The Self-Assembling Process and Applications in Tissue ...€¦ · engineering exist, termed the...

The Self-Assembling Process and Applicationsin Tissue Engineering

Jennifer K. Lee,1,3 Jarrett M. Link,1,3 Jerry C.Y. Hu,1 and Kyriacos A. Athanasiou1,2

1Department of Biomedical Engineering, University of California, Davis, California 956162Department of Orthopaedic Surgery, University of California, Davis, California 95616

Correspondence: [email protected]

Tissue engineering strives to create neotissues capable of restoring function. Scaffold-freetechnologies have emerged that can recapitulate native tissue function without the use of anexogenous scaffold. This review will survey, in particular, the self-assembling and self-orga-nization processes as scaffold-free techniques. Characteristics and benefits of each processare described, and key examples of tissues created using these scaffold-free processes areexamined to provide guidance for future tissue-engineering developments. We aim toexplore the potential of self-assembly and self-organization scaffold-free approaches, detail-ing the recent progress in the in vitro tissue engineering of biomimetic tissues with thesemethods toward generating functional tissue replacements.

The field of tissue engineering aims to reca-pitulate native tissue function toward re-

placing damaged or diseased tissues and organs.The tissue-engineering paradigm is traditional-ly composed of living cells, scaffolds, and sig-nals. The scaffolds used in tissue engineering arehighly diverse, ranging from synthetic to naturalpolymers and hydrogels to woven meshes. Theemergence of scaffold-free processes—tissue-engineering platforms that do not require theaddition of an exogenous scaffold—have ex-panded the capabilities of the field. Scaffold-free techniques have been successfully used inengineering musculoskeletal (DuRaine et al.2015) as well as cardiovascular, metabolic, andcorneal tissues (Athanasiou et al. 2013). Withinscaffold-free approaches, two primary, thermo-

dynamically driven modalities have been de-scribed: self-organization and self-assembly (Fig.1) (Athanasiou et al. 2013).

In this review, a summary of the progress intissue engineering will be covered. Although awide variety of cell sources and stimuli can beapplied in tissue engineering, the focus of thiswork will be related to scaffold-free approaches.Herein we focus specifically on the in vitro tis-sue-engineering techniques that generate bio-mimetic tissues (i.e., those that recapitulate na-tive tissue). In vivo tissue engineering can alsoinclude the injection of cell suspensions andmatrix-associated cells for continued develop-ment and maturation in an in vivo environ-ment, but are not extensively covered here.Finally, the progress in engineering functional

3These authors contributed equally to this work.

Editor: Joseph P. Vacanti

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tissues with a particular emphasis on self-as-sembling and scaffold-free techniques to treata wide range of diseases will be highlighted.

TISSUE ENGINEERING

Classical tissue-engineering approaches com-bine cells, biomaterials, and bioactive stimulito generate robust implants capable of restoringthe structure and function of tissues damagedby trauma, pathology, or age. Often referred toas the tissue-engineering “triad,” this founda-tional concept of cells, scaffolds, and signalshas informed strategies for numerous out-comes, such as bone regeneration followingcomplex fractures or the development of vascu-lature in vitro to replace diseased vessels (Cha-parro et al. 2015; Shimizu et al. 2015). Signifi-

cant advances in the field have resulted from thisparadigm. However, scaffold-free techniqueshave emerged, which may better apply to cer-tain tissues in which cells may not require exog-enous scaffolds. In this manner, biomimeticand functional tissues of clinically relevant di-mensions may be created. Scaffold-free tech-niques may thus improve the likelihood for clin-ical translation, which remains the ultimate goalof the field.

SELF-ASSEMBLING PROCESS

One promising tissue-engineering technique,especially in cartilage tissue engineering, is theself-assembling process (Hu and Athanasiou2006; Athanasiou et al. 2013). Without the in-fluence of external energy, self-assembly mimics

SAPLoad

SignalsCells

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Figure 1. Scaffold-free tissue engineering. The tissue-engineering paradigm typically consists of cells, scaffolds,and signals. The benefits of scaffold-free approaches have motivated the use of only cells and signals. Depictedhere are example modalities within this paradigm, using homogeneous or heterogeneous cell populations inconcert with mechanical (e.g., compressive loading) and/or biochemical stimuli (e.g., transforming growthfactor b1 [TGF-b1] or sucrose) to enhance neotissue properties. Two distinct forms of scaffold-free tissueengineering exist, termed the self-assembling process (SAP) and the self-organization technique (SOT). Al-though self-organization requires the exogenous input of energy, self-assembly occurs in a closed system.

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developmental events to generate functionalcartilaginous tissue with characteristics remi-niscent of native tissue (Fig. 2). Nonadherentculture substrates—typically agarose—supporthigh-density chondrocyte seeding, prevent cellattachment, and encourage cell–cell interac-tions, facilitating the chondrogenic phenotype.Indeed, cell adhesion is up-regulated in chon-drocytes (Ofek et al. 2008) during the initialphase of the self-assembling process, reminis-cent of mesenchymal condensation during car-tilage morphogenesis (Tavella et al. 1994; De-Lise et al. 2000). For example, increased levels ofN-cadherin on the cell surface can minimizefree energy, according to the differential adhe-sion hypothesis as described below. Critically,no external energy is provided to the systemduring self-assembly (i.e., it is a closed system).Since the development of the self-assemblingprocess, substantial efforts toward understand-ing the mechanisms of action have been inves-

tigated to refine the technique further. In par-ticular, the differential adhesion and differentialinterfacial tension hypotheses have been used todescribe the self-assembling process.

Informing the mechanism of the self-as-sembling process, the differential adhesion hy-pothesis posits that tissues minimize free energyvia cell–cell binding. The type and number ofadhesion proteins present on a cell surface giverise to cell–cell interactions. Correspondingly, amass of cells behave analogously to a liquid andwill minimize its surface tension, known as tis-sue surface tension. This tension will determinethe sorting behavior of cells in a mixed popula-tion, as cells with higher surface tension will sortto the center, maximizing intercellular adhe-sion. Consequently, tissue surface tension willbe minimized. Similarly, in the self-assemblingprocess, the nonadherent substrate forces a ho-mogeneous cell population to minimize freeenergy via cell–cell adherence, facilitated by in-

A B C D

E F G H

Figure 2. The phases of self-assembly and cartilage development. Self-assembling articular cartilage forms in amannerreminiscent ofcartilagemorphogenesis. In thefirstphaseof self-assembly (A),ahigh-densitycell solutionis seeded in a nonadherent well. During phase 2 (B), minimization of free energyoccurs as cells bind to one anoth-er via cell-adhesion receptors like N-cadherin. In phase 3 (C), extracellular matrix synthesis is up-regulated.Finally, the engineered tissue matures as distinct regions develop and native tissue-like functional properties areapproached (D). Similarly, the process of long bone formation is mediated first by mesenchymal condensation(E). Robust matrix deposition begins as cells differentiate (F), following chemotactic agents to elongate the bonein opposite directions (G). Over time, the core forms a site for vascularization to become bone (H ).

Self-Assembly and Tissue Engineering

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creased levels of N-cadherin and other adhesionmolecules (Ofek et al. 2008; Raghothaman et al.2014). The development of this continuous ag-gregate is critical to neotissue development, re-flects the process of mesenchymal condensa-tion, and can potentially drive chondrogenicgene expression (Ofek et al. 2008; Raghothamanet al. 2014). The mechanism of self-assemblycan thus be partly explained by the differentialadhesion of surface-bound molecules.

Another mechanism that may contribute tothe self-assembling process is differential inter-facial tension. Cortical cell tension, driven bycontractility of the actin cytoskeleton and cellsurface tension, has been implicated in cell sort-ing (Brodland 2002; Krieg et al. 2008; Manninget al. 2010). As in differential adhesion, theminimization of free energy drives the cellularbehavior in the differential interfacial tensionhypothesis, with cell sorting dictated by forcesgenerated by the cell cytoskeleton and at the cellmembrane. Specifically, cells generating similartensions will tend to aggregate as comparedwith those showing different tensions. The dif-ferential adhesion and differential interfacialtension hypotheses may be related (Manninget al. 2010). Increased understanding of the rel-ative contributions and/or interactions of theseprocesses would help elucidate self-assemblymechanisms.

