the immune response.The breakdown of ECM components, either through interaction with adhered pathogens or byspecific proteases such as matrix metalloproteinases (MMPs), exposes cryptic binding sites thatfacilitate the activation of the innate immune response.[29] The detection of these sites by patternrecognition receptors on immune cells leads to the initiation of an inflammatory response thatenables infiltration of immune cells to the injured region. Among the major contributors are lowmolecular weight hyaluronan (LMWHA) fragments, which induce the release of proinflammatory cytokines TNF- and IL-1 by macrophages.[29] Fibronectin fragments expressingan extra domain A have demonstrated activation of receptors that induce responses similar tothose triggered by bacteria.[30] In addition to initiating the inflammatory response, ECM fragmentsalso act as chemoattractants that further promote cell infiltration.[31] For example, specificcleavage of collagen I by MMPs generates fragments that contribute to neutrophil recruitment inthe early stages of inflammation. Elastin fragments demonstrate similar chemotactic propertiesfor monocytes. The release of cytokines by infiltrating cells also helps to tailor the production ofMMPs and subsequent generation of ECM fragments, re-illustr

ii ABSTRACT The extracellular matrix (ECM) is a complex component of tissue that includes collagens, glycoproteins, proteoglycans, and elastic fibers.