Drawing from knowledge of developmentalbiology, biomedical engineers can use the self-assembling process to drive cell sorting, geneexpression, and tissue formation in a mannersimilar to morphogenesis. Our enhancedunderstanding of underlying mechanisms inself-assembly will drive the rational selectionof agents that can positively modulate the for-mation of tissues with increased functionalproperties.

OTHER SCAFFOLD-FREE TECHNIQUES

Although promising, self-assembly is but oneexample of a variety of scaffold-free tissue-engi-neering methodologies that have gained tractionand present unique advantages. For instance,scaffold-free systems do not produce syntheticdegradation by-products, can maintain the

rounded phenotype of cells such as chondro-cytes, and do not require harsh processingchemicals involved in scaffold production(Huey et al. 2012). Many of these alternatetechniques can be grouped within the self-orga-nizing tissue-engineering paradigm, which is adistinct subset of scaffold-free tissue engineer-ing that requires external energy input into thesystem for tissue formation (Fig. 1). Moreover,these varied techniques are able to form tissueswith a range of dimensions (Fig. 3). Within thisparadigm exist methods such as pellet culture,aggregate culture, and cell-sheet engineering.

Pellet culture is a fundamental method ofscaffold-free tissue engineering. Requiringsubstantial external energy, pellet culture is me-diated by centrifugation of cells inside a coni-cal-shaped tube. Subsequently, cell pellets arecultured in medium specific to a certain tissue,driving cellular differentiation to achieve tissue-specific gene expression and extracellular matrix(ECM) deposition. Bone, liver, and cartilage tis-sues have all been formed in this manner (Onget al. 2006; Gurkan et al. 2011; Diekman et al.2012). Despite the ability to create tissues withrelevant ECM components, pellet culture fails tomeet many clinical translation criteria, such asrobust mechanical properties and anatomicallyrelevant geometries and dimensions. Thus, bio-medical engineers interested in translationalmedicine typically focus on strategies otherthan pellet culture for use in in vitro biomimetictissue engineering.

Avariety of methods exist to generate cellu-lar aggregates or spheroids, and these terms areoften used interchangeably in the literature.Similar to pellet culture, aggregate culturemaintains cells in a 3D environment and canbe used to enhance tissue-specific gene and pro-tein expression. Multiple methods are used toinduce aggregate formation: hanging drop,round, or v-bottom well-plates, and rotationalculture. In hanging-drop culture, cell suspen-sions are placed in droplets on the lid of a cul-ture plate; after inverting, gravity assists cells incoalescing at the base of the drop, formingan aggregate. Similar to self-assembly, thewell-plate method uses nonadherent round-or v-bottom plates to statically induce aggregate

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formation. Finally, by subjecting a cell popula-tion to rotational culture in the presence of tis-sue-specific growth factors, aggregation and dif-ferentiation is encouraged through cell–cell andcell–matrix interactions. Subsequently, relevantECM proteins are synthesized and neotissue be-gins to form. Although these methods are ableto form aggregates that can serve as importanttools in understanding mechanisms of differen-tiation and phenotypic maintenance, they maynot be suitable for in vitro biomimetic tissueengineering.

Aggregate or spheroid culture, like pelletculture, produces small-diameter cellular aggre-gates and may not be a feasible approach toengineer mechanically functional tissues whenused alone, likely because of the limited numberof cells in each aggregate. If the approach is partof a larger tissue-engineering effort (Murphyet al. 2013a), however, then it can be used toengineer anatomically relevant tissues. For in-stance, aggregate culture can encourage bothdifferentiation of stem cells and redifferentia-tion of passaged primary cells, followed by their

application in other tissue-engineering meth-ods, such as self-assembly, to create larger con-structs (Murphy et al. 2015). Bioprinting hasemerged as a method to use directly these smalldiameter aggregates as “bioink,” which are fedthrough a small nozzle and deposited in specificlocations during 3D printing. Additionally, withfusion of multiple spheroids, larger continuousconstructs can be generated. Indeed, applicationof compression to mesenchymal stem cell(MSC) aggregates within a mold has been ableto generate large and continuous cartilage con-structs (Bhumiratana et al. 2014). Thus, al-though the tissues formed by both pellet andaggregate/spheroid culture may not reach clin-ically relevant dimensions, these methods areimportant for the phenotypic maintenance ofmany cell types and can be integrated as part of alarger tissue-engineering process.

Cell-sheet engineering is a scaffold-free ap-proach using external manipulations and ther-mal energy to form 3D tissues. Cells are culturedin monolayer on functionalized substrates oron a thermoresponsive polymer. In the case of

Pellet

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Figure 3. Energy usage and achievable dimensions in scaffold-free processes. Scaffold-free processes differ withrespect to the energy required for tissue formation and the dimensions that can be achieved. Spheroid oraggregate formation based on the methods of hanging drop, round, or v-bottom well plates, or rotationalculture requires minimal energy and forms aggregates of small dimensions. Pellet culture requires substantialenergy in the form of centrifugation and similarly forms small-diameter cellular aggregates. Finally, self-assem-bly and larger self-organization techniques such as cell-sheet engineering are able to generate sizeable constructsof clinically relevant dimensions.



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direct cellular attachment, cells are removedvia enzymatic cleavage of their cell–matrix at-tachments to the surface; to avoid enzymaticdetachment, cell scraping can also be used. Thecell sheet can be draped over a mandrel to form,for example, a hollow vascular structure(L’Heureux et al. 1998). Alternatively, thermo-responsive polymers have been developed inwhich, subject to a change in temperature,the polymer changes conformation and inducesdetachment of the cell layer (Nishida et al.2004a). The polymer method avoids the use ofmechanical or enzymatic cell detachment topreserve the cell-matrix-binding interactions.Further, fusion of multiple cell-sheet layers canbe used to generate tissues of greater thicknessto recapitulate the zonal architecture of targettissues (Shimizu et al. 2006). As comparedwith pellet and aggregate culture techniques,cell-sheet engineering can form substantiallylarger structures.

ADVANTAGES OF SCAFFOLD-FREETECHNIQUES

The process of engineering tissues using scaf-fold-free techniques shows distinct advantages.For instance, scaffold-free methods may pro-mote native tissue integration, facilitate en-hanced matrix deposition and, thus, more directmechanotransduction, and avoid the release ofharmful by-products. In the case of self-assem-bled articular cartilage, the neotissue has anabundance of cells at the construct edge, whichlikely encourages tissue growth into native car-tilage and promotes integration (Athens et al.2013). Direct cell–ECM interactions in self-as-sembled cartilage prevent cells from experienc-ing stress shielding, which has been shown toimpede matrix production and remodeling(Hu and Athanasiou 2006). Scaffold-free ap-proaches can avoid issues of cytotoxicity causedby the harsh processing conditions—particu-late-leaching polymerizing chemicals and plas-ticizers, for example—required for manufac-turing of some biomaterials (Vunjak-Novakovicet al. 1999). Without the use of foreign materials,scaffold-free processes can reduce the likelihoodof a foreign material-associated immune re-

sponse; this response is known to limit the du-rability of the implanted construct and poten-tially compromise the health of the patient(Avula et al. 2014). Biocompatibility issues, as-suming a cell source is chosen appropriately, aremitigated in the scaffold-free paradigm becausesynthetic materials are avoided. Scaffold-freetechniques thus possess several advantages foruse in tissue-engineering strategies.

Yet, there are limitations associated withthe scaffold-free paradigm. If scaffold-free tech-niques cannot match native tissue functionalproperties at implantation, clinical translationis complicated. For instance, if engineered car-tilage with inferior mechanical properties wereplaced into a femoral focal defect, stress concen-trations could develop within the engineeredcartilage and at the native-engineered tissueinterface. Especially in the case of load-bearingtissues, mismatch in functional properties couldresult in destruction of the implant if not ap-propriately unloaded postsurgery. Physiciansmay have to devise rehabilitation regimens spe-cific to scaffold-free constructs to improve theclinical viability of these approaches. To ensureclinical success of scaffold-free approaches, ap-plication of biomimetic stimuli (Huey et al.2012; Athanasiou et al. 2015; Makris et al.2015) is crucial to driving the development offunctionally relevant neotissues.

Scaffold-free approaches often require ahigh cell-seeding density, which brings intoquestion the issue of cell sourcing. It is impor-tant to note that certain cell types are anchor-age-dependent and require the presence of anexogenous scaffold at seeding; these cells maynot be suitable for a scaffold-free approach. Pri-mary autologous cell-harvesting techniques of-ten do not meet cell number requirements andcan be associated with donor site morbidity(Amini et al. 2012). Passaged primary autolo-gous cells are available in higher quantities, butmay be limited by expansion potential and donot address the issue of donor site morbidity.Furthermore, passaged primary cells may ex-perience epigenetic changes that affect geneexpression (Darling and Athanasiou 2005),leading to an altered ECM profile and, poten-tially, reduced functional properties. Stem cells,

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such as MSCs and dermis-derived stem cells,represent an attractive cell source as they aremore widely available and have shown the abil-ity to differentiate into many different cell andtissue types. In many cases, however, stem cellsdo not fully differentiate into the target cell,which may affect the ultimate properties of gen-erated neotissue. Moreover, differentiation pro-tocols are often complex and may result in non-homogeneous cell populations. Cell sourcingremains a critical issue in tissue engineeringand must be solved to improve the prospectsof clinical translation for scaffold-free ap-proaches.

Tissue-specific design criteria must beconsidered when choosing a particular tissue-engineering approach. In some cases, a self-assembly model may be ideal. For example,chondrocytes are particularly amenable to theself-assembling process: passaged rabbit cellstreated with a combination of bioactive (i.e.,transforming growth factor b1 [TGF-b1] andchondroitinase-ABC) and mechanical (i.e., hy-drostatic pressure) stimuli can create tissueswith clinically relevant dimensions with a ten-sile stiffness reaching 6.3 MPa (Arzi et al. 2015).On the other hand, a scaffold may be necessaryfor recapitulating the structure–function rela-tionship in a large segmental defect of bone, asosteoblasts require a scaffold for survival. Re-searchers must continue to refine these tech-niques and further define native tissue structureand function to develop the most effectivetissue-engineering strategies. We would like toemphasize the importance of biomimetic, func-tional tissue engineering, which will be dis-cussed at length later in this article.

SCAFFOLD-FREE TISSUE ENGINEERINGIN THE CLINIC

Although obstacles still exist, promisingly, a fewscaffold-free processes have reached the clinic.Autologous chondrocyte implantation (ACI),although not an in vitro biomimetic tissue-en-gineering method, was established in 1994 as aclinical treatment for focal articular cartilagedefects (Brittberg et al. 1994). Requiring twosurgical steps—one to harvest tissue, the other

to implant cells into a defect—ACI has beenshown to be superior to other surgical tech-niques such as mosaicplasty (Bentley et al.2012), although other studies have shown thatACI is no better functionally than other cheaperorthopedic procedures like microdrilling or mi-crofracture (Tuan 2007; Lim et al. 2012). In ACI,the defect may be filled with reparative tissuesynthesized by implanted articular chondro-cytes; this fibrocartilaginous tissue shows func-tional properties inferior to native hyaline artic-ular cartilage. Thus, ACI may lead to a limitedrepair response. Furthermore, this techniquemay be less effective in older patients, who aremore likely to suffer from musculoskeletal mal-adies as a result of the reduced proliferative andregenerative capabilities of aged cells. However,the clinical development pathway for ACI caninform the translation of scaffold-free in vitrobiomimetic tissue-engineering techniques.

In a biomimetic scaffold-free approach,cell-sheet-engineered vasculature for end-stagerenal disease patients undergoing hemodialysishas shown promise in clinical trials (McAllisteret al. 2009). Scaffold-free techniques for vascu-lar tissue engineering have shown a higher pro-pensity for achieving native tissue structure andcan withstand higher burst pressures (L’Heu-reux et al. 2006). Additionally, removing theinfluence of a biomaterial reduces the chanceof dangerous thrombosis formation and leuko-cyte activation. In this clinical trial, concernsrelated to unrolling of the cell sheet arose(McAllister et al. 2009). Reported cases of un-rolling identify a critical shortcoming of cell-sheet engineering for vascular tissue engineer-ing; methods to ensure fusion of the cell sheetvia improved nutrient transport may promotelong-term closure (McAllister et al. 2009). If theissues of extended culture times and the poten-tial for unrolling can be solved, in vitro cell-sheet engineering for diseased vasculature mayexperience broad clinical use.

In a biomimetic tissue-engineering ap-proach akin to self-assembly, ISTO Technologiesin concert with Zimmer Biologics has producedscaffold-free neocartilage that successfully com-pleted phase I/II trials and continues on thepath of commercialization. The RevaFlex prod-



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uct is generated from a high-density cell culturederived from juvenile donors; the investigatorspreviously determined that chondrocytes de-rived from younger donors possessed enhancedneocartilage-generation potential (Adkissonet al. 2001). RevaFlex was implanted into 12patients in a phase I/II clinical trial initiatedin late 2006 (McCormick et al. 2013). In thisclinical study, clinical efficacy was assessed viapatient-reported outcome measures, magneticresonance imaging (MRI), and elective second-look arthroscopy and biopsy (McCormick et al.2013). Of second-look arthroscopies (nine of 12patients), RevaFlex reportedly resulted in“�66% of lesions show[ing] gross anatomicalcartilage characteristics with adequate fill withpromising histologic characteristics” (Mc-Cormick et al. 2013). Additionally, no immuno-logical response to allograft neocartilage wasfound; these findings of a lack of graft rejectionaddress many clinical concerns associated withusing juvenile cells from an allogeneic source forgraft production (Lu et al. 2005; McCormicket al. 2013). RevaFlex received Food and DrugAdministration (FDA) approval for an investi-gational new drug (IND) application beforeclinical trials and is thus regulated as a biologic,rather than a device. Ultimately, the commercialsuccess of RevaFlex paves the way in regulatoryaspects of scaffold-free tissues—particularly interms of cell sourcing and immune rejection—and will inform the clinical development of car-tilage constructs generated from self-assemblingor self-organization techniques.

Although translation of scaffold-free tech-niques to the clinic is limited, these advancesillustrate the required regulatory path for clini-cal success. For instance, cell-based therapieslike ACI may provide some insight into clinicaltranslation of scaffold-free tissue-engineeredtherapies. Although these cell therapies do notfall into the scope of biomimetic in vitro tissueengineering, they are informative examples ofclinical translation of cell-derived products. Re-vaFlex is an example of an in vitro engineered,biomimetic articular cartilage tissue replace-ment; the path to FDA approval of RevaFlexhighlights the challenges of clinical translationof such a product. Given the similarities between

the self-assembling process and the methodsused to generate the RevaFlex graft, the RevaFlexpathway provides guidance for clinical trans-lation of a self-assembled articular cartilagereplacement. Although continued refinementof these scaffold-free tissue-engineering tech-niques is required, lessons from approved, clin-ically available cell-based and biomimetic tis-sue-engineered therapies should be noted.

FUNCTIONAL TISSUE ENGINEERING

Biomimetic tissue engineering aims to generatefunctional tissues in vitro to achieve certainproperties of target tissues; these properties de-pend on the tissues’ primary roles in vivo. Thebenchmarks for engineered tissues are derivedfrom their native counterparts. For instance,musculoskeletal tissue engineers focus onforming tissues with appropriate mechanicalstrength and stiffness to function in the tissues’native load-bearing capacities. Hepatic tissueengineers focus on forming tissues able to ex-press specific enzymes and proteins necessaryto mimic liver function. Some of these com-monly reported parameters are presented inTable 1. Scaffold-free tissue-engineering tech-niques aiming to recapitulate native tissuefunction should bear in mind these reportedproperties and strive to bring them closer tonative tissue values. The following sectionshighlight recent progress in engineering mus-culoskeletal, cardiovascular, liver, and cornealtissues using scaffold-free biomimetic in vitrotissue engineering.

PRIMARILY MECHANICALLY FUNCTIONALTISSUES

Bone

Bone is a mineralized collagen matrix responsi-ble for primary load bearing in the musculo-skeletal system. In addition, bone serves as ananchorage point for ligaments, tendons, andmuscles to facilitate movement. Clinical appli-cations of tissue-engineered bone often relate tothe repair of critical-sized defects that cannotheal on their own. The primary objective of tis-

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sue engineers, therefore, is to engineer bonewith compressive and tensile stiffness andstrength reminiscent of native bone. Addition-ally, indicators of vascularization and minerali-zation are used to assess bone quality. Scaffold-free approaches used in bone engineering areprimarily cell-sheet-based, with few using ag-gregate culture.

Osteogenic cell sheets are used to generatetissue-engineered bone, targeting the compres-sive and mineral properties of native bone. Os-teogenically differentiated MSC sheets rolledinto cylindrical constructs showed mineraliza-tion and a compressive strength of �1.6 MPa(Ma et al. 2010). The cell-sheet structure canbe placed into critical-sized defects to facilitatenew tissue formation, although additional val-idation of the mechanical properties of the new-ly formed bone is needed (Akahane et al. 2010).In vivo implantation promoted expression ofbone genetic markers (i.e., collagen type I, os-teocalcin, and osterix) in cell-sheet cocultures ofosteogenic stromal cells and endothelial cells ascompared with monocultures of osteogeniccells (Pirraco et al. 2014). Although bone-tissueengineering with scaffold-free cell sheets hasbeen attempted, most of these studies do notassess the mechanical properties of formed tis-sues. As such, additional work analyzing thefunctional mechanical properties of cell-sheet-based, in vitro tissue-engineered bone is needed.

Few studies examine aggregate or spheroidculture for engineering large bone-tissue con-structs, as aggregate studies are primarily usedfor differentiating progenitor cells into osteo-

genic cells for future application in larger scaf-fold-free or scaffold-based techniques. Variousscaffold-free aggregate techniques are able togenerate mineralizing spheroids, but larger con-structs are not often formed in subsequent steps(Hildebrandt et al. 2011). Studies using scaf-fold-free processes from cellular differentiationto large construct formation would improve ourunderstanding of the potential applicability ofscaffold-free techniques in bone-tissue engi-neering.

Cartilage

Articular cartilage is a load-bearing tissue thatalso serves to facilitate the smooth translation ofdiarthrodial joints. Unlike bone, however, artic-ular cartilage does not heal itself; clinical appli-cation of tissue-engineered articular cartilage,then, serves to replace degenerated cartilage tis-sues. The primary benchmarks of articularcartilage tissue engineering are sufficient com-pressive and tensile properties. In addition, lu-brication properties and integration ability aredesirable. Scaffold-free methods, including theself-assembling process, cell-sheet engineering,and aggregate culture, have been applied in ar-ticular cartilage tissue engineering towardachieving the mechanical properties of nativetissues.

The self-assembling process as a scaffold-free tissue-formation technique has been exten-sively studied (Hu and Athanasiou 2006) forengineering of articular cartilage. With thisscaffold-free process, primary articular chon-

Table 1. Advantages of scaffold-free techniques

Target tissues Examples of commonly reported parameters

Primarily mechanical function (e.g., musculoskeletal, cardiovascular)Bone Compressive and tensile stiffness, strengthArticular cartilage Compressive and tensile stiffness, strengthTendon, ligament Tensile stiffness, strength, maximum forceHeart Electrical conductance, contractilityVessel Burst pressure, recapitulation of layered structures

Nonmechanical tissues (e.g., metabolic, corneal)Liver Albumin, a-1 antitrypsin, P450 cytochromeCornea Transparency, refractive power



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drocytes treated with matrix cross-linking en-zyme lysyl oxidase-like protein 2 (LOXL2) cangenerate tissues achieving compressive andtensile stiffnesses up to 220 kPa and 2.3 MPa,respectively (Makris et al. 2014). Using combi-nations of bioactive stimuli (i.e., TGF-b1,chondroitinase-ABC) and/or mechanical stim-uli (i.e., hydrostatic pressure), tissues with ten-sile stiffnesses up to 6.3 MPa and 2.1 MPa canbe generated from passaged leporine and por-cine cells, respectively (Murphy et al. 2013b;Arzi et al. 2015). Self-assembled articular carti-lage derived from primary chondrocytes cul-tured in chondrogenic-inducing medium ex-pressed superficial zone protein, imparting thetissue with lubrication capacity and frictionalproperties approaching those of native articularcartilage (Peng et al. 2014). The self-assemblingprocess, thus, can be used to engineer nativetissue-like neocartilage. Future work investigat-ing the implantation of these lubricated andmechanically robust tissues in an orthotopiclocation would lead the field in generating func-tional tissue-engineered cartilage.

Other scaffold-free methods for cartilage tis-sue engineering include cell-sheet engineeringand aggregate culture. In a technique similar toself-assembly, chondrocytes placed in a nonad-herent well self-aggregate into “cartilage tissueanalogs” expressing collagen type II and nativetissue levels of glycosaminoglycan (Kraft et al.2011). Moreover, these tissues were reported tohave an equilibrium compressive Young’s mod-ulus on par with native tissue (Mohanraj et al.2014). Cell-sheet engineering has also been usedto engineer cartilage tissues; contraction of anMSC-derived cell sheet led to a tensile strengthof �1.2 MPa (Ando et al. 2008). Scaffold-freeaggregate and cell-sheet culture methods areable to generate cartilage tissues that are me-chanically viable. Additional investigation usingthese methods to achieve native tissue-like me-chanical properties as well as lubrication wouldbenefit efforts to engineer scaffold-free cartilage.

Ligaments and Tendons

Ruptured ligaments and tendons are oftenrepaired with autologous or cadaver-derived

grafts, which are limited in supply. Tissue engi-neering of ligaments and tendons thus aims tofill this need for mechanically robust replace-ments. Engineered ligaments and tendons arecommonly assessed for various tensile testingcriteria, such as tensile stiffness, strength, andforce. Scaffold-free methods used in formingthese tissues are akin to cell-sheet engineering,whereas spheroids have been used to a limitedextent.

Cell-sheet methods in ligament and tendonengineering rely on the strong contractile forcesof seeded cells. Monolayers of stromal cells cul-tured on laminin-coated substrates detachedand organized into rod-like tissues anchoredby silk sutures; these tissues reached a tensileforce of 0.26 N, a tangent modulus up to15.4 MPa, and a tensile stress of 2.11 MPa(Hairfield-Stein et al. 2007). Rolling of atenocyte-derived cell sheet stimulated withascorbic acid and connective tissue growthfactor achieved a reported tensile stiffness of�200 N/mm2 (MPa) and strongly expressedcollagen type I and tenomodulin (Ni et al.2013). Additional work exploring layering orbundling of scaffold-free ligaments and tendonsto achieve mechanically robust tissues with larg-er geometries would move the field closer to areplacement ligament or tendon.

The use of spheroid culture in tendon andligament engineering is limited primarily to dif-ferentiation and phenotypic maintenance ofcells for seeding on woven scaffolds. Scaffold-free spheroids derived from anterior cruciateligament cells became smaller over time, butincreasingly expressed collagen and tenascin Cand could colonize scaffolds (Hoyer et al. 2015).Hanging-drop spheroid culture of tenocytessimilarly enhanced expression of tendon-asso-ciated genes (e.g., collagen type III, scleraxis) ascompared with monolayer cultures, with ascor-bic acid, insulin, and TGF-b1 achieving higherexpression levels than TGF-b1 and insulin-likegrowth factor 1 (IGF-1) use (Theiss et al. 2015).Although the use of spheroids in tendon andligament engineering shows the ability of scaf-fold-free culture to enhance relevant gene ex-pression, these studies did not examine thefunctional mechanical properties of engineered

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tissues. As such, additional work exploring themechanical properties achievable through acombination of scaffold-free and scaffold-basedmethods for tendon and ligament engineeringwould benefit the field.

Cardiac

Cardiac tissues function primarily in contrac-tion, relying on rapid electrical conductanceto synchronize the heartbeat. In cases of myo-cardial infarction, large portions of the heartare damaged and cannot properly conduct theseelectrical signals. Cardiac tissue engineeringaims to repair and replace damaged tissue torestore electrical conductivity and contractilitytoward reestablishing normal heart function.Cell-sheet engineering as a scaffold-free methoddominates cardiac tissue engineering and aimsprimarily to achieve electrical conductance forsynchronous contractility. More recently, effortsto vascularize engineered cardiac tissues beforeimplantation have emerged.

Cell-sheet engineering techniques have beendeveloped to form 3D cardiac tissues capable ofelectrical communication. Synchronous andspontaneous beating was achieved by layeringcardiac cell sheets derived from embryonic car-diomyocytes (Shimizu et al. 2002). Electricalconnectivity via the formation of gap junctions,as indicated by connexin43 staining, has beenobserved in layered cardiac tissues (Shimizu etal. 2006; Matsuura et al. 2011). Because of themetabolic requirements of cardiac cells, vascu-larization of layered cardiac sheets is importantfor in vivo survival of the graft (Dilley and Mor-rison 2014). Multistep transplantation of tenthree-layer cardiac sheets cocultured with endo-thelial cells promoted vascularization in vivo,resulting in a fused, 30-layer-thick cardiac tissuebeating simultaneously (Shimizu et al. 2006).Electrical conductivity and synchronous beatingin engineered cardiac tissues can thus be achievedwith cell-sheet engineering. These studies showthat in vivo tissue engineering of cell sheets in-duces vascularization. Additional studies explor-ing in vitro vascularization and in vivo integra-tion of vascular cardiac tissues are needed topromote repair of damaged heart tissues.

Vascular

Diseases affecting the vascular system can leadto myocardial infarction, stroke, or peripherallimb ischemia. Vascular tissue engineering aimsto replace segments of diseased vessels. To reca-pitulate native tissue function, engineered ves-sels should be able to withstand physiologicalburst pressures; additionally, they should resistcyclic loading fatigue and maintain an endothe-lium layer (Seifu et al. 2013). In the last twodecades, vascular tissue engineering has diver-sified to include scaffold-free systems that in-clude self-assembly, cell-sheet, and spheroid-based techniques.

The self-assembling process has seen lim-ited application in vascular tissue engineering.Vascular rings were formed through self-as-sembly of smooth muscle cells before theywere placed sequentially on a silicone mandreland underwent fusion (self-organization)(Gwyther et al. 2011). Although the tensilemechanical properties of individual vascularrings were assessed (ultimate tensile strengthof 100–500 kPa), the functional properties ofthe fused tubular structure were not. In addi-tion to examining burst pressure, future workusing both self-assembly and self-organizationcould control cell placement (e.g., endothelialcells at the vascular ring center, smooth mus-cle cells in the media layer, and fibroblasts atthe outermost edge) to create a tissue-engi-neered vessel with structural morphology andmechanical properties reminiscent of vasculartissues.

Vascular tissue formation using cell-sheetengineering is the most popular scaffold-freesystem used. Sequential sheets of human vas-cular smooth muscle cells and fibroblastswrapped around a mandrel fused to form atubular vessel capable of endothelializationand showed a reported “burst strength”.2000 mmHg (�265 kPa) (L’Heureux et al.1998). Subsequent iterations of this methodproduced fibroblast- and endothelial-cell-basedvessels with reported burst pressures of morethan 3500 mmHg (�465 kPa) (L’Heureuxet al. 2006). Ascorbic acid-treated MSCs canalso be used in a similar process to generate a



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cell-sheet-based vascular tissue with suitablesuture loading strength (Zhao et al. 2012). Fi-nally, cell-sheet methods can be combined withscaffold-based technologies to enhance func-tional properties. Primary smooth muscle–cell-derived sheets seeded onto electrospun col-lagen/poly(1-caprolactone) scaffolds achievedadditional increases in tensile strength of ves-sels compared with the scaffold alone (Ahnet al. 2015). These increases in tensile proper-ties are encouraging, although burst pressurewas not assessed in this study (Ahn et al.2015). These studies show that cell-sheet-basedvascular tissue engineering can achieve func-tional burst pressure properties exceeding thoseof native tissue. Toward potentially engineeringcontractile arterial vessels, future work shouldinclude smooth muscle cell phenotypic main-tenance and/or differentiation and their in-corporation into a mechanically robust vascu-lar graft.

Finally, a combination of scaffold-freespheroid formation and the self-organizingtechnique of bioprinting has been used to alimited extent in vascular tissue engineering.This process used agarose as a mold to supportthe build process in bioprinting spheroids(formed via pellet culture) composed ofsmooth muscle cells and fibroblasts (Norotteet al. 2009). Layer-by-layer composition in thisstudy allowed for the design of a double-layeredvascular wall showing patterns of smooth mus-cle cell and fibroblast organization (Norotteet al. 2009). Interestingly, placement of stem-cell-based spheroids on a prestretched electro-spun scaffold resulted in incomplete fusion andhole formation in tissue-engineered vessels,suggesting that the scaffold may impede fusion(Beachley et al. 2014). Although burst pressureas a functional parameter was not assessed inthese spheroid-based studies, the results showthe ability to finely control structural architec-ture in vascular tissue engineering and achievesmall-diameter vessels (,5 mm). Additionalwork to enhance fusion of spheroid structuresand produce mechanically viable vessels, towardachieving branching vasculature capable ofwithstanding burst pressures, would greatlybenefit the field.

NONMECHANICAL TISSUES

Liver

Liver diseases including fibrosis and viral infec-tions have driven the need for alternativesources of healthy liver tissue because donorsare limited. The most successful option forcomplete liver failure remains liver transplanta-tion. Primary hepatocyte transplantation in-volving injection of a cell suspension has beenused, but transplantation of engineered tissuesis still under development. Liver tissue engi-neering is meant to both maintain hepatocytephenotype in culture and to differentiate pro-genitor cells into mature hepatocytes. To createa functional tissue-engineered solution, engi-neers focus on protein and metabolite secretion,primarily production of albumin, a1 antitryp-sin (A1AT), and the P450 cytochrome enzyme.Scaffold-free methods of aggregate or spheroidculture and cell-sheet techniques have beenused to achieve these objectives.

Scaffold-free spheroids are formed from avariety of cell sources and are the primary scaf-fold-free method used in liver tissue engineer-ing, as spheroids provide a means to maintainthe phenotype of liver cells. The dimensions ofpelleted aggregates can influence both the im-mediate and long-term expression levels of liv-er-specific albumin (Gevaert et al. 2014) andshould be considered in liver-tissue engineer-ing. Additionally, hepatocyte spheroids havebeen shown to survive and maintain their phe-notype at least 3 days when implanted in vivo(Ota et al. 1997). Coculture of hepatocytes andother cell types can further enhance the pheno-typic maintenance of hepatocytes. For instance,cocultures of hepatocytes and hepatic stellatecells induced increased expression of albuminand cytochrome P450 compared with hepato-cytes alone (Wong et al. 2011). Similarly, aggre-gates formed via coculture of hepatocytes andpancreatic islet cells not only maintained he-patocytic (and pancreatic) phenotypes but en-hanced expression of liver-specific proteinsover hepatocyte aggregates (Jun et al. 2013).These studies thus show the importance of scaf-fold-free spheroid culture not only to maintainbut also to enhance hepatocyte phenotype, es-

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pecially when hepatocytes are cultured withsupport cells. Future work should validate thelong-term phenotypic stability of hepatocytescultured as spheroids and their potential inlong-term restoration of liver function whenimplanted in vivo.

Cell-sheet culture in liver-tissue engineeringinvolves coculture of hepatocytes with an addi-tional cell source or layering of multiple hepa-tocyte sheets. Hepatocyte cell sheets showed ro-bust expression of albumin as evaluated viaimmunohistochemistry; increases in proteinproduction correlated with enhanced liver-tis-sue volume as a result of layering multiple cellsheets (Ohashi et al. 2007). In addition to robustalbumin and A1AT production, cell sheets de-rived from hepatocytes cocultured with fibro-blasts promoted enhanced vascularization aftersubcutaneous implantation when comparedwith hepatocyte-derived sheets (Sakai et al.2015). This scaffold-free coculture techniquecould address the pressing need for vasculariza-tion after transplantation to ensure survival ofengineered liver tissues. It is important to notethat the in vivo vascularization of engineeredliver tissues is encompassed within the in vivotissue-engineering methodology; successful invitro engineering of liver vasculature wouldgreatly advance the field. Scaffold-free cell sheetsof hepatocytes alone or in coculture are thusable to express proteins indicative of liver func-tion and induce vascularization of implantedtissues.

Cornea

The cornea is a transparent and avascular ocularstructure that provides physical protection tothe eye and serves as an optical interface. Anepithelium and endothelium layer comprise acombined �10% of the corneal thickness andact primarily as a barrier and integration pointto the remainder of the eye, respectively. Struc-turally, aligned collagen fibrils, termed lamellae,comprise the bulk of the corneal stroma, whichrepresents 90% of corneal thickness (Ghezziet al. 2015). Proteoglycans between lamellae lay-ers contribute to corneal transparency, a uniqueattribute of this tissue. Diseased or damaged

corneal tissues can lead to vision loss and blind-ness, creating a clinical need for tissue-engi-neered corneas for transplantation. The prima-ry challenge of engineering the cornea is tocreate a transparent structure with suitable ma-trix organization that confers substantial refrac-tive power and mechanical protection (Ghezziet al. 2015). Full-thickness corneal tissue engi-neering has primarily been completed with scaf-fold-based methods, although cell-sheet sys-tems have been used to engineer certain layers.

Cell-sheet engineering is the predominantscaffold-free method studied for corneal regen-eration. Researchers created autologous epithe-lial cell sheets that were able to restore the cor-nea’s transparency and natural barrier functionand improve visual acuity in human patients(Nishida et al. 2004b); this technique was pre-viously shown in a rabbit corneal model (Ni-shida et al. 2004a). Although these studies suc-cessfully created an epithelial cell sheet, they didnot create the stroma. Other cell-sheet work at-tempted to create thicker corneal tissues byaltering the cell-sheet growth substrate (Teich-mann et al. 2013) or by engineering specificallythe endothelium layer (Madathil et al. 2014).Despite this exciting work in stroma or endo-thelium engineering, these studies have not yetexamined the functional parameters of trans-parency and physical protection. Cell sheetsare promising for the creation of transparentand protective corneal layers. Additional studiescombining various cell-sheet layers may eluci-date the potential of generating full-thicknesscorneal transplants.

CONCLUSIONS AND FUTURE DIRECTIONS

In the last few decades, the tissue-engineeringfield has made tremendous strides toward cre-ating functional tissues able to replace thosedamaged by disease, trauma, or age. Scaffold-free tissue engineering recently emerged as analternative approach that uses only cells and sig-nals, aiming to exploit the benefits of scaffold-free systems. Within scaffold-free systems, self-organization and self-assembling processes canbe defined based on whether external energy isapplied (Fig. 1). As the field progresses in the



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continued use of scaffold-free systems, it willbecome critical to create stricter definitionsfor terminology used to denote various tech-niques; this issue is particularly important inthe development of spheroid, aggregate, andpellet-based technologies, terms often used in-terchangeably. Depending on the target tissue ofinterest, a given scaffold-free (or even scaffold-based) method may be preferable. This reviewsummarizes the current progress in the tissue-engineering field, focusing primarily on scaf-fold-free techniques. Specifically, we highlightthe recent advances and existing limitations inbiomimetic in vitro tissue engineering, towardcreating tissues that truly restore the function ofthe intended tissue targets. Although the fieldhas seen expansive growth with the advent ofnew technologies, this review highlights re-maining hurdles that need to be addressed forclinical translation.

Extensive progress in tissue engineering hasresulted in tissues that recapitulate certain met-rics of target tissues; to assess the long-termfunctionality and facilitate clinical translation,however, increased development and standard-ization of appropriate animal models are need-ed. These tissue-engineering models shouldmatch not only the disease characteristic, butthe defect characteristics as well. For example,osteoarthritis models should aim to better reca-pitulate not only the inflammatory environ-ment, but also the size and shape of a cartilagedefect. In addition to appropriately modelingthe disease state, standardization of animalmodels across research groups would assist indirect comparisons of studies. Use of selectFDA-approved models may guide animal selec-tion. In scaffold-free systems, specifically,achievement of sufficient mechanical propertiesis critical to the survival of the implant withinthe host environment. In the case of articularcartilage replacement, until mechanically bio-mimetic tissues can be engineered using scaf-fold-free systems, it may be advisable to explorerehabilitation techniques that use unloading ofthe patient’s joint until the neotissue has ma-tured mechanically. Because of the dependenceon high numbers of cells, scaffold-free tissue-engineered constructs may need to address the

permanence of these cells within the constructto determine that cells do not leave the implant-ed neotissue and elicit adverse effects elsewherein the host. Continued improvements to animalmodels will facilitate the translation of engi-neered tissues to human patients. Until trulybiomimetic tissue is created, use of scaffold-free techniques in the clinic may progress withappropriate rehabilitation and postoperativeprocedures in place to ensure neotissue matu-ration and development.

Cell sourcing is arguably the most limitingstep of tissue engineering—both scaffolds andsignals can be synthetically created, whereascells must be derived from a natural sourceand are thus a limited resource. The high cellnumbers needed for scaffold-free techniquesrender them particularly vulnerable to cell-sourcing issues. Using aggregate culture proto-cols, cell-sourcing limitations can be addressed,as these approaches can be an effective means topromote the desired cell phenotype in a 3D en-vironment. Because primary cells are limited inavailability, most tissue engineers select progen-itor cells that can be differentiated using aggre-gate culture. Once the desired phenotype hasbeen obtained, these cells can be used in a sub-sequent scaffold-free or scaffold-based methodto generate constructs of clinically relevant di-mensions. Increased development of our abilityto differentiate progenitor cells or maintain pri-mary cells in 3D culture can ultimately addressthe significant cell numbers needed in scaffold-free in vitro biomimetic tissue engineering.

Although this review focuses on scaffold-free systems, continued progress in tissue engi-neering may require the simultaneous use ofscaffold-free and scaffold-based techniques asthe complexity of engineered tissues increases.As mentioned previously, many cells are an-chorage-dependent and are not suitable foruse in scaffold-free systems. For instance, oste-oblasts require a scaffold for their survival.Therefore, to form a biphasic osteochondralgraft, a cell-laden bone scaffold may be used inconjunction with scaffold-free neocartilage.Identification of the appropriate system—scaf-fold-free or scaffold-based—for independentcell types will further the field in developing

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more complex tissues through the combinationof various systems.

Finally, enhanced understanding of the de-velopment of various tissue types will aid inidentification of whether scaffold-free or scaf-fold-based systems are most appropriate. In thisarticle, we highlight the parallels between scaf-fold-free neocartilage generation and in vivodevelopment of articular cartilage (Fig. 2). Mes-enchymal condensation of cartilaginous pre-cursors occurs in the absence of a scaffold; tissueengineering with chondrocytes in a scaffold-free process thus reflects the developmental en-vironment. The work in developmental biologycan thus inform the selection of tissue-engi-neering modalities. Increased understandingof the processes by which various tissues andorgans develop will aid the selection of scaf-fold-free or scaffold-based techniques.

A key focus of this work is the self-assem-bling process, which results in functional tissueformation in a cell-driven manner that requiresno external input of energy. In some tissue types(i.e., cartilage), the self-assembling processmimics natural mechanisms of developmentalbiology. By studying the self-assembling plat-form, enhanced understanding of developmentmay be achieved. Conversely, our current under-standing of developmental processes may be ap-plied to self-assembling techniques, toward dis-covering methods to enhance the functionalproperties of neotissue. Ultimately, engineeredneotissue will reach a level of complexity reca-pitulating native tissue functions, allowing neo-tissue not just to repair, but also to regeneratediseased tissues.

REFERENCES

Adkisson HD, Gillis MP, Davis EC, Maloney W, Hruska KA.2001. In vitro generation of scaffold independent neo-cartilage. Clin Orthop Relat Res 391: S280–S294.

Ahn H, Ju YM, Takahashi H, Williams DF, Yoo JJ, Lee SJ,Okano T, Atala A. 2015. Engineered small diametervascular grafts by combining cell sheet engineeringand electrospinning technology. Acta Biomater 16:14–22.

Akahane M, Shigematsu H, Tadokoro M, Ueha T, Matsu-moto T, Tohma Y, Kido A, Imamura T, Tanaka Y. 2010.Scaffold-free cell sheet injection results in bone forma-tion. J Tissue Eng Regen Med 4: 404–411.

Amini AR, Laurencin CT, Nukavarapu SP. 2012. Bone tissueengineering: Recent advances and challenges. Crit RevBiomed Eng 40: 363–408.

Ando W, Tateishi K, Katakai D, Hart DA, Higuchi C, NakataK, Hashimoto J, Fujie H, Shino K, Yoshikawa H, et al.2008. In vitro generation of a scaffold-free tissue-engi-neered construct (TEC) derived from human synovialmesenchymal stem cells: Biological and mechanicalproperties and further chondrogenic potential. TissueEng Part A 14: 2041–2049.

Arzi B, DuRaine GD, Lee CA, Huey DJ, Borjesson DL, Mur-phy BG, Hu JC, Baumgarth N, Athanasiou KA. 2015.Cartilage immunoprivilege depends on donor sourceand lesion location. Acta Biomater 23: 72–81.

Athanasiou KA, Eswaramoorthy R, Hadidi P, Hu JC. 2013.Self-organization and the self-assembling process in tis-sue engineering. Annu Rev Biomed Eng 15: 115–136.

Athanasiou KA, Responte DJ, Brown WE, Hu JC. 2015.Harnessing biomechanics to develop cartilage regenera-tion strategies. J Biomech Eng 137: 020901.

Athens AA, Makris EA, Hu JC. 2013. Induced collagencross-links enhance cartilage integration. PLoS ONE 8:e60719.

Avula MN, Rao AN, McGill LD, Grainger DW, Solzbacher F.2014. Foreign body response to subcutaneous biomate-rial implants in a mast cell-deficient Kitw-Sh murine mod-el. Acta Biomater 10: 1856–1863.

Beachley V, Kasyanov V, Nagy-Mehesz A, Norris R, OzolantaI, Kalejs M, Stradins P, Baptista L, da Silva K, Grainjero J,et al. 2014. The fusion of tissue spheroids attached to pre-stretched electrospun polyurethane scaffolds. J Tissue Eng5: 1–11.

Bentley G, Biant LC, Vijayan S, Macmull S, Skinner JA,Carrington RWJ. 2012. Minimum ten-year results of aprospective randomised study of autologous chondro-cyte implantation versus mosaicplasty for symptomaticarticular cartilage lesions of the knee. J Bone Joint Sur Br94: 504–509.

Bhumiratana S, Eton RE, Oungoulian SR, Wan LQ, AteshianGA, Vunjak-Novakovic G. 2014. Large, stratified, andmechanically functional human cartilage grown in vitroby mesenchymal condensation. Proc Natl Acad Sci 11:6940–6945.

Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O,Peterson L. 1994. Treatment of deep cartilage defects inthe knee with autologous chondrocyte transplantation. NEngl J Med 331: 889–895.

Brodland GW. 2002. The differential interfacial tension hy-pothesis (DITH): A comprehensive theory for the self-rearrangement of embryonic cells and tissues. J BiomechEng 124: 188–197.

Chaparro FJ, Matusicky ME, Allen MJ, Lannutti JJ. 2015.Biomimetic microstructural reorganization during su-ture retention strength evaluation of electrospun vascularscaffolds. J Biomed Mater Res B Appl Biomater 104: 1525–1534.

Darling EM, Athanasiou KA. 2005. Rapid phenotypicchanges in passaged articular chondrocyte subpopula-tions. J Orthop Res 23: 425–432.

DeLise AM, Fischer L, Tuan RS. 2000. Cellular interactionsand signaling in cartilage development. OsteoarthritisCartilage 8: 309–334.



ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Diekman BO, Christoforou N, Willard VP, Sun H, Sanchez-Adams J, Leong KW, Guilak F. 2012. Cartilage tissue en-gineering using differentiated and purified induced plu-ripotent stem cells. Proc Natl Acad Sci 109: 19172–19177.

Dilley RJ, Morrison WA. 2014. Vascularisation to improvetranslational potential of tissue engineering systems forcardiac repair. Int J Biochem Cell Biol 56: 38–46.

DuRaine GD, Brown WE, Hu JC, Athanasiou KA. 2015.Emergence of scaffold-free approaches for tissue engi-neering musculoskeletal cartilages. Ann Biomed Eng 43:543–554.

Gevaert E, Dolle L, Billiet T, Dubruel P, van Grunsven L, vanApeldoorn A, Cornelissen R. 2014. High throughput mi-cro-well generation of hepatocyte micro-aggregates fortissue engineering. PLoS ONE 9: e105171.

Ghezzi CE, Rnjak-Kovacina J, Kaplan DL. 2015. Cornealtissue engineering: Recent advances and future perspec-tives. Tissue Eng Part B Rev 21: 278–287.

Gurkan UA, Kishore V, Condon KW, Bellido TM, Akkus O.2011. A scaffold-free multicellular three-dimensional invitro model of osteogenesis. Calcif Tissue Int 88: 388–401.

Gwyther TA, Hu JZ, Billiar KL, Rolle MW. 2011. Directedcellular self-assembly to fabricate cell-derived tissue ringsfor biomechanical analysis and tissue engineering. J VisExp 57: e3366.

Hairfield-Stein M, England C, Paek HJ, Gilbraith KB, Den-nis R, Boland E, Kosnik P. 2007. Development of self-assembled, tissue-engineered ligament from bone mar-row stromal cells. Tissue Eng 13: 703–710.

Hildebrandt C, Buth H, Thielecke H. 2011. A scaffold-free invitro model for osteogenesis of human mesenchymalstem cells. Tissue Cell 43: 91–100.

Hoyer M, Meier C, Breier A, Hahner J, Heinrich G, DrechselN, Meyer M, Rentsch C, Garbe LA, Ertel W, et al. 2015. Invitro characterization of self-assembled anterior cruciateligament cell spheroids for ligament tissue engineering.Histochem Cell Biol 143: 289–300.

Hu JC, Athanasiou KA. 2006. A self-assembling process inarticular cartilage tissue engineering. Tissue Eng 12: 969–979.

Huey DJ, Hu JC, Athanasiou KA. 2012. Unlike bone, carti-lage regeneration remains elusive. Science 338: 917–921.

Jun Y, Kang AR, Lee JS, Jeong GS, Ju J, Lee DY, Lee SH. 2013.3D co-culturing model of primary pancreatic islets andhepatocytes in hybrid spheroid to overcome pancreaticcell shortage. Biomaterials 34: 3784–3794.

Kraft JJ, Jeong C, Novotny JE, Seacrist T, Chan G, DomzalskiM, Turka CM, Richardson DW, Dodge GR. 2011. Effectsof hydrostatic loading on a self-aggregating, suspensionculture-derived cartilage tissue analog. Cartilage 2: 254–264.

Krieg M, Arboleda-Estudillo Y, Puech PH, Kafer J, Graner F,Muller DJ, Heisenberg CP. 2008. Tensile forces governgerm-layer organization in zebrafish. Nat Cell Biol 10:429–436.

L’Heureux N, Paquet S, Labbe R, Germain L, Auger FA.1998. A completely biological tissue-engineered humanblood vessel. FASEB J 12: 47–56.

L’Heureux N, Dusserre N, Konig G, Victor B, Keire P, WightTN, Chronos NA, Kyles AE, Gregory CR, Hoyt G, et al.

2006. Human tissue-engineered blood vessels for adultarterial revascularization. Nat Med 12: 361–365.

Lim HC, Bae JH, Song SH, Park YE, Kim SJ. 2012. Currenttreatments of isolated articular cartilage lesions of theknee achieve similar outcomes. Clin Orthop Relat Res470: 2261–2267.

Lu Y, Adkisson HD, Bogdanske J, Kalscheur V, Maloney W,Cheung R, Grodzinsky AJ, Hruska KA, Markel MD. 2005.In vivo transplantation of neonatal ovine neocartilageallografts: Determining the effectiveness of tissue trans-glutaminase. J Knee Surg 18: 31–42.

Ma D, Ren L, Liu Y, Chen F, Zhang J, Xue Z, Mao T. 2010.Engineering scaffold-free bone tissue using bone marrowstromal cell sheets. J Orthop Res 28: 697–702.

Madathil BK, Kumar PRA, Kumary TV. 2014. N-isopropy-lacrylamide-co-glycidylmethacrylate as a thermorespon-sive substrate for corneal endothelial cell sheet engineer-ing. Biomed Res Int doi: 10.1155/2014/450672.

Makris EA, Responte DJ, Paschos NK, Hu JC, AthanasiouKA. 2014. Developing functional musculoskeletal tissuesthrough hypoxia and lysyl oxidase-induced collagencross-linking. Proc Natl Acad Sci 111: E4832–E4841.

Makris EA, Gomoll AH, Malizos KN, Hu JC, AthanasiouKA. 2015. Repair and tissue engineering techniques forarticular cartilage. Nat Rev Rheumatol 11: 21–34.

Manning ML, Foty RA, Steinberg MS, Schoetz EM. 2010.Coaction of intercellular adhesion and cortical tensionspecifies tissue surface tension. Proc Natl Acad Sci 107:12517–12522.

Matsuura K, Masuda S, Haraguchi Y, Yasuda N, Shimizu T,Hagiwara N, Zandstra PW, Okano T. 2011. Creation ofmouse embryonic stem cell-derived cardiac cell sheets.Biomaterials 32: 7355–7362.

McAllister TN, Maruszewski M, Garrido SA, WystrychowskiW, Dusserre N, Marini A, Zagalski K, Fiorillo A, Avila H,Manglano X, et al. 2009. Effectiveness of haemodialysisaccess with an autologous tissue-engineered vasculargraft: A multicentre cohort study. Lancet 373: 1440–1446.

McCormick F, Cole BJ, Nwachukwu B, Harris JD, AdkissonIv HD, Farr J. 2013. Treatment of focal cartilage defectswith a juvenile allogeneic 3-dimensional articular carti-lage graft. Oper Tech Sports Med 21: 95–99.

Mohanraj B, Farran AJ, Mauck RL, Dodge GR. 2014. Time-dependent functional maturation of scaffold-free carti-lage tissue analogs. J Biomech 47: 2137–2142.

Murphy MK, Masters TE, Hu JC, Athanasiou KA. 2013a.Engineering a fibrocartilage spectrum through modula-tion of aggregate redifferentiation. Cell Transplant 24:235–245.

Murphy MK, DuRaine GD, Reddi A, Hu JC, Athanasiou KA.2013b. Inducing articular cartilage phenotype in costo-chondral cells. Arthritis Res Ther 15: R214.

Murphy MK, Huey DJ, Hu JC, Athanasiou KA. 2015. TGF-b1, GDF-5, and BMP-2 stimulation induces chondro-genesis in expanded human articular chondrocytes andmarrow-derived stromal cells. Stem Cells 33: 762–773.

Ni M, Rui YF, Tan Q, Liu Y, Xu LL, Chan KM, Wang Y, Li G.2013. Engineered scaffold-free tendon tissue produced bytendon-derived stem cells. Biomaterials 34: 2024–2037.

J.K. Lee et al.


ww

w.p

ersp

ecti

vesi

nm

edic

ine.

org



Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N,Watanabe H, Yamamoto K, Nagai S, Kikuchi A, Tano Y, etal. 2004a. Functional bioengineered corneal epithelialsheet grafts from corneal stem cells expanded ex vivoon a temperature-responsive cell culture surface. Trans-plantation 77: 379–385.

Nishida K, Yamato M, Hayashida Y, Watanabe K, YamamotoK, Adachi E, Nagai S, Kikuchi A, Maeda N, Watanabe H,et al. 2004b. Corneal reconstruction with tissue-engi-neered cell sheets composed of autologous oral mucosalepithelium. N Engl J Med 351: 1187–1196.

Norotte C, Marga FS, Niklason LE, Forgacs G. 2009. Scaf-fold-free vascular tissue engineering using bioprinting.Biomaterials 30: 5910–5917.

Ofek G, Revell CM, Hu JC, Allison DD, Grande-Allen KJ,Athanasiou KA. 2008. Matrix development in self-assem-bly of articular cartilage. PLoS ONE 3: e2795.

Ohashi K, Yokoyama T, Yamato M, Kuge H, Kanehiro H,Tsutsumi M, Amanuma T, Iwata H, Yang J, Okano T, et al.2007. Engineering functional two- and three-dimension-al liver systems in vivo using hepatic tissue sheets. NatMed 13: 880–885.

Ong SY, Dai H, Leong KW. 2006. Inducing hepatic differen-tiation of human mesenchymal stem cells in pellet cul-ture. Biomaterials 27: 4087–4097.

Ota K, Saito S, Fujisawa K, Tanaka N. 1997. Xenotransplan-tation of spheroidal aggregate-cultured hepatocytes.Transplant Proc 29: 912–913.

Peng G, McNary SM, Athanasiou KA, Reddi AH. 2014. Sur-face zone articular chondrocytes modulate the bulk andsurface mechanical properties of the tissue engineeredcartilage. Tissue Eng Part A 20: 3332–3341.

Pirraco RP, Iwata T, Yoshida T, Marques AP, Yamato M, ReisRL, Okano T. 2014. Endothelial cells enhance the in vivobone-forming ability of osteogenic cell sheets. Lab Invest94: 663–673.

Raghothaman D, Leong MF, Lim TC, Toh JK, Wan AC, YangZ, Lee EH. 2014. Engineering cell matrix interactions inassembled polyelectrolyte fiber hydrogels for mesenchy-mal stem cell chondrogenesis. Biomaterials 35: 2607–2616.

Sakai Y, Yamanouchi K, Ohashi K, Koike M, Utoh R, Hase-gawa H, Muraoka I, Suematsu T, Soyama A, Hidaka M, etal. 2015. Vascularized subcutaneous human liver tissuefrom engineered hepatocyte/fibroblast sheets in mice.Biomaterials 65: 66–75.

Seifu DG, Purnama A, Mequanint K, Mantovani D. 2013.Small-diameter vascular tissue engineering. Nat Rev Car-diol 10: 410–421.

Shimizu T, Yamato M, Akutsu T, Shibata T, Isoi Y, Kikuchi A,Umezu M, Okano T. 2002. Electrically communicatingthree-dimensional cardiac tissue mimic fabricated by lay-ered cultured cardiomyocyte sheets. J Biomed Mater Res60: 110–117.

Shimizu T, Sekine H, Yang J, Isoi Y, Yamato M, Kikuchi A,Kobayashi E, Okano T. 2006. Polysurgery of cell sheetgrafts overcomes diffusion limits to produce thick, vas-cularized myocardial tissues. FASEB J 20: 708–710.

Shimizu T, Akahane M, Morita Y, Omokawa S, Nakano K,Kira T, Onishi T, Inagaki Y, Okuda A, Kawate K, et al.2015. The regeneration and augmentation of bone withinjectable osteogenic cell sheet in a rat critical fracturehealing model. Injury 46: 1457–1464.

Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P.1994. N-CAM and N-cadherin expression during in vitrochondrogenesis. Exp Cell Res 215: 354–362.

Teichmann J, Valtink M, Gramm S, Nitschke M, Werner C,Funk RH, Englemann K. 2013. Human corneal endothe-lial cell sheets for transplantation: Thermo-responsivecell culture carriers to meet cell-specific requirements.Acta Biomater 9: 5031–5039.

Theiss F, Mirsaidi A, Mhanna R, Kummerle J, Glanz S, Bah-renberg G, Tiaden AN, Richards PJ. 2015. Use of biomi-metic microtissue spheroids and specific growth factorsupplementation to improve tenocyte differentiation andadaptation to a collagen-based scaffold in vitro. Bioma-terials 69: 99–109.

Tuan RS. 2007. A second-generation autologous chondro-cyte implantation approach to the treatment of focal ar-ticular cartilage defects. Arthritis Res Ther 9: 109.

Vunjak-Novakovic G, Martin I, Obradovic B, Treppo S,Grodzinsky AJ, Langer R, Freed LE. 1999. Bioreactor cul-tivation conditions modulate the composition and me-chanical properties of tissue-engineered cartilage. J Or-thop Res 17: 130–138.

Wong SF, No DY, Choi YY, Kim DS, Chung BG, Lee SH.2011. Concave microwell based size-controllable hepato-sphere as a three-dimensional liver tissue model. Bioma-terials 32: 8087–8096.

Zhao J, Liu L, Wei J, Ma D, Geng W, Yan X, Zhu J, Du H, LiuY, Li L, et al. 2012. A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesen-chymal stem cells. Artif Organs 36: 93–101.



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published online March 27, 2017Cold Spring Harb Perspect Med Jennifer K. Lee, Jarrett M. Link, Jerry C.Y. Hu and Kyriacos A. Athanasiou The Self-Assembling Process and Applications in Tissue Engineering

Subject Collection Tissue Engineering and Regenerative Medicine

Craniofacial Tissue EngineeringWeibo Zhang and Pamela Crotty Yelick Blood Vessel Developments

Historical Perspective and Future Direction of

Sashka Dimitrievska and Laura E. Niklason

Bone and Cartilage Tissue EngineeringHoning Cell and Tissue Culture Conditions for

Johnny Lam, Esther J. Lee, Elisa C. Clark, et al.Tissue EngineeringThe Self-Assembling Process and Applications in

Jennifer K. Lee, Jarrett M. Link, Jerry C.Y. Hu, et al.

CellsRegions: The Importance of Stem and Progenitor Tissue Engineering Functional Gastrointestinal

Andrew Trecartin and Tracy Grikscheit

Biologic Scaffolds

Londono, et al.Alessandra Costa, Juan Diego Naranjo, Ricardo

The Heart and Great VesselsEkene Onwuka, Nakesha King, Eric Heuer, et al. Tissue Engineering

Three-Dimensional Bioprinting Strategies for

Dokmeci, et al.Yu Shrike Zhang, Rahmi Oklu, Mehmet Remzi

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